GENE CLONING, CHARACTERIZATION AND
THERMODYNAMIC STUDIES OF HIGHLY THERMOSTABLE
CELLULOLYTIC ENZYMES FROM GENUS THERMOTOGA
A THESIS TITLED
NAME: FATIMA AKRAM
Registration No.
2013–PhD–BIOT–12
INSTITUTE OF INDUSTRIAL BIOTECHNOLOGYGC
UNIVERSITY LAHORE
Gene Cloning, Characterization and Thermodynamic
Studies of Highly Thermostable Cellulolytic Enzymes
from Genus Thermotoga
A THESIS TITLED
Submitted to GC University Lahore
in partial fulfillment of the requirements
for the award of degree of
Doctor of Philosophy
In
Biotechnology
By
FATIMA AKRAM
Registration No.
2013–PhD–BIOT–12
INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY
GC UNIVERSITY LAHORE
IN THE NAME OF
WHO HAS CREATED US
AND
MADE FOR US THE FACULTIES OF
HEARING, SEEING, FEELING
AND
UNDERSTANDING
ALLAH
Declaration
i
DECLARATION
I, Ms. Fatima Akram Registration No. 2013-PhD-BIOT-12 hereby declare that the matter
printed in the thesis titled Gene Cloning, Characterization and Thermodynamic
Studies of Highly Thermostable Cellulolytic Enzymes from Genus Thermotoga is my
own work and has not been submitted and shall not be submitted in future as research
work, thesis for the award of similar degree in any University, Research Institution etc in
Pakistan or abroad.
At any time, if my statement is found to be incorrect, even after my Graduation, the
University has the right to withdraw my PhD Degree.
Dated: 02-06-2017
Signature of Deponent
Plagiarism Undertaking
ii
PLAGIARISM UNDERTAKING
I, Ms. Fatima Akram Registration No. 2013-PhD-BIOT-12 solemnly declare that the
research work presented in the thesis titled “Gene Cloning, Characterization and
Thermodynamic Studies of Highly Thermostable Cellulolytic Enzymes from Genus
Thermotoga”is solely my research work, with no significant contribution from any other
person. Small contribution/help wherever taken has been acknowledged and that
complete thesis has been written by me.
I understand the zero tolerance policy of HEC and Government College University
Lahore, towards plagiarism. Therefore I as an author of the above titled thesis declare that
no portion of my thesis has been plagiarized and any material used as reference has been
properly referred/cited.
I understand that if I am found guilty of any formal plagiarism in the above titled thesis,
even after the award of PhD Degree, the University reserves the right to withdraw my
PhD Degree and that HEC/University has the right to publish my name on
HEC/University website, in the list of culprits of plagiarism.
Dated: 02.06.2017
Signatures of Deponent
Research Completion Certificate
iii
RESEARCH COMPLETION CERTIFICATE
Certified that the research work contained in this thesis titled “Gene Cloning,
Characterization and Thermodynamic Studies of Highly Thermostable Cellulolytic
Enzymes from Genus Thermotoga”has been carried out and completed by Ms.Fatima
Akram Registration No. 2013-PhD-BIOT-12 under my supervision. This Thesis has
been submitted in partial fulfillment of the requirements for the award of Degree of
Doctor of Philosophy in Biotechnology.
Certificate of Approval
iv
Dedication
v
DEDICATED TO
My Supervisor
DR. IKRAM-UL-HAQ (SI), FPAS,
Professor Emeritus, ISESCO Laureate and
Distinguished National Professor,
for his support, encouragement
and help throughout my research work.
Acknowledgements
vi
ACKNOWLEDGEMENTS
Myriads of thanks to Almighty ALLAH, the plenipotent who endowed upon me
potential, courage, resoluteness and enabled me to accomplish this research work, I invoke
peace for Holy Prophet Hazrat Muhammad (Sallaho Alahe Wasallam), who is
forever a torch of guidance for humanity as a whole and showing us the path of knowledge.
There are no words to express sincerest gratitude to my great supervisor Prof. Dr.
Ikram-ul-Haq (Sitara-e-Imtiaz), FPAS, Distinguished National Professor,
Emeritus Professor Institute of industrial Biotechnology, GC University Lahore and
principle investigator of the project entitled “Production of bioenergy from plant
biomass” funded by Ministry of Science & Technology (MoST), Government of Pakistan.
Present research work is the part of this project. I would like to pay my best regards and
deepest gratitude to my impressive supervisor for grant me the opportunity to work in his
project under his dynamic supervision, consolatory behavior and fatherly affection during
the course of research work. His untiring attitude towards work has given me enthusiasm
to follow this approach towards life. I am really fortunate to be a part of his research group.
I could not have imagined having a better supervisor and mentor for my Ph.D. I am highly
thankful to ORIC, GC University Lahore to allot me funds for the completion of my
research work.
I wish to express my gratitude to respected Prof. Naeem Rashid, Director of School of
Biological Sciences (SBS), University of the Punjab Lahore, for his constructive advice,
directions, and guidance during purification of cloned enzymes. I wish to express my
gratitude to respected Prof. Dr. Hamid Mukhtar, Director of Institute of industrial
Biotechnology, GC University Lahore, for his constructive advice, support and professional
guidance. I express my humble gratitude to all my teachers. I would like to thank Mr. Ali
Nawaz, lecturer IIB and express my special thanks to all the authors of the references. I
would also like to thank Ms. Sumra, Ms. Sana, Ms. Saira, Ms. Madeeha Ahmad and Ms.
Iqra (research officers), my research student Muhammad Kaleem deserve special thanks as
Acknowledgements
vii
he was used to stay and work for me till late while I was optimizing the culture conditions.
I would also like to express warm thanks to all the clerical staff of ORIC and IIB specially
Mr. Abdul Sitar and Mr. Habib, and all lab attendants of IIB.
Words are inadequate to express my warmest gratitude to my grandfather, parents,
whose support, motivation and encourage me at each step of life and whose prayers made
it possible for me to carry out my work progressively. My special thanks to my brother Mr.
Wajid Ali, sweet sister and loving cousins for their moral support, always being available
to give positive suggestions and help. Special thanks to all my friends and well-wishers.
Fatima Akram.
Table of Contents
viii
TABLE OF CONTENTS
Declaration………………………………….…………………………………….. i
Plagiarism Undertaking……………………………….………………………...... ii
Research Completion Certificate………….……………………………………... iii
Certificate of Approval…………………….……………………………………... iv
Dedication………………………………….……………………………………... v
Acknowledgements………………………………….……………………………. vi
Table of Contents………………………………….……………………………… viii
List of Tables………………………………….………………………………….. x
List of Illustrations…………………………….………………………………….. xii
List of Abbreviations……………………….…………………………………….. xvii
Abstract………………………………….……………………………………….. xx
CHAPTER NO. 01: INTRODUCTION
1.1. Introduction……….……………………….………………………………….. 01
1.2. Objective……………………………………………………………………… 19
CHAPTER NO. 02: REVIEW OF LITERATURE
2. Review of Literature………………………….………………………………. 20
CHAPTER NO. 03: MATERIAL AND METHODS
3.1. Materials……………………….……………………………………………… 45
3.2. Cloning and Sequencing of TnbglA and TnbglB Genes……………………… 54
3.3. Sub-cloning of TnbglA and TnbglB in Expression Vector…………………… 67
3.4. Heterologous Expression and Production of TnBglA and TnBglB…………… 71
3.5. Purification of Recombinant TnBglA and TnBglB…………………………… 81
3.6. Characterization of Recombinant TnBglA and TnBglB………………………. 85
CHAPTER NO. 04: RESULTS
4.1. β-1,4-glucosidase genes (bglA and bglB) from T. naphthophila …………….. 93
Table of Contents
ix
4.2. Gene Sequence of TnbglA…………………………………………………….. 94
4.3. Gene Sequence of TnbglB…………………………………………………….. 95
4.4. Cloning and Sequencing of TnbglA and TnbglB Genes……………………… 97
4.5. Sub-cloning of TnbglA and TnbglB in Expression Vector…………………… 127
4.6. Expression Analysis of Recombinant Proteins……………………………….. 131
4.7. Preliminary Characterization of TnBglA and TnBglB………………………… 132
4.8. Production of Recombinant TnBglA and TnBglB…………………………….. 137
4.9. Purification of Recombinant TnBglA and TnBglB……………………………. 148
4.10. Characterization of Purified TnBglA and TnBglB…………..……………….. 153
4.11. Structure Analysis of TnBglA and TnBglB………………………………….. 174
4.12. Enhanced Production of Recombinant TnBglA and TnBglB………………... 190
CHAPTER NO. 05: DISCUSSION
5. Discussion………………………………….……………………………….. 204
CHAPTER NO. 06: CONCLUSION
6. Conclusion………………………………….……………………………….. 232
CHAPTER NO. 07: REFERENCES
7. References………………………………….……………………………….. 233
ANNEX
Research Publication and Patent Application...….…………………………. 258
List of Tables
x
LIST OF TABLES
Table Titles Pages
3.1 Composition of amplification reaction mixture. 59
3.2 Compositions of different media. 73
3.3 SDS-PAGE resolving gel composition. 76
3.4 SDS-PAGE Stacking gel composition. 76
3.5 Native resolving/separating gel composition 78
3.6 Native stacking gel composition 79
4.1 Characteristics details of TnbglA and TnbglB genes of T. naphthophila. 94
4.2 (a) List of Non-cutter enzymes for TnbglA.
(b): List of Non-cutter enzymes for TnbglB
99
100
4.3 Oligonucleotides for PCR amplification of TnbglA and TnbglB genes. 101
4.4 (a) Nucleotide sequence BLAST result of TnbglA.
(b) Nucleotide sequence BLAST result of TnbglB.
111
115
4.5 (a) Output data of SignalP 4.1 for TnBglA Signal peptide prediction.
(b): Output data of SignalP 4.1 for TnBglB Signal peptide prediction.
121
122
4.6 Theoretical Properties of TnBglA and TnBglB Protein using ProtParam
tool.
123
4.7 (a) Purification summary of recombinant TnBglA
(b) Purification summary of recombinant TnBglB
150
151
4.8 Substrate specificity of the recombinant TnBglA and TnBglB 166
4.9 (a) Kinetic parameters of TnBglA
(b) Kinetic parameters of TnBglB
168
169
4.10 (a) Kinetic parameters of TnBglA for the hydrolysis of pNPG substrate.
(b) Kinetic parameters of TnBglB for the hydrolysis of pNPG substrate
169
169
4.11 (a) Thermodynamic parameters of TnBglA for pNPG hydrolysis.
(b) Thermodynamic parameters of TnBglB for pNPG hydrolysis.
171
172
4.12 (a) Thermodynamic parameters for denaturation of recombinant TnBglA
(b) Thermodynamic parameters for denaturation of recombinant TnBglB
175
175
List of Tables
xi
4.13 (a) Statistics of Ramachandran Plot. Results computed after Procheck
analysis of TnBglA protein structure using PDBsum
(b) Statistics of Ramachandran Plot. Results computed after Procheck
analysis of TnBglB protein structure using PDBsum
178
180
4.14 (a) Comparison of TnBglA production, induced with 0.5 mM IPTG
(b) Comparison of TnBglA production, induced with 150 mM lactose
201
201
4.15 (a) Comparison of TnBglB production, induced with 0.5 mM IPTG
(b) Comparison of TnBglB production, induced with 150 mM lactose
202
202
List of Illustrations
xii
LIST OF ILLUSTRATIONS
Figures Title Page
1.1 Structure of lignocellulosic plant biomass 04
1.2 Hierarchical structure of cellulose extracted from plants 04
1.3 Schematic representation of biodegradation of cellulosic substrate by using
cellulases
07
1.4 The catalytic retaining two steps mechanism of β-glucosidase for the hydrolysis
of β-glycosidic bond
13
1.5 (a) General structure of Thermotoga
(b) Octopus Spring, water from the main spring supports the growth of a variety
of Thermotoga species
17
1.6 Electron micrographs of stained ultrathin section of Thermotoga naphthophila
RKU-10T strain. Arrows indicate the cell wall (cw) and toga (t)
17
3.1 GeneRuler DNA Ladder Mix, ready-to-use #SM0333 (0.1µg µL-1) 58
3.2 (a) PCR cycling conditions for bglA
(b) PCR cycling conditions for bglB
60
3.3 Restriction map of pTZ57R/T 62
3.4 Map of pET-21a(+) expression vector with cloning and expression sites 68
3.5 Standard curve of Bovine Serum Albumin (BSA) 75
3.6 Novagen Perfect Protein Marker (10-225 kDa), Cat # 69079-3 77
3.7 Standard curve of para-nitrophenol (pNP) 81
3.8 Standard curve of Glucose 89
3.9 Standard curve of Xylose 90
4.1 (a) Linear sequence of TnbglA gene with NEB single cutter endonucleases
(b) Linear sequence of TnbglB gene with NEB single cutter endonucleases
98
99
4.2 Agarose gel of isolated genomic DNA from Thermotoga naphthophila 102
4.3 (a) Analysis of TnbglA amplified product resolved on 0.8% agarose gel
(b) Analysis of TnbglB amplified product after agarose gel electrophoresis
103
103
4.4 Analysis of TnbglA and TnbglB purified PCR product 104
List of Illustrations
xiii
4.5 Blue-white sereening 105
4.6 Randamly selected well-isolated white colonies from master plates 105
4.7 (a) Screening of positive transformants of E. coli DH5α harboring pTZ57R/T–
TnbglA plasmids by colony-PCR
(b) Screening of positive transformants of E. coli DH5α harboring pTZ57R/T–
TnbglB by colony-PCR
106
106
4.8 Isolated recombinant plasmids pTZ57R/T–TnbglA and pTZ57R/T–TnbglB 107
4.9 (a) Restrition analysis of pTZ57R/T–TnbglA plasmid using single restriction
enzyme
(b) Restrition analysis of pTZ57R/T–TnbglB plasmid using single restriction
enzyme
108
108
4.10 (a) Sequence alignment between cloned TnbglA and NCBI retrieved sequence
(b) Sequence alignment between cloned TnbglB and NCBI retrieved sequence
111
114
4.11 (a) Multi-alignment of β-glucosidase from T. naphthophila (TnBglA) with some
other GH1 family β-glucosidases
(b) Multi-alignment of β-glucosidase from T. naphthophila (TnBglB) with some
other GH3 family β-glucosidases
117
120
4.12 (a) Signal peptide prediction graph of TnBglA
(b) Signal peptide prediction graph of TnBglB
121
122
4.13 (a) Analysis of recombinant pTZ57R/T–TnbglA plasmid after double digestion
using two restriction endonucleases
(b) Analysis of recombinant pTZ57R/T–TnbglB plasmid after double digestion
using two restriction endonucleases
124
125
4.14 Streak plate of E. coli DH5α harboring pET-21a(+) 126
4.15 (a) Isolation of plasmid pET-21a(+)
(b) Double restricted pET-21a(+) using restriction endonucleases
126
126
4.16 (a) Purified double restricted pET-21a(+)
(b) Purified double digested DNA fragments (TnbglA and TnbglB)
127
127
4.17 Several white bacterial colonies were appeared on LB-agar plates supplement
with ampicillin after transformation of E. coli DH5α with constructed
expression plasmids
128
List of Illustrations
xiv
4.18 Colony PCR for the screening of positive transformants of E. coli DH5α
harboring constructed recombinant plasmids
128
4.19 Isolated recombinant plasmids treated with RNase 129
4.20 Analysis of recombinant constructed expression plasmids after double digestion
using restriction endonucleases
130
4.21 Several white bacterial colonies were appeared on LB-agar plates supplement
with ampicillin and chloramphenicol after transformation of E. coli BL21
CodonPlus (DE3)-RIPL with recombinant circular plasmids
130
4.22 Colony PCR for the screening of positive transformants of E. coli BL21
CodonPlus harboring constructed recombinant plasmids
131
4.23 SDS-PAGE analysis of recombinant proteins expression in E. coli BL 21
CodonPlus DE3-(RIPL)
132
4.24 Effect of pH and various buffer system on the activity of TnBglA 134
4.25 Effect of temperature on TnBglA and TnBglB 135
4.26 Effect of time course profile of recombinant TnBglA and TnBglB 135
4.27 Cell fractionation analysis for cloned TnBglA and TnBglB 136
4.28 SDS-PAGE analysis of cell lysate (intracellular) and supernatant (extracellular)
fractions of recombinant proteins expressed in E. coli BL 21
137
4.29 Effect of Pre-induction optimal cell density of cloned bacterial on recombinant
enzymes expression
138
4.30 Effect of heat shock treatment on recombinant TnBglA and TnBglB expression 139
4.31 Analysis of different concentration of IPTG as inducer on TnBglA and TnBglB
expression
140
4.32 Effect of different concentration of IPTG on recombinant enzymes expression 141
4.33 Effect of different induction temperature on recombinant TnBglA and TnBglB
expression
142
4.34 Effect of agitation speed on culture growth and recombinant enzymes
expression
143
4.35 Effect of medium pH on the recombinant protein expression 145
4.36 Analysis of TnBglA and TnBglB expression after induction 146
4.37 Effect of incubation time on recombinant enzymes expression 147
List of Illustrations
xv
4.38 (a) SDS-PAGE analysis of TnBglA from pET-21a–bglA in E. coli BL21
CodonPlus (DE3)-RIPL
(b) SDS-PAGE analysis of TnBglB from pET-21a–bglB in E. coli BL21
CodonPlus (DE3)-RIPL
150
151
4.39 Native PAGE analysis of TnBglA and TnBglB 152
4.40 Effect of pH on the activity of purified TnBglA and TnBglB 154
4.41 Effect of temperature on the activity of purified TnBglA and TnBglB 155
4.42 Effect of time course profile of recombinant TnBglA and TnBglB 156
4.43 Thermal stability of TnBglA and TnBglB 157
4.44 pH stability of TnBglA and TnBglB 158
4.45 Effect of metal ions and different inhibitors on purified TnBglA and TnBglB
activity
160
4.46 Effect of organic solvent on purified TnBglA and TnBglB activity 162
4.47 Effect of short chain alcohol on purified TnBglA and TnBglB activity 163
4.48 Effect of glucose and xylose on purified TnBglA and TnBglB activity 165
4.49 (a) Lineweaver-Burk plot for the hydrolysis of various substrates using TnBglA
(b) Lineweaver-Burk plot for the hydrolysis of various substrates using
TnBglB.
167
168
4.50 Arrhenius plot of recombinant enzymes of T. naphthophila 171
4.51 Pseudo-first-order plots for irreversible thermal denaturation of recombinant
thermostable enzymes
173
4.52 Arrhenius plot for irreversible thermal inactivation of purified recombinant
enzymes
174
4.53 (a) Secondary structure of TnBglA
(b) Secondary structure of TnBglB
176
177
4.54 (a) Ramachandran Plot for TnBglA on Procheck using PDBsum
(b) Ramachandran Plot for TnBglB on Procheck using PDBsum
178
179
4.55 (a) Three-dimensional (3-D) model structure of TnBglA
(b) Catalytic triad of TnBglA
(c) Three-dimensional (3-D) model structure of TnBglB
(d) Catalytic triad of TnBglB
181
182
183
184
List of Illustrations
xvi
4.56 Ligplot output for H-bond interactions of amino acid residues of TnBglA
(a) Involved in binding with cellobiose
(b) With the p-phenyl sugars as substrates
(c) Surface view of 3-D structure of TnBglA
185
186
187
4.57 Ligplot output for H-bond interactions of amino acid residues of TnBglB
(a) Involved in binding with cellobiose
(b) With the p-phenyl sugars as substrates
(c) Surface view of 3-D structure of TnBglB
188
189
189
4.58 Effect of media on recombinant enzymes activity induced with 0.5 mM IPTG 191
4.59 Effect of media on recombinant enzymes activity induced with 150 mM lactose 192
4.60 SDS-PAGE analysis of recombinant TnBglA in E. coli BL 21 grown in various
0.5 mM IPTG inducing media
193
4.61 SDS-PAGE analysis of TnBglB in E. coli grown in various IPTG inducing
media
194
4.62 SDS-PAGE analysis of TnBglA in E. coli grown in various lactose inducing
media
195
4.63 SDS-PAGE analysis of TnBglB in E. coli grown in various lactose inducing
media
196
4.64 Effect of IPTG on recombinant enzymes expression in 4×ZB medium 198
4.65 Effect of lactose on the expression of recombinant TnBglA and TnBglB
enzymes in 4×ZB medium
199
4.66 SDS-PAGE analysis of recombinant purified TnBglA and TnBglB after lactose
(150 mM) induction in 4×ZB medium
203
List of Abbreviations
xvii
LIST OF ABBREVIATIONS
AEC Anion exchange chromatography
Ag Silver
ANOVA Analysis of variance
Aͦ Angstrom
APS Ammonium per sulphate
Asp/D Aspartic acid
α Alpha
Ba Barium
β Beta
BGL/Bgl Beta-glucosidase/β-glucosidase
BLAST Basic Local Alignment Search Tool
BME Beta-mercaptorthanol
Bp Base pair
BSA Bovine serum Albumin
Ca Calcium
CaCl2 Calcium chloride
Cal Calorie
°C Degrees Celsius
CAPS 3-(cyclohexylamino)-1-propanasulfonic acid
CH3CO2K Potassium acetate
CMC Carboxymethyl cellulose
Conc. Concentration
C-Score Raw cleavage site score (for signal peptide determination)
Cu Copper
Da Daltons
ddH2O Double distilled water
dH2O Distilled water
DCW Dry cell weight
DNA Deoxyribonucleic acid
DNS 3,5-Dinitrosalicylic acid
dNTPs Deoxyribonucleoside triphosphates
D-Score Discrimination score
DTT Dithiothreitol
EDTA Ethylene diamine tetraacetic acid
ExPASY Expert Protein Analysis System
Fe Iron
G Gibb’s free energy
g/L Grams per liter
GHs Glycosyl hydrolase(s)
List of Abbreviations
xviii
Glu/E Glutamic acid
H Enthalpy
h Hours(s)
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIC Hydrophobic Interaction chromatography
His/H Histidine
IEC/IEXC Ion exchange chromatography
IPTG Isopropyl-β-D-thiogalactopyranoside
J Joule
K Kelvin
kcat Turnover number
kcat/Km Catalytic efficiency
kb Kilo base pair/1,000 bp
Kd Dissociation constant
kDa Kilo Daltons
Ki Inhibitor constant
Km Michaelis-Menten constant
L Liter
LB Luria-Bertani
lb/in2 Pounds per square inch (psi)
Max/max Maximum
MCS Multiple cloning sites
MES 2-(N-morpholino)-ethanesulfonic acid
Mg Magnesium
mg/mL Milligram per milliliter
MgCl2 Magnesium chloride
MgSO4 Magnesium Sulphate
Millivolt(s) mV
min Minute(s)
mL Milliliter
mM Millimolar
µg Microgram
µL Microliter
mol Mole
MOP 3-(N-morpholino)-propanesulfonic acid
MW Molecular weight
MWCO Molecular weight cutoff
NaCl Sodium chloride
NCBI National Center for Biotechnology Information
ng Nanogram
Ni Nickel
Ni-column Nickel column
List of Abbreviations
xix
nm Nanometer
OD Optical density
oNPG o-nitrophenyl-β-D-glucopyranoside
oNPGal o-nitrophenyl-β-D-galactopyranoside
ORF Open reading frame
Pb Lead
% Percentage
PCR Polymerase Chain Reaction
pH potential of Hydrogen/ power of Hydrogen
pI Isoelectric point
pM picomole
PMSF Phenylmethylsulfonyl fluoride
pNPF p-nitrophenyl-β-D-fucopyranoside
pNPG/p-β-NPG p-nitrophenyl-β-D-glucopyranoside
pNPGal p-nitrophenyl-β-D-galactopyranoside
pNPMan p-nitrophenyl-β-D- mannopyranoside
pNPX p-nitrophenyl-β-D-xylopyranoside
Rb Rubidium
RPM/rpm Revolution per minute
S Entropy
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
sec Second (s)
SPSS Statistical package for the social sciences
S-Score Signal peptide score (for signal peptide determination)
TAE Tris acetic acid EDTA
Taq Thermus aquaticus
TCP Total Cell Protein
TE Buffer Tris EDTA Buffer
TEMED N,N,N',N'-Tetra methyl ethylene diamine
Tm Melting temperature
U/mL Units per milliliter
U/mL/min Units per milliliter per minute
Vmax Maximal velocity
w/v Weight per volume
X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
Y-Score Combined cleavage site
Zn Zinc
ABSTRACT
Abstract
xx
Abstract
With a paradigm shift in industry, moving from natural fuels to alternative renewable
resource utilization, concisely the growing demands of bioenergy has led to the emphasis
on novel cellulolytic enzymes to improve efficiency of bioconversion process of
lignocellulosic plant biomass. Currently, biodegradation is an area of extensive research,
the cynosure of biofuel industry is on the utilization of non-edible lignocellulosic biomass
(feedstock, agricultural and municipal residues) as an exploitable, inexpensive and
potential source of alternative renewable energy in the form of bioethanol, thus the need of
efficient thermostable cellulases are expected to increase in the future. β-glucosidase is an
essential member of cellulase enzyme system that plays a critical role in cellulosic substrate
hydrolysis and in many other biological processes.
Therefore, the present study describes cloning of two novel highly thermostable
cellulolytic enzymes β-1,4-glucosidases (TnBglA and TnBglB) from a bacterium
Thermotoga naphthophila RKU-10T and overexpressed in Escherichia coli BL21
CodonPlus (DE3)-RIPL. Purification and biochemical characterizations together with
kinetic and thermodynamic analysis give insights about the thermostability of both
enzymes. Various cultivation and induction strategies were applied to enhance the
production of engineered host cells density and expression of highly efficient
thermotolerant TnBglA and TnBglB, induced individually with IPTG and an alternative
inducer lactose. Culture conditions and other parameters including media compositions,
pre-induction optical density, agitation, inducer concentrations, temperature and time of
induction were optimized to achieve maximum yield of heterologous proteins.
Genomic DNA of T. naphthophila was used as template to amplify two cellulolytic genes
TnbglA and TnbglB (ADA66698.1 and ADA66752.1) of 1.341 and 2.166 kb, which
encoded β-1,4-glucosidase proteins of 446 and 721 amino acid residues, respectively.
Amplicons of genes were cloned initially in pTZ57R/T vector by employing dA×dT tailing
technique and consequently subjected to sequence analysis. Sequence homology analysis
demonstrated that TnBglA and TnBglB belong to glycoside hydrolase family 1 (GH1) and
family 3 (GH3), respectively. Further, both genes were sub-cloned in pET-21a(+) vector
Abstract
xxi
and over-expressed in E. coli BL21 under the control of inducible T7 lac promoter.
Initially, LB medium was used for the production and optimization of various cultivation
parameters to get the high level expression of both recombinant enzymes. However,
optimal expression and activity of both enzymes were observed when culture induced with
0.5 mM IPTG after heat shock treatment (42°C, 1 h) at 0.6 pre-induction optical density
(OD600nm) followed by incubation at 22°C for 72 h in a shaking incubator at 200 rpm.
Both extracellular TnBglA and TnBglB with a molecular weight of 51.50 and 81.14 kDa,
respectively were purified to homogeneity by ion-exchange and hydrophobic interaction
chromatography after heat treatment at 70°C for 1 h. Purified enzymes TnBglA and TnBglB
displayed optimal activity at pH 7.0 (95°C) and pH 5.0 (85°C temperature), respectively.
Both enzymes were quite stable over a broad range of pH (6.0-8.5) and temperature (60-
90°C), fairly stable up to 8 h at 80°C. GH1 TnBglA activity was stimulated in the presence
of glucose and xylose, with a Ki value of 1200 and 1300 mM, respectively. Whereas, GH3
TnBglB revealed Ki value of 150 mM for glucose and 200 mM for xylose, both enzymes
displayed affinity towards p-nitrophenyl substrates and cellobiose.
Km, Vmax and kcat values of TnBglA, using pNPG as substrate, were 1.5 mM, 297 mmol mg-
1min-1, and 1527778 s-1, respectively. Whereas, TnBglB showed Km, Vmax and kcat values of
0.45 mM, 153 mmol mg-1min-1 and kcat 1214285.7 s-1, respectively using pNPG as
substrate. Thermodynamic parameters as ∆H*, ∆G* and ∆S* for pNPG hydrolysis by
TnBglA were calculated at 95°C as 25.7 kJ mol-1, 47.24 kJ mol-1 and -58.6 J mol-1 K-1,
respectively. Thermodynamic parameters for pNPG hydrolysis by TnBglB like ∆H*, ∆G*
and ∆S* were calculated at 85°C as 24.1 kJ mol-1, 46.55 kJ mol-1 and -62.74 J mol-1 K-1,
respectively. TnBglA displayed a half-life (t1/2) of 5.21 min at 97°C with denaturation
parameters of enzyme including ΔH*D, ΔG*D and ΔS*D were 662.04 kJ mol-1, 110.10 kJ
mol-1 and 1.491 kJ mol-1 K-1, respectively. TnBglB showed a half-life (t1/2) of 4.44 min at
94°C with denaturation parameters of enzyme including ΔH*D, ΔG*D and ΔS*D were
283.78 kJ mol-1, 108.7 kJ mol-1 and 0.477 kJ mol-1 K-1, respectively.
Generally, inadvertently preparing medium and unintentional induction of engineered E.
coli BL21, give poor or variable yields of heterologous proteins. Therefore, to enhance the
Abstract
xxii
activity and production of an industrially relevant cloned enzymes through various
cultivation and induction strategies. High-cell-density and optimal heterologous proteins
expression were obtained in 4×ZB medium after 72 h inducement at 22°C when culture
gave heat shock (at 42°C for 1 h) at 0.6 OD600nm and induced either with 0.5 mM IPTG/150
mM lactose. TnBglA and TnBglB activities were enhanced 3.8 and 0.096 fold in 4×ZB
medium with 150 mM lactose, respectively under optimal conditions. However,
considerably greater dry cell weight of TnBglA and TnBglB cultures were 11.30 g DCW
L-1 and 11.08 g DCW L-1 in 4×ZB medium, respectively induced with 150 mM lactose.
Use of the inexpensive and non-toxic inducer lactose, and an effective process strategy is
essential to achieve a high level of enzyme expression.
The expression and purification scheme, presented here, has a potential of scaling up to
obtain pure and active enzymes, relatively economical for further studies and other
applications. Finally, this is a report on enhanced production of highly heat active cloned
β-glucosidases from T. naphthophila (TnBglA and TnBglB) with high catalytic efficiency
and low product inhibition, which have excellent tolerance against glucose and xylose, and
also exhibit independence of detergents, chemical inhibitors and metal cations. All these
significant features make both TnBglA and TnBglB appropriate candidates for
biotechnological and industrial applications.
Keywords: thermostable; β-glucosidase; thermodynamics; glucose tolerance;
heterologous protein expression; induction strategy; Thermotoga naphthophila.
CHAPTER-I
INTRODUCTION
Introduction
1
1.1. Introduction
The widening gap between energy demand and supply has led to power outages, and has
caused disruption in the economy and development of countries. Despite having abundant
renewable energy resources, the sharp growing demand has led to countrywide energy
crisis which threatens the economic growth. Overall energy consumption, impending
shortages of natural fossil fuels and global warming are expected to increase in near future.
Every year world energy demand is rapidly increasing, and in 2012 energy consumption
has increased by 1% and by this carbon dioxide (CO2) emissions has led to an augmented
by 1.4% (Enerdata, 2013). The petroleum-based fossil fuels are the main resource of energy
in this world for the industrialization and economic growth of states. Globally, all known
and anticipated reserves of oil will not significantly run out for more than 100 years because
of exceed production and high consumption rates (Saratale and Oh, 2012).
The use of fossil fuels have various multi-faceted problems like supply instability, rising
cost, and global warming by a steady rise in environmental pollution which may adversely
affect Earth planet in near future. These economic and geopolitical factors have played a
significant role in reviving an interest in renewable energy resources that can decrease and
displace our reliance on uncertain and unstable sources of petroleum-based liquid fuels for
world-wide major energy demand, and also decline change in global climate (Kuhad et al.,
2016). Therefore, the search for more sustainable energy sources which will be ecofriendly,
inexpensive and high in yield, is now no longer something of a luxurious (Khelil and
Cheba, 2014). These concerns have shifted researcher’s efforts to develop technologies for
the production of ‘greener energy’ known as bioenergy that is an alternative, sustainable,
clean and renewable energy source, which can effectively use in place of fossil fuel to meet
rising energy demands of the world (Maki et al., 2009; Raghuwanshi et al., 2014).
Biofuels (bioenergy) are the only substitute source of energy for the predictable future and
can form the basis of sustainable development in terms of environmental and
socioeconomic concerns (Demirbas, 2007). Biofuels are solid, liquid (bioethanol,
biobutanol and biodiesel) or gaseous (hydrogen and methane) fuels acquired relatively
from recently dead biological material mainly from plant biomass (agricultural byproducts
Introduction
2
and dedicated energy crops) while fossil fuels are derived from long dead biological
material (Mohanram et al., 2013). Firstly, the idea of converting natural plant biomass-
derived carbohydrate sugars to biofuel was proposed in the 1970s (Lamers et al., 2008). In
recent time, this idea is being considered again seriously and biofuel has received
considerable attention because it offer a means to minimize dependence on natural crude
oil and to reduce emissions of greenhouse gases (CO, CO2 and CH4) which affect our
environment inadequately. Policy intercessions, in the form of supports and mandated
biofuels blending with fossil fuels are driving the rush to liquid biofuels.
Biofuel can contribute to develop an economic, and to make the environment better as it
contains higher octane ratings ethanol which combust in a more efficient and clean way
than gasoline, consequently their atmospheric carbon footprint is integrally decreased as it
offers carbon neutral alternative (Mohanram et al., 2013). Many developed as well as
developing countries (states) have made huge investments in infrastructure, process
development and biofuel (bioethanol) production facilities (Koppram et al., 2014). In 2008,
1.8% world transport fuel has provided by biofuel (Bringezu et al., 2009). However,
petroleum consumption can immediately be decreased by blending the fuel with
bioethanol, and the use of blended gasoline can cut down CO2 emissions by 25-30%
(Mohanram et al., 2013). In US, Canada, and Brazil renewable fuel standard has been
boosted, in these days, 10-25% ethanol successfully mixed with fuel (Maki et al., 2009).
According to an international report, Pakistan can instantaneously reduce its petrol
consumption by at least 30-40 million barrels per year, by blending fuel with 10% ethanol.
On the Earth's surface, plants use solar energy to fix atmospheric CO2 and collectively
resolved to recycle an estimated 1011 tons of carbon per year, which is used in the formation
of complex carbohydrates and their derivative via photosynthesis (Yeoman et al., 2010).
For the commercial production of biofuels, several plants and plant-derived materials
(sugars) are used as substrates including grains for ‘first generation’, and lignocellulosic
waste biomass for ‘second generation’ biofuels. Currently in biofuel industries, sucrose-
and starch- containing agricultural crops (sugarcane, corn, maize and wheat) are used as
substrates for production with conventional technologies, known as first generation
biofuels.
Introduction
3
However, edible raw materials are a controversial resource for biodegradation and will not
be adequately sufficient to meet the raising demand of biofuel (Greene et al., 2004;
Mohanram et al., 2013). First generation biofuels production, neither economically nor
ecologically sustainable, as grain substrates require large land areas for cultivation and
compete with crops (edible plant materials) meant for human consumption (Yeoman et al.,
2010). Therefore, there is a robust necessity to look at non-consumable lignocellulosic
plant biomass (feedstock, municipal and agricultural wastes) that can use effectively as
cellulosic substrates for the production of second generation biofuels and commodity
chemicals, this renewable natural carbohydrates will provide us a sustainable and
economical alternative energy in the form of bioethanol (Klose et al., 2012).
‘Second generation’ of biofuel is highly imperative because they are based on highly
abundant and inexpensive lignocellulosic biomass; a complicated composite with three
main biopolymers i.e., approximately 40-55% cellulose, 25-50% hemicellulose (xylan) and
10-40% of lignin, although individual compositions vary among species. (Vanitha et al.,
2011; Mohanram et al., 2013). Non-edible cellulosic biomass is a significant and
noteworthy energy source to cope with the food problem and shortage of energy with
explosive or abrupt increase in human population. Cellulosic wastes are the chief source of
biomass which constitutes switch grasses, miscanthus, forestry and agricultural residues
(sugarcane bagasse, corn fiber, rice hulls and wheat bran etc.), municipal and industrial
superfluous materials. These raw materials are cheap, unexploited, environmentally sound,
easily accessible, and inexhaustible biological resource which could be proficiently utilized
as substrate for bioethanol production instead of edible grain crops (Maki et al., 2009;
Yennamalli et al., 2013; Koppram et al., 2014).
Cellulose is the most abundant (profuse) organic compound on Earth, with the formula
(C6H10O5)n, virtually inexhaustible and a great potential renewable bioenergy source
(Teugjas and Väljamäe, 2013); major constructive component of algae and plant cell walls,
although some bacteria and marine invertebrate animals (tunicates) also produced cellulose
(Väljamäe et al., 2001; Lynd et al., 2002; Tang et al., 2014). Cellulose, a bio-polymer is
mingled with hemicellulose and lignin elements which make a complex architecture of
plant cell wall (Figure 1.1). Cellulose exist in the form of microfibrils, a compact inflexible
Introduction
4
highly organized unbranched homo-polysaccharide composed of D-glucose units linked
via β-1,4-glycosidic bond (Figure 1.2) (Artzi et al., 2014). Many of these polysaccharide
linear chains are bound together through Van der Waals forces and hydrogen bonding,
arranged in a parallel arrays and in turn form macrofibrils. Cellulose is different in their
configuration has crystalline domain which intermixed with less ordered amorphous
regions, where these forces are loosen (Fernandes et al., 2011; Endler et al., 2011).
Figure 1.1: Structure of lignocellulosic plant biomass (Tomme et al., 1995).
Figure 1.2: Hierarchical structure of cellulose extracted from plants (Rojas et al., 2015).
Introduction
5
Cellulose degradation is challenging due to its multifaceted and recalcitrant nature (Dong
et al., 2010). Generally, production of bioethanol involves pretreatment of cellulosic plant
biomass and its enzymatic conversion to fermentable sugars (glucose) molecules which
can be converted to bioethanol (Chan et al., 2016). Bioethanol has been conceived of as
the future source of green alternative for the development of transportation fuels and
various other chemicals (Kuhad et al., 2016). This process has many other application,
such as treating waste water from textile, laundry detergents, dying and pulping industries.
(Wang et al., 2015). The limiting factor of bioethanol production is the efficient conversion
of biomass to fermentable sugars; however, complex and rigid structure of cellulose is a
leading bottleneck to preclude the development of a lucrative production method.
Initially, hydrolysis of cellulosic material was performed chemically at high temperature
using various acids but this approach was often non-specific, slow and some time the
process became uncontrolled which is the major barrier to its widespread application (Kim
et al., 2001). In nature, cellulosic material is hydrolyzed mostly by the hydrolytic and
oxidative biocatalysts of bacteria and fungi (Lynd et al., 2002; Horn et al., 2012). Though,
several conventional (thermal and chemical) and non-conventional (biochemical and
microbial) strategies have been suggested and employed, either alone or in combination
but none have proven appropriate and satisfactory as a standalone strategy because of the
intricacy and complexity of feedstock (biomass). This obstacle needs robust technologies
for bioconversion which are insensitive to fluctuation (variation) in bio-feedstock,
adequately vigorous to face biologically challenging process operating conditions and can
significantly increase the yield (Sohel and Jack, 2012).
Keeping all these specifications in mind, plausibly it is better to consider thermophilic and
hyperthermophilic microorganisms (fungi and bacteria that natively live at extremely high
temperatures) and their biocatalysts for decisive roles in saccharification process
(hydrolysis of a polysaccharides or cellulose into soluble sugar components). Recently a
large number of thermophiles and hyperthermophiles are available having an extensive and
competent glycosidic hydrolase (GH) inventory (VanFossen et al., 2008). Consequently,
biodegradation using highly effective thermo- efficient cellulolytic enzymes is the most
promising conversion technology. Thermostable cellulases require less energy and mild
Introduction
6
environmental conditions to convert biomass into fermentable sugars that are used to make
many important bio-based industrial products, which can replace fossil fuels (Zhang et al.,
2007; Vanitha et al., 2011).
Cellulases are the third largest group of industrial biocatalysts, have been commercially
available for more than 30 years. Applied and basic studies on cellulases have
demonstrated their industrial and biotechnological applications in various fields.
Commonly, cellulases have been extensively used in textile (for biofinishing, biopolishing
of fabrics and biostoning of jeans), food processing, animal feed, fruit juices, brewing (beer
purification) and wine making, to enhance flavor and aroma in brewery products (beer,
wine, fruit juices, tea and coffee), in agriculture to control plant diseases and pests, to
enhance plant growth and seed germination, to enhance oil extraction yields, to improve
the nutritive quality of bakery products, in laundry detergents, in paper and pulp (to
increase the brightness of paper and to decrease the viscosity of pulp), cosmetic and
pharmaceutical industries. Most attractively have developed significant interest due to their
auspicious and promising applications in biofuel production (Kuhad et al., 2011; Ferreira
et al., 2014).
Enzymatic saccharification of cellulosic substrates is a complex and intricate process which
requires the synchronized (coordinated) action of three main cellulolytic enzymes: (i) endo-
1,4-beta -glucanases (EGs, EC 3.2.1.4), (ii) cellobiohydrolases also called exo-1,4-beta-
glucanase (CBHs, EC 3.2.1.91), and (iii) beta-1,4-glucosidases (Bgls, EC 3.2.1.21), these
three hydrolytic enzymes are usually grouped as cellulase system (Colussi et al., 2012;
Singh et al., 2016). Firstly, endo-1,4-β-glucanases (EGs) randomly cleave the linear chains
of cellulosic fibers in amorphous regions (middle portion of cellulose molecules), where
EGs are easily accessible, act on the inner β-1,4-glucosidic bonds and convert cellulosic
polysaccharide chain into short fragments of different lengths. Then, cellobiohydrolases
(CBHs) act at the exposing sites of both reducing and non-reducing ends of these short
chains in less-accessible crystalline regions to release the cellodextrin and cellobiose as
major products, which finally hydrolyze by β-1,4-glucosidases (Bgls) to produce glucose
monomers (Figure 1.3) as the final product of biodegradation (Sørensen et al., 2013;
Introduction
7
Dantur et al, 2015; Pereira et al, 2015; Singh et al., 2016). During hydrolysis, activities of
endo-1,4-β-glucanase and cellobiohydrolase are inhibited by cellobiose (product inhibitor),
therefore, β-glucosidase stimulates and regulate the cellulose hydrolysis more efficiently
by relieving cellobiose inhibition (Liu et al., 2012; Pei et al., 2012; Schroder et al., 2014).
Figure 1.3: Schematic representation of biodegradation of cellulosic substrate by using cellulases. During
hydrolysis cellobiose and cello-oligosaccharides are produced by synergistic action of cellobiohydrolases
(exo-acting enzyme, CBHs) and endo-1,4- β-glucanases (endo-acting enzyme, EGs), which are subsequently
hydrolyzed by β-glucosidases to produce glucose units (monomers) as a final product of complete cellulose
metabolism (Singh et al., 2016).
β-1,4-glucosidases (β-D-glucoside glucohydrolase) are ubiquitous enzymes, widely
distributed in nature, can be found in simple microbes (fungi and bacteria) to highly
evolved plants and animals, and considered to be a significant component of cellulase
system (Krisch et al., 2010). It acts on cellobiose, alkyl- and aryl-β-D-glucosides in β-1,4-
glucosidic linkage, exhibits transglycosylase and alkyl transferase activities, and catalyzes
the synthesis of glycoconjugates and alkyl glucosides (Teugjas and Väljamäe, 2013). β-
glucosidases are broadly used in several fundamental processes ranging from
Introduction
8
developmental regulation (metabolic pathways) to chemical protection against pathogen
(insects and microbes) attack, and a number of biotechnological and industrial applications,
including bioethanol production from agricultural cellulosic wastes, host-pathogen
interaction, oncogenesis, cellular signaling, synthesis of valuable β-glucosides and
production of biodegradable nonionic surfactants (Bhatia et al., 2002; Chuankhayan et al.,
2007; Li et al., 2013).
β-glucosidase has achieved significant attention due to their diverse and essential
physiological roles in various biological system that depend on the enzyme location in
which they present (Singhania et al., 2012). In cellulolytic microorganisms, β-glucosidase
is the main biocatalyst for the metabolism of carbohydrate (grow on various glucose
conjugates or disaccharides and oligosaccharides sugars) and cellulase induction, plays
crucial role in cell wall mechanism, symbiotic association and host-pathogen (Singh et al.,
2016). In plants, β-glucosidase plays pivotal roles in several essential biological (natural)
processes such as in pigment metabolism, cell wall development, fruit ripening, seed
development, detoxification of cyanogenic glycosides, regulating chemical defense against
pathogen attack by releasing pathogen-defending compounds from glycosidic bonds,
production and activation of phytohormones, and exhibits a vital role in hydrolysis of
glycosylated flavonoids (Belancic et al., 2003; Marotti et al., 2007; Krisch et al., 2010;
Mehmood et al., 2014). In mammals, β-glucosidase involves in degradation of glycolipids
in mammalian lysosomes and hydrolysis of glucosyl ceramides, thus deficiency of this
enzyme causes a non-neuropathic lysosomal storage disorder in human known as
Gaucher’s disease (Lieberman et al., 2007; Singhania et al., 2012).
β-glucosidase has large potential applications in various industries, gained more attention
due to liberation of aromatic compounds and flavor from tasteless glycosylated precursors
present in fermenting products, musts, fruits juices and wine (Su et al., 2010; Fan et al.,
2011). β-glucosidases are extensively used in food processing, dairy, beverage and flavor
industries. It is significantly used in wine making, production of fruit juices, and
extensively applied to improve the flavor and aroma of fruit juices, wines and tea (Pei et
al., 2012; Keerti et al., 2014; Cota et al., 2015). During juice extraction, hydrolysis of
naringenin to pruning by enzyme reduced the bitterness of juices and liberate aroma from
Introduction
9
grapes of wine (Harhangi et al., 2002). Aside from the enhancement of flavor and aroma,
β-glucosidases may increase the nutritional value of beverages, food and feed by release
of anti-oxidants, vitamins and other valuable compounds from their glycosides (Singh et
al., 2016).
In tea industry, the essential oil contents has been improved by treating with β-glucosidase
and enhanced the quality of beverages products (Krisch et al., 2010; Su et al., 2010). In
rice, enzymatic action of β-glucosidase can be released vitamin B6 (pyridoxine) from
pyridoxine glucoside (Opassiri et al., 2004). It mostly used to extract the medically
essential compounds from vegetables and fruits by the hydrolysis of phenolic and
phytoestrogen glucosides (Schroder et al., 2014). In cellulosic-based feeds used as an
additives, supplementation of β-glucosidase is highly beneficial for single-stomach animals
like chickens and pigs which enhanced the digestibility of feed and lead to better utilization
of nutrient (Krisch et al., 2010).
Production of aglycone by the breakdown of soybean isoflavone glycosides via enzyme β-
glucosidase is most desired application of this enzyme in industries. Isoflavones are
recognized to prevent several chronic diseases (improve bone health and lowers risks of
cardiovascular diseases) and certain cancers because they potently inhibit the growth of
cancer cells. Isoflavones, in soy-based foods are chiefly present in inactive form of
glycosides, and has been proved that the biological effects occur due to aglycone form
rather than their glycosylated form because aglycone forms quickly absorbs by intestine
(Izumi et al., 2000; Hu et al., 2009). This enzyme can be used in the manufacturing of
cosmetics products for melanogenesis inhibitory activities, arbutin-β-glycosides was
synthesized through the transglycosylation reaction using β-glucosidase to make a new
skin whitening agent (Jun et al., 2008).
β-glucosidase is highly employed for the synthesis of oligosaccharides (short chain sugars)
and alkyl-glycosides; oligosaccharides can be applied as growth promoting agent,
diagnostic tools and therapeutic agents, have imperative functions in various biological
systems such as cell proliferation, fertilization, and embryogenesis. Alkyl-glycosides have
several antimicrobial properties and prospective application in cosmetic, chemical,
Introduction
10
pharmaceutical, detergent and food industries as these hydrolyzed and catalyzed by β-
glucosidase enzyme (Bankova et al., 2006; Singh et al., 2016). Furthermore, this versatile
enzyme can be employed in many synthetic reactions because of its wide and varied roles
in nature (Bhatia et al., 2002).
β-glucosidases are a large group of versatile and flexible hydrolytic biocatalysts. According
to different criteria, these heterogeneous enzymes have been categorized into various
groups because there is no single precise way for their classification. Generally, two well-
known strategies have been reported in the literature for the classification of β-glucosidases
by which they can be divided, on the basis of their substrate specificity and
nucleotides/amino acids sequence identity (Henrissat and Bairoch, 1996). Substrate
specificity classifies β-glucosidases into three main groups: (i) aryl-β-D-glucosidases, have
great affinity to aryl-β-D-glucosides, (ii) cellobiases, preferably hydrolyze only
oligosaccharides, and (iii) broad specific β-glucosidases, can hydrolyze various types of
substrates. Most of the characterized β-glucosidases are assembled in third group due to
their catalytic activity towards various substrates (Pei et al., 2012; Fang et al., 2014; Chan
et al., 2016). The most accepted method of classification is on the basis of amino acid
sequence and structural folding similarity (homology), therefore β-glucosidases have been
sub-divided into various glycoside hydrolases (GHs) families 1, 3, 5, 9, 30 and 116
(Sørensen et al., 2013; Lombard et al., 2014).
However, β-glucosidases are commonly found in either the glycoside hydrolase (GH)
family 1 or family 3 (Opassiri et al., 2007; Cantarel et al., 2009). Currently, 133 GH
families are listed (registered) in the regularly updated website (http://www.cazy.org)
Carbohydrate Active enZyme (CAZy) (Cantarel et al., 2009). Structural features and amino
acid sequence of enzymes are the more informative and important tool for classification,
therefore International Union of Biochemistry and Molecular Biology categorized GH
families into structurally determined groups (Harris et al., 2010). Enzyme’s structural
features can be applied to analyze and study the topographies of other group members of
family by using various bioinformatics tools and approaches. Particularly, tertiary structure
of enzyme at active site dictates their catalytic behavior (mechanism) and affinity towards
various substrates. Enzymes which have similar sequence and conserved motifs, are
Introduction
11
assemble in the same GH family. Additionally, GH families are categorized into various
clans, families with conserved catalytic amino acid residues, analogous catalytic
mechanism and domain structures, suggestive and evocative of a mutual ancestry (lineage),
are gathered in single clan (Singh et al., 2016).
Large number of families have grouped in Clan GH-A including β-glucosidase GH family
1, GH family 5 and GH family 30, they all have a conserved (α/β)8 triose phosphate
isomerase (TIM) barrel scaffold or protein domain that has catalytic site. Maximum
number of characterized β-glucosidases so far contains a classic (α/β)8 barrel structure that
is a key feature of GH family 1 (GH1) and their catalytic active site have two conserved
carboxylic acid (glutamic acid, Glu166 and Glu351) residues on four and seven β-strands,
acting as catalytic acid/base (proton donor) and nucleophile, respectively (Cairns and Esen,
2010; Vuong and Wilson, 2010). The structure of β-glucosidases from Phanerochaete
chrysosporium (a basidiomycete) and human cytosolic have been studied by x-ray
crystallography and finally illustrated the presence of (α/β)8 barrel folds (Nijikken et al.,
2007; Tribolo et al., 2007).
Structural data of GH family 3 (GH3) β-glucosidases are still scarce, since only few have
been characterized thoroughly up till now. β-glucosidases from GH3 have two conserved
domains, an (α/β)8 barrel and α/β sandwich domain including a six stranded β-sheet
sandwich between three α-helices on both/either side (α/β)6. Conserved active site of GH3
β-glucosidases are located at the interface of both sandwich domains, however each domain
contributes one carboxylic acid catalytic amino acid residue, at the N-terminal domain an
aspartic acid (Asp285) and at the C-terminal domain a glutamic acid (Glu491) act as a
catalytic nucleophile and a proton donor, respectively (Singhania et al., 2012; Colussi et
al., 2015).
An alternative β-glucosidases classification system have been established on sequence
homology, and allocated these enzymes into two sub-families: (i) β-glucosidase family A
contains phospho-β-glucosidases and β-glucosidases from microorganisms to mammals,
and (ii) β-glucosidase family B comprises enzyme from rumen bacteria, molds and yeasts.
GH family 1 comprises approximately 62 β-glucosidases from archaebacteria, eubacteria,
Introduction
12
mammals and plants origin, this family also contains thioglucosidases and 6-
phosphoglycosidases. GH family 1 β-glucosidases are highly active with broad substrate
specificity and also exhibited the activity of β-galactosidase. Approximately 44 β-
glucosidases are belonged to GH family 3, mostly isolated from mold, yeast and some
bacterial source, whereas some hexosaminidases also include in this family. Majority of
fungal β-glucosidases studied and characterized are the members of GH family 3 (Cantarel
et al., 2009; Krisch et al., 2010; Singh et al., 2016). The classification of a family also
demonstrates the glycoside hydrolases evolutionary relationship (Singh et al., 2016).
Generally, β-glucosidases exhibited tremendous diversity in terms of substrate specificity
but little is known about their catalytic mechanism acceptably. Numerous techniques for
example inhibition, pH dependence, essential residues labeling with fluorosugars, isotopic
effect, structure-reactivity studies (with deoxy- substrate analogues) and site-directed
mutagenesis have been applied for elucidating the catalytic behavior and active site
topology of enzyme (Kempton and Withers, 1992; Street et al., 1992; Withers et al., 1992;
Wang et al., 1995). Enzyme hydrolyze the β-glycosidic bonds efficiently between a
polysaccharide and non-polysaccharide moiety.
Most β-glucosidases characterized to date, have been placed in GH family 1 and GH family
3, which are all retaining enzymes, perform their catalytic mechanism in two steps, initially
glycosylation and followed by deglycosylation occurs. Among all reported β-glucosidases,
a conserved glutamic acid (Glu) is the main active site residue (Wang et al., 1995). The
retaining mechanism of β-glucosidase for the catalysis of β-glycosidic bond is completely
demonstrated in figure 1.4. In first step, a glucose-enzyme intermediate complex is formed
by the action of a glutamic acid (Glu) on the anomeric carbon which acts as a nucleophile,
and after that in deglycosylation step, another acid/base catalytic residue activates a water
molecule which hydrolyzes the glycosidic bond to liberate free glucose (monosugar)
molecule (Litzinger et al., 2010).
Introduction
13
Figure 1.4: The catalytic retaining two steps mechanism of β-glucosidase for the hydrolysis of β-glycosidic
bond. Glycosylation, the first step in which a conserved glutamic acid (Glu) residue attacks on the glycosidic
bonds and acts as nucleophile, and by the hydrolytic action of cellobiohydrolase (other enzyme of cellulase
system) cellobiose released. An enzyme-substrate intermediate complex is formed as a result of this. Second
step is known as deglycosylation, by general acid/base catalyst reaction another conserved glutamic acid
(Glu) residue activates a water molecule which further acts on the intermediate complex to release the glucose
molecule.
β-glucosidases are widely found in all form of life and involved in their various essential
bioconversion processes as well as these enzymes have various industrial applications.
Thermotolerant and highly active cellulases, especially β-glucosidases have received
considerable attention, in these days, are extremely desirable to attain a higher degree of
efficiency on the industrial scale and for biotechnological applications. Heat resistant, and
solvent-tolerant enzymes commonly known as ‘extremozymes’ are the valuable tools for
industrial processes in which hardly degradable polymers and compounds need to be
decomposed and liquefied (Elleuche et al., 2014). The quest for hyperstable and acidic-
alkali resistant GHs, mainly cellulolytic enzymes have led the way towards extremophiles
which are adapted to survive at extremely harsh conditions of ecological niches such as
high salt concentrations, extreme of pH, high pressure and temperature. These
microorganisms are the valuable home of unique biocatalysts that can withstand harsh
Introduction
14
conditions of current industrial processes as compare to the prevailing enzymes derived
from mesophiles (mesozymes) (Elleuche et al., 2015).
In recent time, applications and needs of cellulases are completely different with respect to
the various industrial processes such as for biofuel industry prefers thermostable and acidic
resistant enzymes, whereas detergent industry favors enzymes with broad range of pH and
temperature stability as well as halo-tolerant (resistant to salts). However, textile and
industrial wastes treatment industries requires cellulolytic enzymes with heat and alkali
stable characteristics (Kumar et al., 2011). Thermal stability is the most common,
conspicuous and noticeable property of cellulases required in industries. Conducting
biotechnological and industrial processes at elevated temperature has numerous advantages
including higher reaction rate, decrease in viscosity, lower the risk of contamination,
increased solubility of organic compounds (hydrophobic substrates) and improved
bioavailability of hardly degradable environmental pollutants (Niehaus et al., 1999; Kuhad
et al., 2016).
Demand of thermostable β-glucosidases is growing rapidly because of its usage in food
processing and biofuel production. Therefore, the search for enzyme, insensitive to product
inhibition (glucose) with high specific activity and thermal stability, has increased
worldwide. Thermostable β-glucosidases, with high specific activity, have the ability to
function under extreme conditions, a longer hydrolysis time due to stability, lesser amount
used simultaneously and directly along with other heat stable cellulases for saccharification
purpose, as this decreases processing time (by eliminating pre-cooling step where steam is
applied to make biomass more accessible to degradation), improves fermentation yields,
reduces contamination risk, less energy consumption, enhances enzyme activity and
solubility of reactants and products (Liu et al., 2012). Thus, the potential commercial
advantages of efficient and lucrative thermostable cellulases with low degree of inhibition
would reduce the cost of bioethanol production significantly in a simultaneous
saccharification and fermentation (SSF) process (Zhang et al., 2012).
Biomass conversion using cellulases has become a major research area and thermophilic
bacteria are the excellent source of these enzymes (Mehmood et al., 2014). Thermophilic
Introduction
15
fungal cellulases had been extensively studied in last few decades for bioconversion
process (Hong et al., 2007; Yoon et al., 2008; Liu et al., 2012; Ramani et al., 2015; Zhao
et al., 2015). But recently, thermophilic and hyperthermophilic bacteria have accomplished
prodigious consideration due to several reasons such as broad natural diversity, easily
cultivated with cheap nutrients, high growth rate, have great ability for the production of
thermal, alkali and acid-stable cellulases. However, high level of expression, extraction
and purification of bacterial biocatalysts done feasibly as compare to fungal cellulolytic
enzymes (Jabbour et al., 2012; Schroder et al., 2014; Gumerov et al., 2015; Yang et al.,
2015; Chan et al., 2016; Peng et al., 2016).
Hyperthermophiles are the most attractive source of extremely thermotolerant enzymes,
optimally grow at higher temperature usually above 80°C, have been isolated and screened
from different hot deep subterranean locations such as marine sediment, hot springs, oil
reservoir and continental solfatatras. These organisms are thought to be the least evolved,
have deeper phylogenetic branches, most of them belong to archaea domain while some
are the members of genera Thermotoga and Aquifex in bacterial domain (Huber et al., 2000;
Takahata et al., 2001). Certain bacterial species across different genus have been reported
to produce highly thermostable cellulases including Aquifex, Alicyclobacillus,
Fervidobacterium, Geobacillus, Thermoanaerobacterium, Caldicellulosiruptor,
Dictyoglomus, Rhodothermus, Anoxybacillus and Thermotoga.
During the last few years many biocatalysts have been successfully isolated from these
exotic microbes which has exceptional features such as resistant to extreme pH,
temperature and chemicals denaturants (organic solvents, detergents and chaotropic
agents). Hence, these biocatalysts can be used as a model for constructing and designing
proteins with unique properties for industrial and biotechnological applications (Barnard
et al., 2010; Chang and Yao, 2011; Jager et al., 2012; Bhalla et al., 2013; Chen et al.,
2013). Only Less than 5% of hyperthermophilic archaea and bacteria are cultivable in the
laboratory due to their unique and exceptional cultivation requirements (Elleuche et al.,
2015). Therefore, to get the enhanced production of heat loving cellulolytic enzymes
particularly β-glucosidases, various approaches have been adopted by researchers in which
the most prominent strategy is cloning of thermophilic genes in suitable mesophilic hosts
Introduction
16
(Bacillus spp., Escherichia coli, Aspergillus sp., Saccharomyces cerevisiae, and Pichia
pastoris) using efficient expression vectors. Mostly, thermophilic heterologous proteins
produced in mesophiles are not hydrolyzed by host proteases; correctly and efficiently
folded at low (37-40°C) temperature, able to maintain their hydrolytic activity and stability,
and can be purified conveniently by using thermal denaturation (precipitation) of host
proteins. Usually, the degree of obtained protein purity is adequate and appropriate for
several industrial applications (Niehaus et al., 1999).
Cloned β-glucosidases exhibited prodigious advantages including hydrolyzing ability of
isoflavone (Kuo and Lee, 2007), maximum expression, thermo-active and more resistant
to end product inhibition (Jabbour et al., 2012). Various mesophilic host system were used
for achieving high level of heterologous protein expression but E. coli species along with
pET expression vectors series has been proved an excellent and well-established expression
platform (Studier, 2005). Some bacterial thermostable β-glucosidases that showed glucose
tolerant behavior have been cloned previously from Thermoanaerobacterium
thermosaccharolyticum (Pei et al., 2012), Fervidobacterium islandicum (Jabbour et al.,
2012), Caldicellulosiruptor bescii (Bai et al., 2013), Acidilobus saccharovorans (Gumerov
et al., 2015) Thermoanaerobacterium aotearoense (Yang et al., 2015) and overexpressed
in E. coli host. However, these β-glucosidases are not as much glucose resistant as
exceedingly required for biomass degradation and there are limited reports on kinetic and
thermodynamics study of β-glucosidases.
The members of genus Thermotoga has distinct features from other bacteria, optimally
grow at 80-90°C, they are obligate anaerobes, fermentative heterotrophs, rod-shaped,
gram-negative, non-spore forming cells with an outer prominent sheath-like structure or an
envelope known as ‘toga’ and having balloon like structure at both ends (Figure 1.5a). The
presence of sheath ‘toga’ is most noticeable characteristic of all the members of order
Thermotogales, include Thermotoga neapolitana, Thermotoga sp. strain RQ7, Thermotoga
sp. strain FjSS3-B.1, Thermotoga subterranea, Thermotoga elfii, Thermotoga thermarum,
Thermotoga hypogea, Thermotoga maritima and Thermotoga lettingae, have been found
naturally in diverse geographical niches (Figure 1.5b), while two novel species of genus
Thermotoga isolated from the Kubiki oil reservoir from Japan, which called as Thermotoga
Introduction
17
petrophila RKU-1T and Thermotoga naphthophila RKU-10T. The genomic sequence and
phenotypic characteristics revealed that both T. petrophila and T. naphthophila have great
similarity with T. neapolitana and T. maritima (Takahata et al., 2001). The species belong
to genus Thermotoga have the ability to degrade simple and complex sugars effectively,
and are believed to be an excellent source of highly active, resistant to denaturing agents
and heat-stable GHs specifically cellulolytic enzymes (Park et al., 2005; Turner et al.,
2007; Xue et al., 2009; Zhao et al., 2013; Mahmood et al., 2014; Xie et al., 2015; Long et
al., 2016).
Figure 1.5: (a) General structure of Thermotoga bacterium. (b) Octopus Spring, water from the main
spring supports the growth of a variety of Thermotoga species.
Figure 1.6: Electron micrographs of stained ultrathin section of Thermotoga naphthophila RKU-10T strain.
Arrows indicate the cell wall (cw) and toga (t) (Takahata et al., 2001).
(a) (b)
Introduction
18
Thermotoga naphthophila (naphthophila is a Greek word means bitumen-loving), a marine
hyperthermophilic eubacterium has all the general characteristic of family
Thermotogaceae such as obligate anaerobe, rod-shaped and non-spore-forming. Optimally
grow at pH 7.0 and 80°C temperature, cells are 0.8-1.2 µm wide and 02-07 µm long,
heterotrophic bacterial cell of T. naphthophila has a notable outer sheath ‘toga’ with several
lateral and subpolar flagella (Figure 1.6). Whereas, T. neapolitana lacks flagella and a
single sub-polar flagellum present in T. maritima (Takahata et al., 2001). Thermotoga
naphthophila RKU-10T (ATCC BAA-489 / DSM 13996 / JCM 10882) contains a large
number of putative cellulolytic genes.
Therefore, in this present research work two hyperthermophilic novel cellulolytic genes of
T. naphthophila are cloned and overexpressed in E. coli BL21 CodonPlus (DE3)-RIPL, a
mesophilic expression host. Various cultivation and induction strategies have been
developed for enhancing the production of engineered E. coli BL21 CodonPlus (DE3)-
RIPL cells and attaining high-level expression of both enzymes. Heat treatment followed
by chromatographic techniques are applied for the purification of thermo-efficient cloned
enzymes. Biochemical characterization of recombinant β-1,4-glucosidases together with
kinetic and thermodynamic analysis is done which give insights about the thermostability
of enzymes. Industrially, they could effectively use for biodegradation of carbohydrates,
moreover this study has proved that highly thermophilic bacteria are the
impending resources for isolating extremely effective glycoside hydrolases for
biochemical and biofuel production.
Introduction
19
1.2. Objectives
The growing demands of biofuel (bioenergy) has led to the emphasis on novel and highly
active cellulolytic enzymes to improve efficiency and rate of bioconversion process.
Therefore, the search of novel highly active thermostable cellulolytic enzymes is the great
need of time but the cultivation conditions of hyperthermophiles are highly demanding.
Hence, with the help of recombinant DNA technology, it is possible to achieve their
hyperstable enzymes in mesophiles; cloning of hyperthermophilic cellulolytic genes into
heterologous mesophilic host provides an effective approach to accomplish the high level
of enzyme expression. Furthermore, cloning of individual gene helps to study the
biochemical characterization of that recombinant enzyme.
The present research work is intended to clone two highly thermostable cellulolytic β-
glucosidase genes from T. naphthophila and overexpress in mesophilic host. Purify the
cloned β-glucosidases to evaluate their properties by complete characterization. The major
goal of this study is to produce potent hyperthermostable β-glucosidase with high catalytic
activity and low product inhibition. The following steps are followed to achieve the aim of
present study:
Amplification of cellulolytic β-glucosidase genes from T. naphthophila.
TA cloning of amplified product of β-glucosidase genes.
Sequencing of genes
Sub-cloning in expression vector.
Expression of heterologous proteins in E. coli BL21 CodonPlus DE3-(RIPL).
Optimization of cultivation conditions.
Analysis of recombinant proteins expression by enzymatic assays and SDS-PAGE.
Purification of β-glucosidases using chromatographic techniques.
Characterization of cloned β-glucosidases.
Kinetic and thermodynamic analysis of proteins.
CHAPTER-II
REVIEW OF LITERATURE
Review of Literature
20
2. Review of Literature
Bioethanol has been widely accepted to be the most appropriate replacement of
petroleum derived from fossil fuels, hence the rapidly increasing demands of bioethanol
has directed to the importance of active, competent, novel and thermo-efficient
cellulolytic enzymes to enhance the productivity of bioconversion process of plant
biomass. During last few years, many cellulolytic genes have been identified, isolated,
expressed, purified and characterized but the appropriate and impervious cellulases have
not been found yet, which can tolerate all biological challenges of the bio-
saccharification process. Therefore, recombinant thermophilic and hyperthermophilic
bacterial cellulolytic biocatalysts that overexpressed in mesophilic hosts, are the most
suitable candidates, because of their tolerance and resistance to the harsh conditions of
biodegradation process. Thermostable cellulases, especially β-glucosidases, which have
the ability to performed catalytic activity under extreme conditions, highly stable, and
effectively used in various cocktail with other cellulases, as this enhances the yields and
decreases the energy cost of the process (Liu et al., 2012).
In the following section, various aspect of thermo-activated beta-glucosidases relating to
cloning, recombinant protein production, purification, structure analysis, characterization
and applications have been reviewed.
Gabelsberger et al. (1993) identified, isolated and cloned a hyperthermophilic β-
glucosidase from Thermotoga maritima. The recombinant enzyme was overexpressed
and purified from a mesophilic host (Escherichia coli). By size exclusion
chromatography, the apparent molecular weight (MW) of dimer enzyme almost 95 kDa
was determined. Enzyme consisted of two similar subunits of 47 kDa, was determined
by SDS-PAGE. Purified β-glucosidase had great specificity of substrate and
commendably hydrolyzed β-glucoside, followed by β-fucoside, β-galactoside, and β-
xyloside substrates. Kinetic parameters for the hydrolysis of 0.05–20 mM oNPG and
0.1–10 mM oNPGal at 75°C, a non-linear Lineweaver-Burk formed. Lactose and
cellobiose did not cause any positive influence on enzyme activity, while 350 mM
lactose inhibited it completely. Enzyme was optimally hydrolyzed pNPG at pH 6.1. It
Review of Literature
21
exhibited high thermo-tolerant behavior, and pure β-glucosidase (50 μg mL-1) retained
60% residual activity without the addition of any additive dissolved in buffer of pH 6.2,
after being the incubated for 360 min at 95°C.
Kengen et al. (1993) revealed that a hyperthermophilic and highly active β-glucosidase
was found in the cell-free extract of Pyrococcus furiosus with high specific activity. The
crude enzyme was purified to homogeneity with the apparent MW of 58 kDa, has 4.40 pI
value; and characterized completely. Enzyme showed its peak catalytic activity with 446
U mg-1 specific activity at 102 to 105°C and at slightly acidic pH 5.0, against the
substrate of pNPG. Purified BglA activity did not depend on thiol groups, nor on high ion
strength and divalent cations. It exhibited affinity with pNPG, pNPGal, pNPX and
cellobiose substrates, while showed no hydrolytic activity against disaccharides or
polysaccharides like cellulose. Kinetic parameters were studies for pNPG and cellobiose
using Lineweaver-Burk plots and determined Km of 0.15 mM and 20 mM, Vmax of 700 U
mg-1 and 470 U mg-1 for pNPG and cellobiose, respectively. Purified enzyme had
remarkable thermal stability with a half-life of 780 minutes at 100°C.
Ruttersmith and Daniel (1993) isolated a thermostable beta-glucosidase gene from a
hyperthermophilic bacterium Thermotoga sp. strain FjSS3-B.1, cultivated at 80°C, and
after production of enzyme, purified to homogeneity using different strategies like anion
exchange followed by chromatofocusing and gel filtration. On SDS-PAGE, a single band
of purified enzyme with a MW of 75 kDa was observed. Enzyme had an optimal activity
of 195 U mg-1 at pH 7.0 and 80°C temperature. Enzyme had broad substrate specificity
and best catalytic activity was found towards pNPG substrate, with Km value of 0.1 mM.
Purified enzyme activity was inhibited by glucose (Ki value of 0.42 mM), and did not
effect by 40 mM xylose. Purified beta-glucosidase did not show any influence or
inhibitory effect in the presence of metal ions in fact, Ag2+ and Co2+ stimulated the
catalytic efficiency of the enzyme, only Cd2+, Ni2+ and Hg2+ obviously repressed the
activity. DTT, ethanol and Triton X–100 highly enhanced the activity of the beta-
glucosidase. Purified enzyme was tremendously thermotolerant with a half-life of 150
min at 98°C temperature and its stability improved in the presence of betaine or trehalose.
Review of Literature
22
According to Voorhorst et al. (1995) a hyperthermophilic cellobiose-hydrolyzing
biocatalyst known as beta-glucosidase (CelB) was cloned from Pyrococcus furiosus and
overexpressed in prokaryotic host E. coli. Deduced CelB sequence of amino acid had
great similarity with beta-glycosidases that were the members of glycosyl hydrolase (GH)
family 1. High-level production of beta-glucosidase (20% from total cell protein) was
successfully purified by two-step method that included thermoincubation for 40 min at
80° followed by anion-exchange chromatography. Purified enzyme had a MW of 58 kDa
estimated by gel electrophoresis (SDS-PAGE). Purified enzyme displayed peak activity
at 105°C and slightly acidic pH 5.0 against pNPG substrate. Km of 0.41 mM and Vmax of
1,169 µmol min-1 mg-1 was determined using pNPG.
Breves et al. (1997) sequenced a genomic DNA fragment of 5.9 kb from a thermophilic
anaerobe Thermoanaerobacter brockii. The DNA fragment had two genes, which located
adjacent to each other. The two different beta-glucosidases, XglS and CglT, belonged to
different glycosyl hydrolases (GHs) families with MW of 81 kDa and 52 kDa,
respectively. Both genes were overexpressed in E. coli, and recombinant CglT was
recognized by N-terminal sequencing and purified by heat denaturation and fast protein
liquid chromatography, after purification MW of 50 kDa was detected by gel
electrophoresis. Enzyme CglT had peak activity with pNPG, at optimal pH of 5.5 and at
75°C temperature. CglT exhibited thermostability for 24 h at 60°C. Enzyme CglT had the
ability to hydrolyzed various disaccharides as well as β-1,4-cello oligomers. CglT had
affinity for pNPGal and pNPF, while XglS cleaved both β-xylosides and β-glucosides as
well. Glucose moderately inhibited the enzyme activity and the Ki value for glucose was
determined to 200 mM.
Zverlov et al. (1997) cloned and sequenced a thermo-tolerant beta-glucosidase (BglB)
from the genomic DNA of Thermotoga neapolitana. Amino acid sequence alignment
showed that the cloned BglB enzyme was a candidate of GH family 3 and belonged to the
class EC 3.2.1.21. The gene was sub-cloned in pTT51 vector, and followed by
overexpressed in E. coli, and completely purified by thermal precipitation of heat labial
host proteins afterward ion exchange (DEAE column) and hydrophobic interaction
chromatography. SDS-PAGE analysis of purified fraction revealed one sharp band of
Review of Literature
23
protein with a monomeric MW of 81 kDa. Purified BglB had 255 U mg-1 specific activity
on pNPG at the optima of pH (5.5) and temperature (90°C). Km values for cellobiose,
laminaibiose and pNPG were 50, 10 and 0.1 mM, respectively.
Li and Lee (1999) studied gene cloning and expression of a beta-glucosidase from
Flavobacterium meningosepticum, which contained 2,178 bp encoded a protein of 726
amino acids. On the base of sequence analysis, the enzyme was classified as a GH3
family member. The recombinant E. coli cells were induced with IPTG, cultivated in LB-
medium, the high-level expression of enzyme was achieved after 6 hours induction. The
cloned beta-glucosidase enzyme was purified using cation-exchange chromatography, the
purity and 79.95 kDa molecular mass was determined by SDS-PAGE. Optimal
temperature and pH to achieved maximum activity of cloned enzyme (2,369 U mg-1) was
50°C and 5.0, respectively. It was remained stable at pH 5.0–8.1 (25°C temperature).
Recombinant enzyme effectively hydrolyzed aryl-beta-D-glycosides whereas, pNPG was
the most preferred substrate, with Km value of 0.68 mM.
Goyal et al. (2001) cloned, overexpressed, purified and characterized a β-glucosidase
(BglB) of a hyperthermophilic eubacterium Thermotoga maritima in an expression host
E. coli to get high level of heterologous active protein. Heat treatment and ion exchange
chromatography techniques were applied to purify recombinant BglB enzyme, and the
final fraction was subjected to analysis of SDS-PAGE which showed a single band of 81
kDa molecular mass. Optimal temperature and pH at which BglB displayed maximum
hydrolytic activity was 85°C and 5.0, respectively whereas at acidic pH (3.5) and lower
temperature (70°C) enzyme also showed high catalytic activity. BglB demonstrated a
broad range of pH stability from 5.0 to 9.0, and retained 68% residual activity at pH 10.
Enzyme exhibited thermo-tolerant behavior and remained stable up to 75°C temperature
for 30 min. BglB had a broad substrate specificity and showed great affinity towards
pNPG substrate with kcat and Km values of 6.34 s−1 and 0.0039 mM, respectively. Purified
BglB hydrolytic activity was activated in the presence of organic compounds and straight
chain alcohols.
Review of Literature
24
Parry et al. (2001) reported a thermo-tolerant extracellular, active and soluble β-
glucosidase enzyme from Thermoascus aurantiacus was purified using ion exchange and
Mono-P column chromatography techniques. The purified enzyme was homogeneous
with a MW of approximately 120 kDa, presented optimal catalytic activity at 80°C
temperature and pH 4.5, however at 90°C β-glucosidase displayed 70% relative activity.
Purified enzyme had high thermal and pH stability, at 70°C (pH 5.2) β-glucosidase
remained stable up to 48 h while above and below 70°C and pH 5.0 enzyme stability
decreased. It had the ability to hydrolyzed substrates efficiently with a β-glycosidic
linkage, cello-oligosaccharides, alkyl and aryl β-D-glucosides. Kinetic studies
demonstrated the lowest Km value of 0.1137 mM for pNPG and maximum kcat value of
17052 min-1 towards cellobiose. Straight chain alcohol activated the β-glucosidase while
glucose and D-δ-gluconolactone competitively repressed the activity with Ki values of
0.29 mM and 8.3 mM, respectively. Transglycosylation and hydrolytic activity was
optimally done in an acidic environment although enzyme catalytic behavior was
enhanced at high concentration of alcohols. The stereochemistry analysis of cellobiose
hydrolysis showed that purified β-glucosidase was a retaining glycosidase, and the
sequence alignment of N-terminal amino acid specified that β-glucosidase from
Thermoascus aurantiacus belonged to GH family 3.
Molecular cloning of a thermo-tolerant gene of beta-glycosidase with 1,311 bp encoding
437 amino acids from a hyperthermostable bacterium Thermus nonproteolyticus HG102
was carried out by Xiangyuan and co-worker (2001). Enzyme was overexpressed in E.
coli host and purified to homogeneity with a MW of 48,900 Da on SDS-PAGE. The
monomeric enzyme exhibited peak activity at 90 degrees C and at 5.6 pH, with high
catalytic activity towards pNPG. It can also effectively hydrolyzed pNPGal, pNPF and
pNPMan substrates. The kinetic parameters exhibited that the kcat/Km ratio for pNPG and
pNPF was higher than for pNPMan. The recombinant enzyme showed high
thermostability at 90 degrees C with a half-life of 2.5 hours, and displayed pH stability
over a wide range.
Zanoelo et al. (2004) characterized an inducible β-glucosidase enzyme from a Scytalidum
thermophilum a thermophilic fungus. The enzyme had pI value of 6.5 and purified by gel
Review of Literature
25
filtration and ion-exchange chromatography, and the final fraction exhibited a single
protein band of 40 kDa. Optima of temperature and pH of purified enzyme were 60°C
and 6.5, respectively against pNPG substrate. It exhibited thermal stability at 50–55°C,
with a 20 min half-life at 55°C temperature. Purified enzyme hydrolyzed various
substrates such as cellobiose, lactose, pNPX, and oNPGal whereas maximum affinity was
observed with pNPG. Km values for cellobiose and pNPG were 1.61 mM and 0.29 mM,
however Vmax for cellobiose and pNPG were 4.12 U mg-1 and 13.27 U mg-1, respectively.
The activity of enzyme was stimulated by the addition of 50 to 200 mM xylose and
glucose concentration and the Ki values of 43.24 mM for xylose and 36.69 mM for
glucose.
Kang et al. (2005) identified and cloned three thermo-tolerant genes in which a β-
galactosidase (bgaA) and two β-glycosidases (bglA and bglB) from a heat loving
bacterium Thermus sp. IB-21. The genes bgaA, bglA and bglB contained 1,938 bp, 1,311
bp and 1,296 bp of nucleotides which encoded 645, 436 and 431 amino acid residues,
respectively. These three genes were sub-cloned in pET-21b(+) expression vector,
propagated and overexpressed in E. coli BL21 (DE3). Heat treatment at 70°C for 40 min
followed by affinity chromatography techniques were applied for the purification of all
recombinant enzymes. Enzymes homogeneity and purity was analyzed by SDS-PAGE.
All purified recombinant enzymes exhibited maximum activity at pH 5.0–6.0.
Park et al. (2005) reported a molecular gene cloning of a hyperthermotolerant beta-
glucosidase (bglA) of 1,350 bp from Thermotoga neapolitana. The bglA gene was sub-
cloned in p6xHis119 vector, and subsequently overexpressed in E. coli. To purify cloned
BglA, crude supernatant was subjected to heat-treatment at 75°C to remove heat-labile
proteins followed by Ni-NTA affinity column chromatography. SDS-Page analysis
exhibited that purified TnBglA had 52 kDa protein. At 95°C recombinant purified BglA
showed peak catalytic activity and enzyme had great thermal stability with a half-life of
12 min at 105°C. TnBglA was optimally active over a range of 5.0–7.0 pH and remained
stable between 4.5–7.5 pH. TnBglA had high substrate specificity towards natural
(lactose and cellobiose) and artificial substrates (pNPG). BglA kinetic studies revealed
the values of Km (0.89 mM), kcat (657.2 s-1) and kcat Km-1 (753.91 mM-1 s-1) for pNPG
Review of Literature
26
substrate. Chemical inhibitors and metallic cations did not significantly influence the
activity of the recombinant enzyme, whereas beta-mercaptoethanol and dithiothreitol
markedly enhanced BglA enzymatic activity.
Hong et al. (2006) cloned a hyperthermophilic β-glucosidase gene (BglB) of 2.9 kb from
a anaerobic bacterium Thermotoga maritima MSB8 into pET-21a(+)vector and
heterologously overexpressed in E. coli BL21. Sequence homology of BglB with other β-
glucosidases from variety of organisms revealed that BglB was a conspicuous member of
GH family 3. After the enzyme inducement and production, crude BglB was purified by
applying affinity column chromatography followed by final eluted protein with a single
band of 81 kDa molecular mass was analyzed on native and SDS-PAGE. Purified BglB
hydrolyzed several substrates effectively such as pNPG, salicin, and arbutin, but peak
activity was found with pNPG substrate at 80°C temperature and 7.0 pH. Purified BglB
remained stable at high temperature of 80°C for up to 7 hours. The divalent cation Hg2+,
Cu2+, and Zn2+ repressed the enzyme activity.
In 2007, Hong and co-worker isolated, cloned and sequenced a thermo-tolerant beta-
glucosidase gene (bgl1) of Thermoascus aurantiacus IFO9748, and overexpressed in
yeast Pichia pastoris. Cloned gene consisted of 2,828 bp and deduced sequence of amino
acids displayed high homology with others GH family 3 glucosidases which confirmed
that this enzyme belonged to family 3. The inducible, active and recombinant enzyme
Bgl1 was purified to homogeneity and subjected to complete biochemical
characterizations. The purified enzyme showed maximum specific activity of 23.3 U mg-1
at 70 degree celsius and pH 5.0 using pNPG as a substate. Bgl1 retained more than 70%
residual activity after 60 minutes incubation at 60°C. Purified enzyme had affinity with
various artificial substrates such as pNPX, pNPC as well as cello-oligosaccharides and
cellobiose. Kinetic parameters were studies for pNPG, pNPX, pNPC and cellobiose. The
recombinant Bgl1 had the ability to utilized cellobiose as a source of carbon.
Kim et al. (2007) isolated a novel β-glucosidase gene (bglA) from uncultured soil
bacteria during the construction of genomic libraries from soil. Amplified product was
cloned in pET-32a(+) vector followed by heterologously expressed in E. coli BL-21
Review of Literature
27
(DE3) host cells. The deduced molecular mass of bglA amino acid sequence was 55-kDa,
which showed 56% similarity with a β-glucosidase of Chloroflexus aurantiacus belonged
to glycosyl hydrolase family 1. The crude extract was purified by applying Ni-NTA
agarose slurry and exhibited optimal pNPG degradation activity of purified enzyme was
observed at fifty five degree celsius and 6.5 pH. Enzyme showed thermal and pH
stability, retained 82% residual activity at 50°C after 60 minutes incubation and broadly
stable at pH 5.5 to 9.0. Its activity was enhanced in the presence of Mn2+, Mg2+, and Cu2+
divalent cations, while Co2+, Zu2+, Ca2+, and Fe2+ had negative effects on enzyme
activity. The purified enzyme had Km and Vmax values using pNPG substrate were 0.16
mM and 19.10 µmol min-1, respectively. Furthermore, purified enzyme displayed broad
substrates specificity and hydrolyzed both natural and synthetic substrate easily.
Kaur et al. (2007) purified and characterized a thermophilic, soluble and active β-
glucosidase from Melanocarpus sp. Enzyme had pI of 4.1, using DEAE-Sepharose (FF)
and Poly-buffer exchanger (PBE 94) column chromatography were used for the
purification of enzyme, 92 kDa molecular mass was determined on SDS-PAGE analysis.
Purified enzyme exhibited activity of 10.04 U mg-1 at the optimal assay conditions 60°C
temperature and pH 6.0. Presence of metallic cations, mercaptoethanol and DDT
influenced the catalytic efficiency of enzyme positively while only Cu2+ repressed the
activity up to 38 %. Enzyme hydrolytic efficiency was evaluated using various substrates,
and found pNPG as the most preferred substrate and low affinity for pNPC. Km (3.3 mM)
and Vmax (43.68 µmolmin-1mg protein-1) of purified β-glucosidase was determined for the
hydrolysis of pNPG, and kcat value of 4×103 min-1 was quantified.
Park et al. 2007 cloned and overexpressed a hyperthermostable beta-glycosidase of an
archaeon, Sulfolobus shibatae in E. coli BL21 (DE3) cells using pRSET-B expression
vector. Maximum expression of enzyme was observed after inducing recombinant
bacterial culture with 1 mM IPTG after that incubated for 3 h at 37°C. The heterologous
protein was efficiently purified using two-step method including heat treatment for 30
min at 70°C followed by affinity chromatography technique. The analysis on SDS-PAGE
demonstrated that enzyme was homogeneous with 57 kDa molecular mass. Purified
enzyme showed activity maximum at pH 5.0 buffer with pNPG substrate and at 95°C.
Review of Literature
28
Enzyme displayed high thermal stability between 70 to 100°C, and had a half-life of 900
minutes at temperature 75°C. It showed broad substrate specificity, mostly metal ions did
not affect the enzyme activity, though 10 mM MnCl2 stimulated its catalytic efficiency.
2-mercaptoethanol and dithiothreitol (DTT) chemicals significantly enhanced the
hydrolysis activity of enzyme.
According to Turner et al. (2007) a novel highly thermo-tolerant gene of β-glucosidase
from Thermotoga neapolitana was cloned and overexpressed in E. coli. Open reading
frame of gene encoded a 721 amino acid residues, the sequence alignment confirmed that
β-glucosidase (TnBgl3B) was a member of glycosyl hydrolase family 3 to be a competent
enzyme in alkyl-glucoside reactions with alcohol as the acceptor molecule using
transglycosylation. Recombinant E. coli cells were induced with IPTG, overexpressed,
active and soluble TnBgl3B was purified to homogeneity with 81 kDa MW using heat
treatment and affinity chromatography. At 90°C and pH 5.6, purified TnBgl3B was
optimally active while alcoholysis/hydrolysis ratio more favorable at 60°C (lower
temperature) where total activity was dropped, however water content and pH were more
heavy effected this ratio. Kinetic study for Km and Vmax were determined 0.11+0.03 mM
and 93+13 U mg-1, using pNPG substrate, respectively.
Kuo and Lee, (2008) studied the genomic sequence of a strain called
Bacillus subtilis natto, on the basis of genomic study two beta-glucosidase genes (yckE
and bglH) were isolated, cloned in E. coli M15 strain and overexpressed after the
induction with IPTG. The recombinant enzymes were active, soluble and highly
expressed in host cells, the enzymes were purified using affinity chromatography. The
YckE and BglH enzymes analyzed by SDS-PAGE which were approximately 54 kDa and
53 kDa, respectively. Recombinant YckE and BglH showed maximum hydrolyzing
activity towards pNPG substrate at 37–45°C, and enzymes displayed thermal stability up
to 45°C, while BglH had more heat stability than YckE enzyme. Both purified enzymes
highly active at pH 6.0, both enzymes fairly stable over a broad pH range, while BglH
was less stable than YckE. By the addition of divalent metallic cations (1 mM) the
enzymes activity were inhibited by only 0-23%, while Cu2+, Fe2+ and Cd2+ ions repressed
the recombinant BglH activity by 43%, 63% and 73%, whereas the activity of YckE was
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moderately repressed (not more than 20%) in the presence of any divalent metal cations.
YckE exhibited great affinity towards pNPF as compared to pNPG, and BglH revealed the
maximum affinity for genistin. The catalytic efficiency (kcat Km-1) of BglH for pNPG,
pNPF and genistin were 3.01, 0.90 and 147.20 mM-1 s-1, respectively. The kcat Km-1 of
YckE for pNPG, pNPF and genistin were 2.44, 306.57 and 19.45 mM-1 s-1, respectively.
Yoon et al. (2008) reported an extracellular and highly active beta-glucosidase enzyme
from Fomitopsis palustris was produced and purified to electrophoretic homogeneity.
The molecular mass of purified beta-glucosidase was determined by using SDS-PAGE,
which displayed a single band of protein to be approximately 138 kDa. Amino acid
sequence analysis was determined that suggested beta-glucosidase from Fomitopsis
palustris had homology with glycosyl hydrolase (GH) family 3 therefore, belonged to
GH3. Purified enzyme displayed prime activity toward pNPG at 70 degrees and pH 4.5.
Enzyme indicated thermal stability, with 97 h and 15 h half-life at 55°C and 65°C,
respectively. The Km value for cellobiose (4.81 mM) and pNPG (0.117 mM) were
calculated along with kcat values for cellobiose and pNPG were 101.8 and 721 sec-1,
respectively. In the presence of gluconolactone and glucose, the purified beta-glucosidase
catalytic efficiency was competitively inhibited and showed Ki values of 0.008 mM and
0.35 mM, respectively using pNPG as a substrate. Enzyme showed high affinity with
cellobiose and pNPG but had no activity toward CMC and xylan substrates.
Bajaj et al. (2009) reported a study on isolation and characterization of β-glucosidase
from a bacterial strain named M+. Enzyme was purified to homogeneity using ion
exchange chromatography, and SDS-PAGE displayed a single band of purified β-
glucosidase with 24 kDa molecular mass. Optimal enzyme activity (2200 U L-1) was
found against pNPG substrate at 50°C temperature in acetate buffer of pH 6.0, while β-
glucosidase showed considerably good catalytic activity at pH 5.0, 7.0–9.0 and also
exhibited good catalytic activity at 60–80°C temperature with 1600–1900 U L-1. Purified
β-glucosidase displayed thermo-tolerant behavior, and was thoroughly active at broad
range of temperature 50–80°C and retained 75–100% activity after 60 min incubation.
Enzyme also revealed pH stability over a range of 5.0–9.0. Enzyme exposed resistance to
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the metal ions and chemical additives while in the presence of Pb2+ enzyme catalytic
behavior boost up 47%.
A hyperthermophilic bacterium producing beta-glycosidase an extracellular active
enzyme, was isolated from hot springs of Japan, and cultured in laboratory at optimal
growth conditions (temperature 80°C and pH 6.5) for 48 h, was carried out by Gu et al.
(2009). The bacterium belonged to genus Thermus, found out after morphological study
and sequencing, bacterium was Thermus thermophilus HJ6 strain with highest cultivation
temperature as compared to others members of genus Thermus. The genomic DNA was
used as template for getting the multiple copies of beta-glycosidase gene with 1,296 bp,
which encoding a protein (431 amino acids) with a predicted MW of 48.7 kDa. The gene
was cloned, sequenced and highly expressed recombinant protein was purified to
homogeneity from the host cells of E. coli BL21 CodonPlus. The heterologous protein
purity was confirmed by SDS-PAGE, which had a single band of 45 kDa. Purified
enzyme showed best activity at pH 8.5 and temperature 90°C with pNPG substrate.
Enzyme had showed great thermal tolerance, with a 30 min half-life of thermal
inactivation at 95°C. Recombinant enzyme displayed Km, kcat and catalytic efficiency kcat
Km-1 values for pNPG was 0.16 mM, 9.24 s-1 and 57.12 mM-1 s-1, respectively. Kinetic
study revealed that pNPF and pNPG substrates were better than the pNPGal. The kcat Km-1
of recombinant enzyme was 70-fold increased at 80°C to that 40°C, which indicated that
high temperature activated the enzyme catalytic efficiency.
Harnpicharnchai et al. (2009) demonstrated the isolation of an endophytic fungal strain,
Periconia sp. BCC2871 for the production of thermo-efficient beta-glucosidase (BGLI).
The complete gene of beta-glucosidase was identified, expressed in the host strain Pichia
pastoris KM71. High-level of expressed heterologous protein was purified to
homogeneity, the purified recombinant enzyme showed the similar biochemical behavior
as the native beta-glucosidase, optimal enzyme activity at 70°C temperature and pH of
5.0–6.0 was observed. Cloned enzyme remained stable even after the incubation at high
temperature for long time period, 60% relative activity was found after 90 min incubation
at 70°C. Purified enzyme was quite stable under neutral to basic conditions, and retained
100% enzyme activity at pH 8.0 after being incubated for 120 min. Heat inactivation of
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enzyme was enhanced remarkably by the addition of sucrose, glucose and cellobiose.
Purified BGLI had maximum affinity towards synthetic substrates having glycosyl
groups, cellobiose and CMC.
According to Hong et al. (2009) a thermo-efficient β-glucosidase gene isolated and
cloned from a thermophilic bacterium Caldicellulosiruptor saccharolyticus DSM 8903,
and was expressed effectively in host cells of E. coli. Heterologous enzyme was
efficaciously purified to homogeneity using heat treatment followed by His-Trap affinity
chromatography, and final fraction presented a single band of 54 kDa on gel. Optimal
temperature 70°C and pH 5.5 of enzyme was found with pNPG substrate. Purified β-
glucosidase showed great thermal stability at 60–80°C, with half-lives of 250, 24.3, and
0.4 hours at 60, 70, and 80°C, respectively. The enzyme exposed no catalytic activity
against aryl-alpha-glycosides while had great affinity towards aryl-beta-glycosides such
as pNPF, pNPG, and pNPGal in decreasing order.
Kim et al. (2009) isolated the genomic DNA of Thermoplasma acidophilum, which
employed as source of bglA gene, a putative β-galactosidase. The bglA gene (1,452 bp)
cloned, expressed, purified and characterized in host E. coli ER2566 strain. The sequence
analysis of amino acid verified that the putative enzyme was a member of GH family 1.
The recombinant enzyme was purified with a single protein band of 57 kDa on SDS-
PAGE and had 82 U mg-1 specific activity by using pNPG. Enzyme maximum catalytic
activity was found at pH 6.0 and 90°C; recombinant enzyme showed high thermal
stability in pH 6.0 buffer, with 0.4 h half-life at 90°C while it displayed less thermostable
behavior below pH 6.0 and showed only 13 h stability at 75°C. Enzyme activity did not
activated or influenced by mono- and divalent metal cations and not effected by EDTA
chelating agent. Recombinant enzyme showed high substrate specificity, and exhibited
maximum towards pNPF and pNPG, with km value of 0.2 mM and 0.4 mM, kcat value of
182 s-1 and 141 s-1, and kcat Km-1 value of 381 s-1 mM-1 and 1,068 s-1 mM-1, respectively.
In 2009, Xue and co-workers reported cloning and overexpression of a hyperthermostable
beta-glycosidase A gene (bglA) from Thermotoga maritima in E. coli BL21 CodonPlus
using pET-20b(+) vector. The expression of soluble heterologous protein (BglA) was
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enhanced from 12.9–15.4% in total soluble cell proteins at 16°C temperature of
induction with low concentration of IPTG inducer. The purified BglA enzyme exhibited
66.8 U mg-1 specific activity at the optimal temperature of 90°C and pH 6.2. BglA
showed stability from pH 4.2–8.2 and heat stability retained almost 90% residual
activity at 90°C after 60 min incubation. Purified BglA exhibited great affinity with
pNPG substrate with Km, kcat Km-1 and Vmax values of 0.43 mM, 589.5 mM-1 s-1 and
323.6 U mg−1, respectively while Km (9.0 mM), kcat Km-1 (16 mM-1 s-1) and Vmax
(183.2 U mg−1) values were found against salicin.
Fang et al. 2010 reported a beta-glucosidase gene (bgl1A) encoded an active protein of
442 amino acids residues, which was isolated and screened from a microbial
metagenomic library of marine. The sequence analysis of Bgl1A protein showed that
enzyme was a member of GH family 1. The gene bgl1A was ligated in pET22b(+) and
transformed into E. coli BL21 (DE3) and recombinant Bgl1A showed high level of
expression in LB-medium after 7 h isopropyl-β-D-thiogalactoside (IPTG) induction. The
enzyme was purified using affinity chromatography with the MW of 51 kDa by SDS-
PAGE analysis. Bgl1A showed great stability towards various chemical inhibitors,
metallic ions and high concentration of sodium chloride (NaCl). It exhibited high Ki
value of 1000 mM with glucose. The kinetic parameters of Bgl1A displayed the Km of
0.39 mM and Vmax of 50.7 µmol mg-1 min-1.
Nam et al. (2010) demonstrated the cloning and sequencing of β-glycosidase gene from a
hyperthermophilic bacterium Thermus thermophilus KNOUC202, and was overexpressed
in a host E. coli JM109 (DE3), the cloned enzyme was completely characterized after
purification. The β-glycosidase gene consisted of 1,296 bp nucleotides encoding an active
protein of 431 amino acid residues, the sequence homology analysis confirmed that
recombinant enzyme was a member of GH family 1. The purified enzyme was
monomeric with a MW of 49 kDa, and exhibited a single band on polyacrylamide gel
electrophoresis. Enzyme had broad range of substrate specificity, Km value of pNPG was
lower as compared for the pNPGal and oNPG, the catalytic efficiency (kcat/Km) for pNPG
hydrolysis higher than those for pNPGal and oPNPG, though kcat value of recombinant
enzyme for the hydrolysis of pNPG was highly less as compared for the pNPGal and
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oNPG. Enzyme revealed optimal activity at 90°C and at the pH of 5.4. The cloned
enzyme presented great stability at 80-100°C temperature, and displayed 100% catalytic
activity after being incubation at 80°C for 120 min in pH 6.8 buffer.
Chang et al. (2011) isolated a bacterium from shrimp shell due to having great β-
glucosidase activity. The gram-negative bacterium belonged to Exiguobacterium genus,
named Exiguobacterium sp. DAU5. The gene comprised 1,350 bp encoded 450 amino
acids, was isolated from the genomic DNA of Exiguobacterium sp. DAU5 and
successfully cloned and expressed in host cells. SDS-PAGE indicated that the purified
β-glucosidase (BglA) enzyme had MW of 52 kDa. The alignment of amino acid
residues sequence homology with other bacterial β-glucosidases showed that enzyme
was a member of family 1. The BglA enzyme revealed that the best substrate for
catalytic activity was pNPG, at optima of temperature 45°C and optima of pH 7.0.
Hydrolytic efficiency was strongly influenced by metallic ions such as Zn2+, Ni2+, K+,
Na2+, Ca2+, Li+ and Mg2+. Chelating agent EDTA partially repressed the catalytic
efficiency of BglA.
Kim et al. (2011) reported a study on cloning of a β-glucosidase gene isolated from
Dictyoglomus turgidum and transformed E. coli strain of ER2566 with recombinant β-
glucosidase gene-pET28a(+) plasmid. This β-glucosidase belonged to GH3 that was
confirmed after the sequence analysis. The enzyme was purified using His-Trap affinity
chromatography, with a specific activity of 31 U mg-1. Purified monomeric enzyme had a
MW of 86 kDa, and showed peak activity at pH 5.0 and 85°C. Enzyme exhibited high
thermostability with a half-life of 334 min at 85°C. Enzyme had a broad substrate
specificity towards aryl-glycoside substrates with maximum hydrolytic activity with
pNPG with a Km value of 1.3 mM and kcat value of 13900-1 and also showed affinity with
oNPG, pNPGal, pNPX and pNPF. The effect of additives revealed that enzyme was
completely independent of metallic monovalent or divalent ions and EDTA.
Xu et al. (2011) cloned, sequenced and expressed a new heat stable β-glucosidase (bgl)
from a thermophilic filamentous fungus Chaetomium thermophilum, bgl gene contained
2,604 bp open reading frame (ORF) which encoded 867 amino acids having a potential
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signal of secretion. Recombinant β-glucosidase (Bgl) was achieved by expression of
cloned gene in Pichia pastoris. Recombinant enzyme was purified to homogeneity with a
MW of 119 kDa by SDS-PAGE analysis. Enzyme showed optimal catalytic activity at
pH 5.0 and 60°C, exhibited a half-life of 55 minutes at 65°C, and was quite heat stable up
to 50–60°C, retained 67.7% and 29.7% remaining activity after being incubated at 60°C
for 60 min and at 70°C for 10 min, respectively.
Haq et al. (2012) cloned a highly thermostable β-glucosidase gene of 1,341 bp from a
heterotrophic, obligate anaerobe Thermotoga petrophila RKU-1 (TpBglA). Heterologous
protein expressed in E. coli strain BL21 CodonPlus, purified monomeric recombinant
TpBglA enzyme displayed a prominent band of 51.5 kDa. The calculated Vmax and Km
values of TpBglA towards pNPG substrate were 42,720 µmol min-1 mg-1 and 2.84 mM,
respectively. Enzyme was optimally active at 80–90°C with a high specific activity of
30,400 U mg-1 against pNPG. TpBglA exhibited a broad substrate specificity towards
other p-nitrophenyl substrates and cellobiose. Conceivable catalytic regions of TpBglA
against nitrophenyl substrates and cellobiose were proposed on the basis of docking
studies.
Jabbour et al. (2012) identified, cloned, sequenced and characterized a thermo- and
glucose tolerant β-glucosidase enzyme from a heat loving bacterium
Fervidobacterium islandicum (FiBgl1A). The bgl1A gene contained 1,380 bp encoding
an enzyme of 459 residues and exhibited high homology to the other proteins of GH
family 1. The gene was expressed in E. coli host after ligated in pET-Blue1 vector, a high
level of FiBgl1A heterologous protein expression was observed, crude cell extract was
purified by heat treatment and gel filtration chromatography. Purified FiBgl1A with a
MW of 50 kDa, displayed optimal catalytic activity towards pNPG at 90°C and at pH
6.0–7.0. FiBgl1A showed remarkable thermostability over a broad range of temperature
(80-100°C). Purified enzyme exposed significant tolerance to glucose with Ki value of
211 mM, no metallic cations were required for the catalytic efficiencies, and not
influenced in the presence of EDTA, dithiothreitol, p-chloromercuribenzoate and urea
with 10 mM concentration. By the addition of various solvents (benzene, n-hexane,
hexadecane, heptane, toluene and alcohol) and surfactants except SDS, FiBgl1A activity
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was enhanced. Enzyme did not display any influence in the presence of different
saccharides especially 50 mM xylose.
Kim et al. (2012) identified and cloned a gene of β-glucosidase (1,467 bp) encoding 489
amino acid residues from Sulfolobus solfataricus, followed by overexpressed in E. coli
ER2566. The enzyme was purified by applying crude sample to a His-Trap High
Performance chromatography, finally the purity of enzyme analyzed on SDS-PAGE with
a band of 57 kDa. The purified enzyme displayed maximum hydrolytic activity at 90°C
and citrate phosphate buffer pH 5.5 with highest specific activity of 179 U mg-1 for
daidzin. Enzyme was highly stable at high temperature and exhibited at 95°C a half-live
of 10 h. With all these features, this cloned β-glucosidase from S. solfataricus was very
useful industrial biocatalyst among other β-glucosidases which can hydrolyzed isoflavone
glycosides.
Liu et al. (2012) cloned an internal sequence of a thermo-tolerant native β-glucosidase
(bgl3) gene from Aspergillus fumigatus Z5, a decomposing fungal strain. The internal
gene sequence contained an ORF of 2,622 bp nucleotides, which encoded a functional
protein, the predicted molecular mass was 91.47 kDa, and deduced protein belonged to
the family 3 of glycoside hydrolase. Cloned β-glucosidase (Bgl3) was successfully
expressed in Pichia pastoris X33, and heterologous protein was purified using affinity
column chromatography. Bgl3 purity was analyzed by SDS-PAGE that showed a band of
130 kDa MW. Characterization of purified recombinant Bgl3 was similar to the native
Bgl3 enzyme, had optimal temperature 60°C and pH 6.0. The cloned Bgl3 was quite
stable at 50-70°C temperature and 4.0-7.0 pH. Recombinant Bgl3 exhibited maximum
hydrolytic activity towards a synthetic substrate pNPG, with 101.7 U mg-1 specific
activity. Activity of enzyme was enhanced in the presence of metal cations; however
EDTA, SDS and Triton X-100 did not cause any obvious influence on enzyme. Km value
of recombinant Bgl3 for pNPG (1.73 mM) and for cellobiose (2.20 mM) were obtained
which was similar with native enzyme while Vmax values acquired towards these
substrates under standard assay conditions were lower than the native enzyme.
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Pei et al. (2012) cloned a β-glucosidase from a thermophilic bacterium
Thermoanaerobacterium thermosaccharolyticum DSM 571. The β-glucosidase (bgl)
gene encoded a protein of 443-amino-acids which commendably overexpressed, purified
and characterized in host cells of E. coli. The recombinant BGL protein expression level
in LB medium was improved from 6.6 to 11.2 U mg-1 by substituting the rare codons of
the N-terminal. Protein was purified by two-step methods (thermal precipitation and
affinity chromatography), and purified BGLA had 52 kDa MW. Optimal pH and
temperature of enzyme was 6.4 and 70°C, respectively. At 68°C, enzyme displayed a
half-life of 60 minutes and pH stability over a range of 5.2-7.6, Mn2+ and Fe2+ cation
significantly enhanced the BGLA activity. The Km of 0.62 mM and 7.9 mM were
observed for pNPG (Vmax of 64 U mg-1) and cellobiose (Vmax of 120 U mg-1), respectively.
Recombinant enzyme exhibited great tolerance to cellobiose and glucose; BGLA activity
was activated lower than 200 mM glucose concentration, while increasing concentration
of glucose, the catalytic activity was gradually repressed with Ki value of 600 mM
glucose.
Zou et al. (2012) reported the cloning and overexpression of a β-glucosidase (DtGH)
from Dictyoglomus thermophilum in E. coli BL21 using pET-21a vector. Induced the
recombinant culture with low concentration of IPTG followed by kept the culture at 37°C
for gene expression. Crude enzyme was purified by heat precipitation and His-Trap
affinity chromatography, and the finally purified fraction exhibited a single band (51.8
kDa) on SDS-PAGE. Purified DtGH biochemical characterization revealed that enzyme
had peak activity at 90°C temperature and neutral pH. DtGH showed great thermal
tolerance at 70-90°C with a high half-lives of 5 h at 90°C. The kinetic parameters of
DtGH towards pNPG were determined, Km and kcat value of 1.15 mM and 218 s-1,
respectively. Due to high thermostability, it was used for direct n-octanol glycosylation at
70°C and increased glucose conversion by 27%. Enzyme showed tremendous stability in
glucose and octanol-aqueous system, and retained 70% of its original activity even after
incubation at 70°C for 7 days.
Bai et al. (2013) reported molecular cloning of a β-glucosidase gene from thermophilic
bacterium Caldicellulosiruptor bescii (CbBgl1A). Purification of recombinant enzyme
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was done using heat treatment and chromatographic techniques, purified CbBgl1A
exhibited peak activity at 85°C and pH 6.8. CbBgl1A belonged to GH family 1, could
hydrolyzed a broad range of substrates such as aryl-β-glycosides and
cellooligosaccharides, and showed maximum affinity towards 4-nitrophenyl β-D-
glucopyranoside (pNPG) with 3.17 mM value of Km and 84.0 s−1 mM−1 value of kcat/Km.
The kinetic assay, molecular modeling and substrate docking of CbBgl1A with pNPG
and cellobiose which elucidated the weak binding ability of CbBgl1A with cellobiose.
Recombinant enzyme had high Ki value (113.8 mM) which revealed its ability to tolerate
the high glucose concentration.
Karnaouri et al. (2013) has been described an attractive report on cloning of a glycosyl
hydrolase (GH) family 3 β-glucosidase gene (bgl3a) from Myceliophthora thermophila.
The bgl3a gene was cloned under the control of a strong promoter and expressed in
Pichia pastoris. The purified fraction displayed a noticeable band of 90 kDa on SDS-
PAGE. The soluble active recombinant protein exhibited specific activity of 41 U mL-1
after 8 days induction. The purified MtBgl3a enzyme had the ability to catalyzed low
MW polysaccharides and substrates. MtBgl3a optimally hydrolyzed p-β-NPG at 70°C
optimal temperature and pH 5.0. Purified enzyme had displayed 143 minutes of half-life
at 60°C. Km value was 0.39 mM and 2.64 mM with p-β-NPG and cellobiose, respectively.
MtBgl3a showed great tolerance against D-gluconic acid, D-glucose and D-xylose sugars,
enzyme exhibited transglycosylation activity in the presence of methanol and its catalytic
activity was increased by short chain alcohol. Enzyme stability in ethanol was highly
prominent, and after 6 h incubation MtBgl3a retained 50% remaining activity as
compared to its original.
Li et al. (2013) described gene cloning of a thermophilic β-glucosidase from an archaeon
Thermofilum pendens (Tpbgl). The Tpbgl gene was overexpressed in an excellent host E.
coli. The purified enzyme had 77.8 kDa MW and exhibited peak catalytic activity with
pNPG substrate at 90°C in pH 3.5 buffer. After 60 minutes incubation at 95°C only 50%
catalytic activity was remained and Tpbgl enzyme showed high thermotolerant behavior
in an acidic conditions (pH 3.5). This extremophilic β-glucosidase included in glucoside
hydrolase family 3, the kinetic studies of recombinant Tpbgl revealed that enzyme had
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optimal catalytic efficiency (kcat/Km) of 3.05 with pNPG, Km value of 0.149 and 0.147
against cellobiose and mannobiose, respectively.
Lu et al. (2013) isolated a gene of β-glucosidase unbgl1A from a metagenomic library.
After amplification, unbgl1A gene was cloned and expressed in E. coli BL21. The
soluble cloned enzyme was purified by one-step method using affinity chromatography, a
band of 52 kDa MW was identified on SDS-PAGE followed by its biochemically
characterization was done. Under optimal temperature 50°C and pH 6.0, the enzyme
unBgl1A had a Km of 2.09 mM, and Vmax of 183.90 μmol min-1 mg-1. UnBgl1A activity
stimulated by NaCl, and had a great stability even at 600 mM of NaCl. The activity was
also enhanced by a variety of monosaccharides and disaccharides, can be tolerate up to
2000 mM glucose concentration, with a Ki value of 1500 mM.
Zhao et al. (2013) cloned a hyperthermostable β-glucosidase gene from Thermotoga
thermarum (Tt-bgl) and overexpressed in E. coli host. Recombinant host cells in LB-
medium induced at 37°C without IPTG, by this the activity Tt-BGL was improved to
13 U mL-1. The crude Tt-BGL was purified by using heat precipitation and affinity
chromatography. The purified enzyme had optimal activity at 90°C temperature and at
pH 4.8, and had a single band of 55 kDa on SDS-PAGE. The Tt-BGL showed high pH
and thermal stability. The Vmax and Km for p-nitrophenyl-β-D-glucopyranoside was 142 U
mg-1 and 0.59 mM, respectively. The enzyme was activated by glucose at ˃400 mM
concentrations, the Tt-BGL activity was progressively inhibited with further increasing
the glucose, but remained 50% of relative activity even in 1500 mM glucose.
Fang et al. (2014) demonstrated gene cloning and characterization of a β-glucosidase
(bglX) from Lactococcus sp. FSJ4. The bglX gene consisted of 1,437 nucleotides which
encoding 478 amino acid residues, purified recombinant monomeric bglX showed a
single band of 54 kDa by SDS-PAGE analysis. The bglX displayed broad substrate
specificity towards pNPG, pNPGal and pNPX which revealed that bglX was a
multifunctional biocatalyst. The enzyme showed great affinity towards aryl glycosides of
glucose and xylose. Optimal temperature and pH of enzyme was 40°C and 6.0 with
pNPG. Substrate docking and molecular modeling showed its multifunctional behavior
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against pNPG, pNPGal and pNPX. The active site pocket revealed the structure-function
relationship of bglX. Docking results elucidated that Glu 180 and Glu 377 were the vital
catalytic amino acids residues of recombinant enzyme, the value of
CDOCKER_ENERGY indicated that bglX has maximum activity towards pNPX
followed by pNPG and pNPGal. The experimental investigation are in conformity with
the obtained docking results.
Mehmood et al. (2014) demonstrated the gene cloning of a β-glucosidase (bglA)
belonging to glycoside hydrolase family 1 (GH1) from Thermotoga maritima and
overexpression in E. coli M15 host strain. Purified heterologous β-glucosidase (BglA)
displayed a prominent single band of 51 kDa on SDS-PAGE. The expression of
recombinant protein was enhanced when lactose used as an inducer, the soluble BglA
produced 40-47% of total cellular proteins which was 440 mg L-1 and 130 mg L-1
recombinant enzyme in Dubos salt medium and LB medium, respectively. The peak
activity of BglA was observed between temperature and pH range of 80 to 100°C and 5.0
to 7.0, respectively. The enzyme exhibited high thermostability up to 140°C. BglA had
Km, kcat and Vmax values of 0.56 mM, 187.1±20 s-1 and 238±2.4 IU mg-1, respectively.
BglA showed a half-life at 80, 90 and 100°C for 136 h, 71 h and 12.6 h, respectively.
Thermodynamic studies of BglA explained the enthalpy (33.73 kJ mole-1), entropy (-
246.46 kJ mole-1), melting temperature (130°C), Gibb’s free energy (127.96 kJ mole-1),
and activation energy for inactivation (36.92 kJ mole-1).
Schroder et al. (2014) characterized a thermotolerant archaeal β-glucosidase (Bgl1)
enzyme belongs to GH family 1 which was isolated from a hydrothermal spring
metagenome. Bgl1 displayed broad substrate specificity with high affinity towards
cellotriose, cellobiose and lactose. Bgl1 showed maximum specific activity 3195 Umg-1
at 90°C and pH 6.5. Enzyme was quite stable for 48 h at pH 4.5-9.5 and at 4°C. Bgl1
exhibited high thermostability, more than 40% activity was observed at 105°C, and after
30 min at 90°C a thermal activation was found. Thermal stability was 5 and 7-fold
enhanced after applying 100 and 200 bar pressure for 120 min at 90°C, respectively.
Enzyme activity was significantly increased by the addition of AlCl3, and Bgl1 also
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showed stability against glucose with Ki value of 150 mM. All these characteristics make
Bgl1 a prominent candidate for numerous applications at high temperature.
In 2014, Tang and co-workers cloned a β-glucosidase gene (bgl) from
Aspergillus oryzae GIF-10, effectively sequenced and overexpressed in Pichia pastoris.
The cDNA gene sequence consisted of 2,586 bp, encoding a protein of 862 amino acids
with a signal peptide chain. The high yield of protein (321 mg mL-1) was obtained after
seven days induction, and after purification recombinant β-glucosidase exhibited MW of
110 kDa on SDS-PAGE with the specific activity of 215 U mg-1. It exhibited broad
substrate specificity, efficiently hydrolyzed 1-4-α-diglycosides and 1-4-β-diglycosides.
Enzyme had 50°C optimal temperature, it displayed 65% remaining catalytic activity
after being incubated for 0.5 h at 60°C. Optimal pH of enzyme was 5.0 and stable over a
broad range of pH from 4.0-6.5, in the presence of Mn2+ cation hydrolytic activity of
enzyme was enhanced.
Yang et al. (2014) has been reported the study of a thermostable β-glucosidase gene of
GH family 3 from Neosartorya fischeri P1 (NfBGL1), and successfully cloned,
overexpressed and characterized in an expression host Pichia pastoris in a 3.7 L
fermentor at high cell density. After purification, SDS-PAGE exhibited a recombinant
NfBGL1 of approximately 80 kDa, enzyme showed maximum specific activity of 2189 ±
1.7 U mg-1 at 80°C and pH 5.0 against pNPG as a substrate. Enzyme NfBGL1 was stable
at 70°C for 2 h, displayed great enzyme catalytic affinity towards xylan, cellobiose,
barley β-glucan, sophorose, salicin, amygdalin, laminiarin, glycitin, lichenin and p-
nitrophenyl substrates. NfBGL1 efficiently hydrolyzed β-1,2 glycosidic bond and
exhibited great bioconversion for soybean isoflavone glycosides into free forms.
Dikshit R and Tallapragada P (2015) characterized a partially purified β-glucosidase from
Monascus sanguineus. Jack fruit seed was found to the best substrate to obtained
maximum yield of enzyme. At 60°C temperature and acidic pH, enzyme was optimally
active. Kinetic studies of enzyme were calculated by using different concentration of
pNPG, and found 0.89 mM of Km value. The enzyme activity was influence by metal
ions, 14.8% activity was inhibited by CaCl2 and 69.8% repressed by KCl. Enzyme
Review of Literature
41
efficiency was inhibited by 100 mM glucose concentration by which pNPG hydrolysis
did not take place. Molecular weight (116 kDa) of partially purified enzyme was
analyzed by SDS-PAGE.
Gumerov et al. (2015) cloned a highly thermo-tolerant multifunctional beta-glycosidase
(a putative β-galactosidase) gene Asac_1390 from a thermoacidophilic crenarchaeon
Acidilobus saccharovorans and was overexpressed in a mesophilic host (E. coli).
Asac_1390 gene encoded 490 amino acid residues and exhibited high sequence
homology with family 1 glycoside hydrolases from the different members of heat loving
archaea. Recombinant enzyme was purified to homogeneity with 55 kDa band, peak
catalytic activity was observed at (pH 6.0) 93°C. Asac_1390 enzyme showed extreme
thermostability, 50% activity was repressed at 90°C after 7 h incubation. Purified
Asac_1390 had a broad range of substrates specificity towards various aryl glycosides
and cellobiose, maximum hydrolytic activity was found for pNPGal (328 U mg−1) and
oNPGal (325 U mg−1) followed by pNPG (246 U mg−1) and pNPXyl (72 U mg−1).
Enzyme revealed great resistance to glucose, and Asac_1390 showed high Km value
(0.24 mM) and kcat/Km (1327 s−1 mM−1) for pNPG which demonstrated that Asac_1390
enzyme was not a β-galactosidase rather than a multifunctional β-glucosidase.
Ramani et al. (2015) identified a β-glucosidase isoform (Bgl4) from Penicillium
funiculosum NCL1, bgl4 gene encoding 857 amino acids which had a glycoside
hydrolase family 3 (GH3) catalytic module; successfully overexpressed in Pichia pastoris
KM71H, cloned Bgl4 protein was a glycoprotein which completely purified with a MW
of ~130 kDa. It had maximum activity at 60°C and pH 5.0 with 1354.3 U mg-1 specific
activity, and remain stable for 1 h at 60°C. Bgl4 displayed high activity towards aryl
substrates with β-xylosidic, β-glucosidic linkages and moderately hydrolyzed salicin and
cellobiose substrates. The enzyme exhibited maximum substrate conversion of p-
nitrophenyl-β-glucoside and cellobiose with the value of 3,332 and 2,083 μmol min-1 mg-
1, respectively. Bgl4 has exposed glucose tolerance up to 400 mM, and increased 2-fold
glucose yield when added with Trichoderma reesei Rut-C30 crude cellulase for cellulose
hydrolysis. Bgl4 was a thermostable and glucose tolerant enzyme, which could efficiently
use for bioethanol production.
Review of Literature
42
Xie et al. (2015) overexpressed a recombinant thermotolerant β-glucosidase gene in E.
coli from Thermotoga petrophila (TpBgl). By SDS-PAGE, it was confirmed that
recombinant purified TpBgl had MW of approximately 81 kDa. It was highly active at pH
5.0 and 90°C with the units of 21 U mL-1. Thermostability was enhanced in the presence
of Ca2+ cation. Enzyme had great selectivity for hydrolyzing at carbon C-20 of
ginsenoside Rb1, the inner and outer glucopyranosyl moieties, and produced ginsenoside
20(S)-Rg3 that was pharmacologically active. A highly thermotolerant β-glucosidase of
GH3-family from T. petrophila had improved thermostability by the addition of Ca2+.
Yang et al. (2015) reported a study on cloning of an intracellular β-glucosidase (BGL)
from Thermoanaerobacterium aotearoense, the gene (bgl) contained 1.314 kb, encoding
a 446 amino acid residual protein which belonged to the family 1 of glycoside hydrolase
(GH1) and had a canonical TIM barrel fold specific for GH1. To enhance the specific
activity of the crude enzyme, recombinant pET-bgl with chaperones propagated in E. coli
host which resulted in 9.2-fold increased expression (256.3 U mg-1) as compared to
without any chaperones. The purified enzyme showed highly thermo- and pH stability
with peak activity at pH 6.0 and 60°C. By the addition of 5 mM K+ or Na+, BGL activity
was significantly stimulated; enzyme exhibited great affinity towards cellobiose substrate
with the Km of 25.45 mM and Vmax of 740.5 U mg-1 values. In the presence of 50-250 mM
glucose concentration, BGL activity was stimulated and displayed the high Ki value of
800 mM glucose.
Zhao et al. (2015) has been cloned a novel GH family 3 β-glucosidase gene of 2.513 kb
from Myceliophthora thermophila (Mtbgl3b), which encoding 870 amino acid residues
and overexpressed in Pichia pastoris an efficient expression host. Highly active at 60°C
and pH 5.0, enzyme remained 90% activity at 50-65°C temperature and showed more
than 70% relative activity at acidic pH 3.5-6.0 and, MtBgl3b displayed psychrophilic
behavior and exhibited 51% relative activity at 40°C. MtBgl3b was quite stable at 60-
65°C, showed great affinity towards pNPG, and also had the ability to hydrolyzed pNPC,
cellobiose, cellotriose and cellotetraose. The Km 2.78 mM and Vmax 927.9 μM mg-1 min-1
kinetic values were determined. Finally, the enzyme catalytic reaction approach was
investigated by molecular docking strategy.
Review of Literature
43
Chan et al. (2016) identified a β-glucosidase gene (1359 bp) in the genomic DNA of
Anoxybacillus sp. DT3-1 (DT-Bgl), subsequently cloned and heterologously
overexpressed in E. coli BL21 (DE3). Phylogenetic study showed that DT-Bgl belonged
to GH family 1, the enzyme was effectively purified using Ni-NTA column
chromatography. DT-Bgl showed a single band of 53 kDa MW after analysis on SDS-
PAGE. Recombinant DT-Bgl enzyme was highly active, displayed peak activity against
pNPG and cello-oligosaccharides. The DT-Bgl exhibited best activity at slightly alkaline
pH 8.5 and 70°C, and did not need any co-factors or metallic ions for activation. The
values of Vmax and Km for DT-Bgl were 923.7 U mg-1 and 0.22 mM, respectively, using
pNPG as substrate. The DT-Bgl was a thermotolerant enzyme with low glucose
inhibition, and retained 93 % activity with the addition of 10 M glucose. It hydrolyzed
cellodextrins (long-chain) to cellobiose, and then converted cellobiose to glucose.
Long et al. (2016) cloned and expressed a 56,000 Daltons (Da) β-glucosidase (TthBgl)
from a hyperthermophilic bacterium Thermotoga thermarum, in a mesophilic host E. coli
BL21. Purified TthBgl showed maximum catalytic activity against pNPG (substrate) at
temperature 85°C and pH 5.0. After being incubated for 2 h at 80°C, enzyme retained
80% activity and displayed H+ ions stability from pH 5.0-6.0. TthBgl showed complete
independence of metallic ions while enzyme activity was considerably repressed by 1
mM Cu2+ and 0.1% sodium dodecyl sulphate (SDS), only 10% and 3% residual activity
was observed, respectively. The Km value was 2.41 mM and Vmax value of TthBgl was
8.79 U mg-1 against pNPG. By the addition of glucose, TthBgl activity was gradually
repressed, and 50% activity was reduced by 500 mM glucose. Phylogenetic analysis
confirmed that β-glucosidase belongs to GH family 3.
Peng et al. (2016) cloned and expressed a multifunctional gene belonging to GH family 1
from Caldicellulosiruptor owensensis (CoGH1A), which showed great activities of β-D-
glucosidase, β-D-galactosidase, β-D-xylosidase and exoglucanase. Enzyme exhibited
great thermostability at 75°C and stable for 12 h at this temperature. The enzyme
catalytic coefficients (kcat/Km) were 2467.5, 7450.0, 90.9, 1085.4, and 137.3 mM-1 s-1,
respectively against pNPG, pNPGal, pNPX, pNPC and cellobiose. CoGH1A was highly
active at pH 5.5 and 85 degree celsius with 1621 U mg-1 of specific activity. Km value of
Review of Literature
44
CoGH1A towards pNPG, pNPGal, pNPC, pNPX and cellobiose were 1.52, 0.61, 0.87,
7.18 and 15.65 mM, respectively along with Vmax value of 4027, 5100, 1065, 736, 2424
μmol mg−1 min−1, respectively. Enzyme showed no apparent requirement for metallic
cation for its hydrolytic activity and remain active in the presence of 5-10 mM cations.
CoGH1A a multifunctional glycoside hydrolase family 1 enzyme has significant
competency in saccharification of lignocellulosic biomass and lactose decomposition.
CHAPTER-III
MATERIAL & METHODS
Material & Methods
45
3.1. Materials
3.1.1. Chemicals, Enzymes and Kits
Mostly, all chemicals used in the present study were of high purity analytical grade
commercially available. Chemical reagents, detergents, inhibitors, salts and buffers were
acquired from Fluka (Switzerland) and Merck (Darmstadt, Germany), while all substrates
employed in the research work were purchased from Sigma chemicals Co. (St. Louis, MO,
USA). Antibiotic chloramphenicol, and cultivation media components were acquired from
Oxoid (Basingstoke, Hampshire, England). Enzymes used in molecular gene cloning, such
as T4 DNA ligase, restriction endonucleases, Polymerase chain reaction (PCR)
components along with Taq polymerase, and antibiotic ampicillin, X-gal, Isopropyl-β-D-
thiogalactopyranoside (IPTG), InsT/A cloning kit (Catalog # K1214), ready-to-use DNA
ladder (GeneRulerTM # SM0333) along with 6X DNA loading dye were acquired from
Thermo Fisher Scientific Inc. (Waltham, USA). QIAprep spin miniprep kit (Catalog #
27104) and QIAquick gel extraction kit (Catalog # 28704) were supplied by QIAGEN
(Hilden, Germany) to purify PCR amplicons and restricted DNA fragments from agarose
and impurities. Protein molecular size marker (Catalog # 69079-3) were obtained from
Novogen (Madison, WI, USA).
3.1.2. Bacterial Strains and Vectors
Hyperthermophilic bacterium Thermotoga naphthophila RKU-10T (ATCC BAA-489 /
DSM 13996 / JCM 10882), employed as a source of genomic DNA having β-1,4-
glucosidases encoding genes, was cultivated anaerobically. PCR amplicons of both TnbglA
and TnbglB genes were initially ligated in pTZ57R/T cloning vector (Thermo Fisher
Scientific Inc. Waltham, USA). Linearized ready-to-use pTZ57R/T vector tailed with 3'-
ddT over hangs, as Taq polymerase adds extra nucleotide adenine (A) 3'-ddA at the end of
amplicons during PCR, by this genes were easily cloned in vector. It was selected due to
one step cloning method, additional modifications of PCR products was not required, high
efficiency yields of recombinants (up to 90%), has multiple cloning site (MCS) designed
for easy manipulation of insert genes, and for effectual blue-white screening. Initially, E.
Material & Methods
46
coli DH5α strain was used as a host for recombinant plasmids propagation and
transformation. For over-expression, pET-21a(+) expression vector and mesophilic host E.
coli BL21 CodonPlus (DE3)-RIPL strain were used, which acquired from Novogen
(Madison, WI, USA).
3.1.3. General Growth Media
Usually, for the cultivating and maintaining E. coli DH5α and CodonPlus (DE3)-RIPL
bacterial strains LB medium was used supplemented with ampicillin (100 µg mL-1) when
required, cultivated at 37°C temperature with 200 revolution minute-1 (rev min-1)agitation.
LB-broth medium composed of 1% (w/v) NaCl, 0.5% (w/v) yeast extract and 1% (w/v)
tryptone, while LB-agar medium contained 1.5% (w/v) agar along with above mention
ingredients. The media components were mixed thoroughly in the double distilled water
(ddH2O) followed by autoclaved at temperature 121°C, pressure 15 lb in-2 for 20 minutes
in Erlenmeyer flasks.
3.1.4. Buffers and Reagents
In the present research work various buffers, reagents and solutions were used. All recipes
of the solutions and reagents are discussed in this section.
3.1.4.1. General stock solutions
Some general stock solutions were prepared in this study, which used to make various other
working solutions.
3.1.4.1.2. Tris–hydrochloride buffers (1 M, pH 7.6 and 8.0)
For the preparation of 1 M Tris-Cl (Tris-HCl), trizma base (molecular weight, 121.14 g
mol-1) dissolved in distilled water, adjusted the required pH (7.6 and 8.0) using 0.2 N HCl,
made the final volume by distilled water (dH2O), filtered the stock solutions.
3.1.4.1.3. Ethylenediaminetetraacetic acid (0.5 M, pH 8.0)
EDTA stock solution was prepared by dissolving EDTA powder salt (MW, 360.32 g mol-
1) in dH2O, slowly added 10 N NaOH in the solution and adjusted the required pH (8.0),
Material & Methods
47
when the solution become clear and pH adjusted, made the final volume by dH2O. The
stock solution was stored at room temperature after wet heat sterilization.
3.1.4.1.4. Potassium acetate (5 M, CH3CO2K)
Weight out required potassium acetate salt (MW, 98.14234 g mol-1), dissolved in some
dH2O initially, and then raised the final volume with dH2O.
3.1.4.2. Solutions for the extraction of genomic DNA
Genomic DNA from T. naphthophila culture was extracted by phenol-chloroform method,
originally demonstrated by Sambrook et al., (1989). For DNA isolation process, following
buffers and reagents were prepared according to the standard protocol.
3.1.4.2.1. TEN Buffer
The buffer was prepared by mixing 10 mM sodium chloride (NaCl), 10 mM Tris-HCl (pH
7.6) and 1 mM EDTA (pH 8.0) in the distilled water. After thoroughly mixing, autoclaved
the buffer at 15 lb in-2 pressure and 121°C temperature, and stored at 4°C.
3.1.4.2.2. SET Buffer
In SET buffer, EDTA (50 mM) and Tris-HCl (50 mM) having pH 8.0 and 7.6, respectively
dissolved in ddH2O and then added 20% sucrose, the final volume was made using ddH2O
and store at 4°C after sterilization.
3.1.4.2.3. Sodium chloride (5 M NaCl)
Weigh out required NaCl salt (MW, 58.44 g mol-1), dissolved in dH2O properly, made the
final volume, and stored at room temperature after autoclaving.
3.1.4.2.4. Sodium dodecyl sulfate (25% SDS)
Weigh out SDS detergent powder, mixed in dH2O carefully and store at room temperature.
3.1.4.2.5. Lysozyme
Always prepared freshly, 10 mg mL-1 solution of lysozyme was prepared in TEN buffer.
Material & Methods
48
3.1.4.3. Solutions for Agarose gel electrophoresis
3.1.4.3.1. Tris-EDTA (TE) buffer
TE buffer mostly used to store genomic DNA and plasmids. To prepare buffer having pH
8.0, dissolved EDTA (1 mM, pH 8.0) and Tris-Cl (10 mM, pH 8.0) in double distilled
water, mixed thoroughly and store 4°C after wet heat sterilization.
3.1.4.3.2. Tris acetate EDTA (50X) buffer
Dissolved trizma base (2 M), EDTA (50 mM, pH 8.0) and acetate (1 M Glacial acetic acid)
in ddH2O, this solution has approximately a pH of 8.6, therefore generally not adjusted.
Filtered the stock solution after that sterilized completely by autoclaving (121°C, 15 lb in-
2 for 15 minutes), and stored at room temperature. Working solution for gel electrophoresis
was 1X TAE, which was mostly prepared freshly.
3.1.4.3.3. DNA loading buffer (6X)
This dye has xylene cyanol FF (0.03% w/v), bromophenol blue (0.03%w/v), EDTA (60
mM), Tris-Cl (10 mM, pH 7.6) and glycerol (60% v/v). Mixed all the components in ddH2O
and stored at -20ºC for further used. Tracking dye is mixed with DNA and RNA samples
during electrophoresis.
3.1.4.3.4. Ethidium bromide
Stock of ethidium bromide (10 mg mL-1) was prepared in double distilled water (ddH2O)
or in TE buffer, with continue stirring to sure that ethidium bromide had completely
dissolved. After proper mixing, transfer the dye solution in a dark bottle or wrap the
container tube with aluminum foil.
3.1.4.4. Solution for Competent Cell Preparation
Bacterial cells were made competent using 50 mM Calcium chloride (CaCl2) chemical
method. After weighing the powder of calcium chloride (MW, 110.98 g mol-1), mixed in
dH2O thoroughly, filtered and stored at 4°C after sterilization.
Material & Methods
49
3.1.4.5. Solutions for plasmid DNA isolation
Plasmid DNA from the harvested bacterial cells was isolated by alkaline lysis method.
Three main solutions were used in this method.
3.1.4.5.1. Alkaline lysis solution-I
Mixed EDTA (10 mM, pH 8.0) and Tris-Cl (25 mM, pH 8.0) in ddH2O, adjusted the pH
of the solution (8.0), autoclaved for 15 minutes followed by the addition 50 mM glucose
using 0.22 µm membrane filter in aseptic conditions and stored at 4°C for used in research
work, when required.
3.1.4.5.2. Alkaline lysis solution-II
This solution must prepared freshly, mixed 1% SDS and 0.2 N NaOH from the stock
solutions in autoclaved dH2O by gentle vortexing. Solution-II has pH around 12.0,
therefore no need to adjust.
3.1.4.5.3. Alkaline lysis solution-III
Mixed potassium acetate (5 M, 60 mL) and glacial acetic acid (11.5 mL) in ddH2O distilled
water to make final volume 100 mL. Adjusted the pH 5.0 of the solution, stored at 4°C
after wet heat sterilization.
3.1.4.6. Buffers and reagents for SDS-PAGE
Following buffers and reagents used for making SDS-PAGE resolving and stacking gel.
3.1.4.6.1. Resolving buffer (pH 8.8)
Required amount of Trizma base was dissolved in ddH2O to prepared 1.5 M Tris-Cl,
adjusted pH to 8.8 with HCl (0.2 N), afterward make the final volume.
3.1.4.6.2. Stacking buffer (pH 6.8)
Weigh out Trizma base for the preparation of 0.5 M Tris-Cl, dissolved in ddH2O, pH was
adjusted to 6.8 with HCl (0.2 N). Sterilization was obtained by autoclaving the solution
and stored at 4°C.
Material & Methods
50
3.1.4.6.3. Ammonium per sulphate (10% APS)
APS (10 % w/v) mixed in ddH2O, freshly made at the time of gel preparation.
3.1.4.6.4. Acrylamide/Bis-acrylamide Solution (30%)
Acrylamide (29 g) and bis-acrylamide (1 g) mixed well in ddH2O, stored in a brown bottle
at 4°C.
3.1.4.6.5. Sodium dodecyl sulfate (10% SDS)
SDS (10%, w/v) powder dissolved in ddH2O, and stored at room temperature.
3.1.4.6.6. Gel loading buffer (5X, pH 6.8^)
Loading dye sample buffer (5X) was prepared by dissolving SDS (10%, w/v),
bromophenol blue dye (0.25%, w/v), glycerol (50%, v/v), Tris-Cl (0.25 M, pH 6.8) and β-
mercaptoethanol (5%, v/v) or DTT (0.5 M Dithiothreitol) in ddH2O. Dye pH was
approximately 6.8 after mixing all the components. After preparation, the dye dispensed
aliquots (1 mL) in sterilized tubes and stored at -40oC. β-mercaptoethanol was added just
before use.
3.1.4.6.7. Tank/ Running buffer (1X, ~pH 8.3)
Also known as Tris-glycine buffer, 1X buffer was prepared by dissolving 1.47% (w/v)
Glycine, 0.3% Trizma base and 0.1% (w/v) SDS in dH2O. Adjusting the pH of the buffer
to 8.3 with 0.2 N NaOH or HCl before making the final volume, then filtered the buffer
and stored at room temperature. Mostly, buffer has ~pH 8.3 so no need to adjust.
3.1.4.6.8. Staining Solution
Acetic acid (10%), methyl alcohol (40%) and coomassie Brilliant Blue R-250 (0.1%)
dissolved in ddH2O. Filtered the solution and stored in an amber or brown colored bottle
at room temperature.
3.1.4.6.9. Destaining solution
Solution was made by mixing 10% acetic acid and 40% methyl alcohol in dH2O, and stored
in a brown bottle at room temperature.
Material & Methods
51
3.1.4.7. Buffers and reagents for Native-PAGE
Mostly same chemical reagents and buffer used in Native-PAGE except that SDS is not
applied during the formation of separating and stacking gel mixtures. Native-PAGE
loading dye buffer and Running buffer recipe is changed from the SDS-PAGE.
3.1.4.7.1. Native-loading buffer (2X, pH 6.8^)
2X native loading dye sample buffer was prepared by dissolving bromophenol blue dye
(1%, w/v), glycerol (25%, v/v), Tris-Cl (62.5 mM, pH 6.8) in ddH2O. Dye pH was
approximately 6.8 after mixing all the components, aliquots (1 mL) in sterilized tubes and
stored at -40oC.
3.1.4.7.2. Tank/ Running buffer (1X, ~pH 8.3)
1X glycine buffer was prepared by dissolving 1.47% (w/v) Glycine and 0.3% Trizma base
in dH2O. Mostly, buffer has ~pH 8.3 so did not adjust but if not then adjusting the pH of
the buffer to 8.3 with 0.2 N NaOH or HCl before making the final volume, then filtered the
buffer and stored at room temperature.
3.1.4.8. Buffers and Reagents for enzymes analysis
After cloning, various buffers used for the analysis and characterization of recombinant
enzymes.
3.1.4.8.1 Sodium acetate buffer (3.7–5.6)
This buffer is also known as Acetate buffer, to make this buffer, stock solution of 0.2 M
sodium acetate (C2H3NaO2) and 0.2 M acetic acid (CH3COOH) were prepared first, having
molecular weight of 82.0343 g mol-1 and 60.05 g mol-1, respectively. To make, 100 mL of
desired pH buffer (4.0–5.5), both stock solutions were adding in different ratio (volume,
mL) and make a final volume of 100 mL.
3.1.4.8.2. McIlvaine buffer (2.2–8.0)
McIlvaine buffer, composed of disodium hydrogen phosphate (0.2 M Na2HPO4) and citric
acid (0.1 M C6H8O7), most commonly known as citrate buffer or citrate phosphate buffer.
Material & Methods
52
Initially, the stock solutions of 0.2 M Na2HPO4 (MW, 141.96 g mol-1) and 0.1 M citric acid
(MW, 192.124 g mol-1) were prepared. After that, citrate phosphate buffer of desired pH
(from 2.5–8.0) was made by mixing both stock solutions to attained a final volume of 100
mL.
3.1.4.8.3. Tris-Cl buffer (7.0–9.0)
Weigh out Tris base and prepared 0.2 M Tris-Cl stock solution which was used later on to
made the required pH and molarity buffer with 0.2 N HCl.
3.1.4.8.4. Sorensen’s phosphate buffer (5.8–8.0)
The required pH buffer was acquired by mixing sodium dihydrogen orthophosphate (0.2
M NaH2PO4, 119.98 g mol-1 MW) and disodium hydrogen phosphate (0.2 M Na2HPO4)
stock solutions in different volumetric ratio and get 100 mL final volume of the buffer.
3.1.4.8.5. HEPES buffer (6.8–8.2)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (C8H18N2O4S, 238.3012 g mol-1 MW),
50 mM buffer was prepared in dH2O, with a pH of 7.0 to 8.0, maintained the required pH
by 10 N NaOH prior to make the final (100 mM) volume.
3.1.4.8.6. MOP buffer (7.0–7.8)
MOP is the famous name of 3-(N-morpholino) propanesulfonic acid (C7H15NO4S, 209.26 g
mol−1 MW), 50 mM buffer was prepared in dH2O, adjusted the pH with 10 N NaOH.
3.1.4.8.7. CAPS buffer (9.0–11.0)
CAPS is the common name of 3-(cyclohexylamino)-1-propanasulfonic acid (C9H19NO3S,
221.32 g mol-1 MW), 50 mM buffer was made by dissolving the required weight of CAPS
in dH2O, desired pH of the buffer was obtained with 10 N HCl.
3.1.4.8.8. MES buffer (5.6–6.6)
MES is the name of 2-(N-morpholino) ethanesulfonic acid (C6H13NO4S, 195.2 g mol-1), 50
mM buffer was prepared in dH2O and maintained pH with 10 N NaOH.
Material & Methods
53
3.1.4.8.9. Bradford’s reagent
To prepare dye-binding reagent G-250 (0.1 g coomassie brilliant blue) dissolved in 95%
ethanol (50 mL) followed by added 85% ortho-phosphoric acid (100 mL), mixed all the
component well and raised the volume up to 1 L with ddH2O. This reagent was kept at low
temperature (4°C) after filtration (twice) in a brown bottle.
3.1.4.8.10. Bovine serum albumin (BSA) stock
To make standard curve of BSA, various dilution were prepared from stock solution of
BSA. Stock solution of BSA (1 mg mL-1) was prepared by mixing BSA powder (100 mg)
in dH2O and make the final volume 100 mL. Filter the solution before used.
3.1.4.8.11 Sodium carbonate (1 M Na2CO3)
Weigh out the required amount to prepare 1 M sodium carbonate (MW 105.98 g mol-1),
mixed well in dH2O, store at room temperature after filtration.
3.1.4.8.12. DNS reagent
3,5-Dinitrosalicylic acid (DNS) reagent, prepared by dissolving all these following
components which were DNS powder (10.6 g), NaOH (19.8 g), Rochelle salts (306 g
sodium potassium tartarate, KNaC4H4O6·4H2O), 8.3 g of sodium metabisulphate
(Na2S2O5), 7.6 mL of phenol (before used melted at 50°C) and 1416 mL dH2O. Mixed
thoroughly, stored at room temperature after filtration in brown or amber bottle to avoid
photooxidation.
3.1.4.8.13. Metallic ion solutions
Metallic salts of sulphate, chloride, nitrate and phosphate, 100 mM solutions were prepared
in buffer and stored at room temperature.
3.1.4.9. Buffers for anion exchange chromatography (Resource Q column)
Two buffers used for anion exchange chromatography, buffer A (binding buffer) and buffer
B (elution buffer) which were prepared as follows:
Material & Methods
54
3.1.4.9.1. Binding buffer (Buffer A)
Tris-Cl (50 mM, pH 8.0) was prepared in deionized water. The buffer was kept at 4°C after
filter sterilization (0.45 µm filter).
3.1.4.9.2. Elution buffer (Buffer B)
The elution buffer B was prepared by dissolving NaCl (1 M) in Tris-Cl buffer (50 mM, pH
8.0). Stored the buffer at 4°C after filtration with 0.45 µm filter.
3.1.4.10. Buffers for Hydrophobic interaction chromatography (Resource ISO
column)
3.1.4.10.1. Binding buffer (Buffer A)
Binding buffer was prepared by dissolving (NH4)2SO4 (1.5 M) in Tris-Cl (50 mM, pH 8.0)
buffer. The buffer was kept at 4°C after filter sterilization (0.45 µm filter).
3.1.4.10.2. Elution buffer (Buffer B)
Tris-Cl buffer (50 mM, pH 8.0) was prepared for elution and stored the buffer at 4°C after
filtration with 0.45 µm filter.
3.2. Cloning and Sequencing of TnbglA and TnbglB Genes
To clone the cellulolytic genes from a hyperthermophilic bacterium T. naphthophila RKU-
10T in mesophilic expression host, first of all two putative cellulolytic (β-1,4-glucosidase)
genes were selected after studying nucleotide and FASTA sequences using online NCBI
database server followed by primers were designed to amplified the specific genes.
Genomic DNA was isolated after the cultivation of T. naphthophila RKU-10T, which was
used as a template for the amplification of β-1,4-glucosidase TnbglA and TnbglB genes.
Multiple copies of specific genes were obtained using PCR strategy, after amplification
PCR products were initially cloned in PTZR/T57 and then analyzed by sequencing of
nucleotides followed by sub-cloning in pET-21a(+) expression vector and propagated in
the mesophilic expression host. All steps related to the cloning are described in the
following sections.
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3.2.1. Sequence of β-1,4-glucosidase Genes
The complete genomic sequence of T. naphthophila was available on NCBI server under
GenBank accession number CP001839.1 and this bacterium has circular DNA with
1809823 base pairs (bp). The two putative β-1,4-glucosidase genes (1,341 and 2,166 bp)
sequence Tnap_0602 and Tnap_0656 locus tags were retrieved from T. naphthophila and
designated as TnbglA and TnbglB. Used gene sequences as query to carried out nucleotide
BLAST from NCBI.
3.2.2. Analysis of Non-Cutter Endonucleases
For gene cloning of TnbglA and TnbglB in pTZR/T57 and pET expression vectors, all the
non-cutter (zero-cutter) restriction endonucleases for both genes were determined using
NEBcutter V2.0 BioLabsInc software (Vincze et al., 2003). The nucleotide sequences of
both genes were uploaded to the online server and a list was retrieved which had all the
non-cutting restriction enzymes. By this analysis, NdeI and HindIII non-cutter restriction
endonucleases were selected for the cloning purpose of both genes. Therefore, NdeI
restriction site was introduced at 5′ end of both forward primers.
3.2.3. Primers Designing
To amplify the coding regions of TnbglA (ADA66698.1) and TnbglB (ADA66752.1), two
sets of primers were designed using Vector NTI Advance TM 10.3 software (Invitrogen
Corporation, California) and OligoCalc software (Kibbe, 2007). NdeI restriction site
upstream (underlined) in forward primer but no restriction site was added in reverse primer
because HindIII restriction site already present in multiple cloning site (MCS) of cloning
vector pTZ57R/T. After primers designing, the order was placed to Macrogen (Korea) after
that lyophilized primers obtained, which were diluted to make stock of 100 µM, and in
polymerase chain reaction (PCR) used 10 pM concentration of each primer.
3.2.4. Cultivation of Thermotoga naphthophila
A hyperthermophilic anaerobic bacterium Thermotoga naphthophila RKU-10T strain was
kindly provided by Prof. Dr. Camella Nesbo (Dalhouse University, Canada), employed as
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a source of two β-1,4-glucosidase genes TnbglA and TnbglB. The bacterium was cultivated
in MMI medium under anaerobic conditions by the method of Haq et al. (2012). The MMI
medium contained KH2PO4 (0.45 g L-1), MgSO4·7H2O (1.4 g L-1), KCl (0.33 g L-1), NH4Cl
(0.25 g L-1), NaCl (20.0 g L-1), soluble starch (1.0 g L-1), yeast extract (0.06 %),
CaCl2.2H2O (0.14 g L-1), MgCl2. 6H2O (0.9 g L-1), trace mineral (1.0 mL) solution (Widdel
et al., 1983), 0.02% resazurin (0.5 mL), and for anaerobic conditioning 0.5 M Na2S (4.0
mL). After mixing all the components thoroughly and adjusted the pH 6.8. The 125-mL
vials were used for the cultivation of T. naphthophila, sealed them tightly with completely
sterile stoppers of butyl-rubber. The culture was incubated at 80°C for 166 hours in a dry
incubator having static condition.
3.2.5. Extraction of Genomic DNA
Genomic DNA of T. naphthophila was isolated by the phenol-chloroform method which
was used as a template for the amplification of genes (TnbglA and TnbglB). All buffers
and reagents for the extraction of DNA were prepared essentially as described by
Sambrook and Russell, (2001). Bacterial culture (10 mL) was harvested in a sterile tube at
6,000×g (at 4°C for 10 minutes), the cell pellet was washed twice with TEN buffer (100
µL) and gently suspended in the same buffer after that centrifuged again (6,000×g at 4°C
for 10 minutes). Discarded the supernatant, dried the pellet then suspended the pellet in
SET buffer (150 μL) followed by added freshly prepared lysozyme (50 μL from the stock
of 10 mg mL-1), mixed gently and subsequently incubated at 37°C for 30 minutes, bacterial
cell wall break by this step. Afterward, 25% SDS (50 μL) was used to completely lyse the
cells. At this time, TEN buffer (100 μL) and 5 M NaCl (50 μL) was added sequentially.
Freshly prepared phenol-chloroform (1:1) was applied next in this mixture, by which
proteins were precipitated out. Now, the working tube was gently inverted to homogenize
all the components of the mixture, followed by centrifuged (6,000×g) at 4°C for
10 minutes. The upper viscous phase was transferred in a fresh sterile tube with the help of
a micropipette, added equal volume of chloroform in the supernatant and mixed it properly,
all residual phenol was removed by this step. The mixture was subjected to centrifugation
at 6,000×g (5 minutes), transferred the resulted upper aqueous layer in another sterile tube.
To precipitate DNA, treated this with double volume of absolute ethanol (chilled), and
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incubated for 15-20 minutes at -20°C to improved precipitation. After incubation, DNA
threads had seen clearly which was spooled out easily and transferred in a fresh tube or
centrifuged (10,000×g) at 4°C for 10 minutes, in order to get a DNA pellet. Then washed
resultant pellet with 70% ethanol and allowed to dry the pellet at 37°C. Resuspended both
spooled out DNA and DNA pellet in TE buffer (100 μL), added Rnase (1 μL) to the sample
tube to remove the RNA from the isolated DNA. Incubated the sample tube at 37°C for 1
h. After incubation, analyzed the DNA by absorbance measurements and quantification
followed by stored DNA at -40°C for further use.
3.2.6. Isolated DNA Analysis
Isolated DNA was analyzed by electrophoresis therefore, 0.8% (w/v) agarose gel was
prepared by mixing agarose with 1X TAE buffer followed by heating the solution in a
microwave oven for 1-2 minutes until it boiled and became transparent or completely clear.
Then cooled in a water bath at about 60-65°C, after that calculated amount of ethidium
bromide (0.5 μg mL-1) was added and mixed well. Poured the solution into a sealed gel-
casting platform followed by placed a suitable gel comb (well-former for sample loading)
in its slot to make wells. Allowed the gel to solidify completely at room temperature for
about half an hour. After solidification, transferred the agarose gel to the running tank of
the electrophoresis containing buffer and removed the comb cautiously. The genomic DNA
sample (5 µL) was mixed with 6X loading dye and electrophoresed through gel along with
DNA ladder (Figure 3.1). In 1X TAE buffer, electrophoresis was accomplished at 70 volts
for 55-60 minutes. When blue dye came near the bottom of the gel, stopped running. DNA
was visualized and analyzed by illuminating the gel on UV-transilluminator followed by
its image captured and saved the record using gel documentation system with GeneSnap
software (SynGene). Exposed the gel for only short time under UV light, to evade DNA
damage.
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Figure 3.1: GeneRuler DNA Ladder Mix, ready-to-use #SM0333 (0.1µg µL-1)
3.2.7. Quantification of DNA
DNA quantification or concentration of isolated DNA was determined by electrophoresis
(intensity of DNA band compared with the known standard amount) as well as
spectrophotometrically measuring absorbance at λ260 using TE buffer as a blank, in a UV-
spectrophotometer. However sample purity was analyzed by a ratio of A260/A280
(Sambrook and Russell, 2001). DNA quantity was obtained by using the following
formula.
Amount of DNA mL-1 = 50 × Optical density (OD260) × dilution factor
However, 1 OD260 = 50 µg mL-1 of DNA
3.2.8. Amplification of TnbglA and TnbglB
Primers were used to amplify TnbglA and TnbglB gene fragments containing 1.341 kb and
2.166 kb nucleotides using polymerase chain reaction (PCR). 50 µL reaction mixture was
prepared in an individual sterile PCR tube (0.5 mL) by placing on ice for each gene
amplification, mixture contained all the necessary components of the PCR along with
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specific concentration of each was stated in table 3.1. All Components of PCR were mixed
thoroughly and consolidated in the bottom of tube by brief centrifugation, and then
transferred to the new sterilized individual PCR tubes and a negative control was also
prepared for PCR analysis, containing all components except DNA template.
Table 3.1: Composition of amplification reaction mixture.
Reaction components Required concentration/Quantity
dNTPs mixture 0.2 mM
10X Taq buffer 1X Taq buffer
Forward Primer 10 pM
Reverse Primer 10 pM
MgCl2 2.5 mM
Template genomic DNA 40 ng
Taq DNA polymerase 1.5 U
Denuclease water to made 50 µL volume
3.2.9. Thermal Cycling Conditions
Placed the reaction mixture tube in a thermocycler for the amplification of DNA fragments.
Closed the lid of thermocycler, set its temperature 105°C, both TnbglA and TnbglB genes
were amplified using optimal thermocycler conditions. Therefore, optimal PCR program
was set according to the fragment length of both genes, and hence the thermal cycling
conditions for TnbglA and TnbglB varied from one another. The PCR optimal conditions
for TnbglA and TnbglB are described in figure 3.2a and 3.2b. To produce 3'-dA overhangs
in amplicons, final extension phase was used which enhanced the efficiency of ligation.
3.2.10. Analysis of PCR Products
To examine the amplicons, mixed the PCR product with 6X DNA loading dye and run on
0.8% agarose gel along with DNA size marker. The amplified DNA bands of genes
(TnbglA and TnbglB), which appeared after gel electrophoresis were visualized under UV
light. Following analysis, the amplicons band corresponding to 1.341 and 2.166 kb were
excised with a sharp sterilized surgical blade (to avoid to get extra agarose gel) and placed
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separately into two sterile pre-weighted 1.5 mL tubes to purify DNA fragments from
agarose gel.
3.2.11. Purification of Amplicons
To purify PCR amplified products from the agarose gel, QIAquick gel extraction kit was
used according to the recommended protocol. To melt the gel slices containing amplified
DNA bands after adding QG buffer (supplied with kit, as a gel melting solution), briefly
incubated the tubes in a pre-heated water bath at 50°C for 10 minutes. During the
incubation period, the tube contents mixed regularly with the intervals of 2-3 minutes by
95°C 95°C
52°C
72°C
4°C 5 min 1 min
1 min
1 min 10 min
30 cycles
Initial
Denaturation Denaturation
Annealing
Elongation Final Extension
72°C
∞
Figure 3.2 (a): PCR cycling conditions for TnbglA
95°C 95°C
54°C
72°C
4°C 5 min 1 min
1 min
2 min 10 min
30 cycles
Initial
Denaturation Denaturation
Annealing
Elongation Final Extension
72°C
∞
Figure 3.2 (b): PCR cycling conditions for TnbglB
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vortexing until the gel contents was completely dissolved in this buffer (did same with both
PCR amplicons). QG buffer solubilized the agarose gel slices and provided a suitable pH
condition (≤ 7.5) and salt for subsequent DNA binding to the silica-membrane. For DNA
binding, poured the sample mixture (700 µL) onto the QIAquick spin column present in a
collection tube (2 mL) and centrifuged (10,000×g) for 60 seconds. During this step, DNA
fragments adsorbed to the silica-membrane whereas all contaminants passed away through
the spin column. After discarding the flow-through, placed the column back, QG buffer
(100 µL) was poured onto the column and centrifuged (10,000×g) again for 60 seconds.
Afterward, bound DNA was washed with the ethanol-containing PE buffer (750 µL), and
centrifuged again for 60 seconds at 10,000×g. Discarded the flow-through, to remove the
residual ethanol (traces of PF buffer) from QIAquick column, that may interfere with the
following enzymatic reactions, centrifugation step was repeated again for 60-120 seconds.
Shifted the column into a fresh sterile eppendrof to elute the adsorbed DNA. Finally,
provided elution buffer was applied to the center of the column, and allowed it to stand at
room temperature for 120-180 seconds. Consequently, the bound DNA was eluted with EB
buffer (50 µL) after centrifuged at 10,000×g for 60 seconds. By measuring absorbance at
λ260 spectrophotometrically, concentration of purified DNA was determined. Visualized
and analyzed by agarose gel electrophoresis as explained earlier. After that stored the gene
clean products at -40°C for further use in TA cloning purpose.
3.2.12. Cloning of TnbglA and TnbglB in pTZ57R/T Vector
Initially, the purified amplicons of both genes were cloned in TA cloning vector pTZ57R/T
by employing tailing technique (Figure 3.3). By using this technique, PCR amplicons with
3'-dA overhangs, were directly ligated into linearized pTZ57R/T vector containing 3’-dT
overhangs. According to the recommended protocol of InsT/A clone kit, ligation mixture
(20 µL) was prepared in a sterile eppendrof tube (1.5 mL) by adding amplified product (50
ng) with linearized pTZ57R/T (100 ng). To break up any concatamers, both vector and
insert DNA were heated for few seconds in a pre-heated water bath at 65°C and quickly
chilled on ice. After chilling, added the 10X ligation buffer (2 µL, to get final concentration
1X) followed by T4 DNA ligase (5 units) and nuclease-free water, for volume made up.
Control reaction was also incubated parallel to the ligation reaction, containing no insert.
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All reaction mixtures were incubated at 16°C for overnight (16–18 hours). When ligation
were done successfully, the recombinant vectors designated as pTZ57R/T–TnbglA and
pTZ57R/T–TnbglB, which were employed to transform competent cells of E. coli DH5α.
Figure 3.3: Restriction map of pTZ57R/T, the vector pre-cleaved with Eco321 restriction enzyme and treated
with terminal deoxynucleotidyl transferase to make tailed with 3'-ddT overhangs at both ends to avoid re-
circulation of pTZ57R/T during ligation.
3.2.13. Preparation of Competent Cells
Component cells of E. coli DH5α were prepared according to the chemical (calcium
chloride) method with some minor modification, originally described by Cohen et al.
(1972). A single well isolated colony of E. coli DH5α was inoculated into sterile LB-broth
medium (10 mL) from freshly streaked LB-agar culture plate followed by kept at 37°C for
16 hours (overnight) in shaker at 200 rev min-1. The sterile LB-broth medium (50 mL) in a
Erlenmeyer flask was seeded with 1% (v/v) overnight (16 hours) culture and incubated at
37°C in a shaking incubator (200 rev min-1) until its OD600nm reached at 0.4-0.5 (mid log
phase). Culture was transferred to a fresh 50 mL sterile falcon tube (pre-chilled) after that
kept on ice bath for 10-15 minutes, E. coli DH5α cells must be keep cold during competent
cells preparation. Then, harvested the culture cells in a pre-chilled (at 4°C) Beckmen
centrifuge at 4,000×g for 10 minutes. After centrifugation, medium supernatant was
discarded and resuspended the pellet in ice-cold 50 mM calcium chloride (CaCl2) solution
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(half of the original culture volume) by gentle mixing. The cells suspension was incubated
on ice for 45–50 minutes, afterward centrifuged again as described above, decanted off the
supernatant and resuspended the resultant pellet in ice-cold 50 mM CaCl2 (1.5–2 mL) by
gentle swirling and kept on ice for further half an hour before transformation. Finally, 100–
200 µL aliquot of competent cells was transferred directly in sterile pre-chilled eppendrof
tube (1.5 mL) for transformation, while the remaining cells were stored at -80°C after
dispensed into aliquot, as 30% glycerol stock.
3.2.14. Transformation of E. coli DH5α
Ligation products (pTZ57R/T–TnbglA and pTZ57R/T–TnbglB) were used to transform
competent cells of E. coli DH5α according to the standard protocol of heat shock method
(Sambrook and Russell, 2001). Chemically prepared competent cells (200 µL) aliquots
were briefly thawed followed by adding overnight ligated DNA sample (5-10 µL), mixed
gently by tapping or pipetting and successively incubated on ice for 30-40 minutes. A
control transformation reaction was run parallel to the ligation product, containing a
circular vector (plasmid) without insert mixed with competent cells, and similarly
incubated on ice as describe above. After that, heat shock at 42°C was given to all mixture
tubes for 1.5 minutes and instantaneously incubated for 3-5 minutes on ice. Poured and
mixed well the sterile pre-heated (37°C) LB-broth medium (800 µL) thereafter to
facilitated the recovery of E. coli DH5α cells in a comfortable growth environment from
transformation shock. Followed by incubated the mixture for 1-2 hours at 37°C with
constant shaking. This incubation prior to spreading mixture on agar plates, allowed the
bacterial cells to recover completely and start to express their genes for antibiotic
resistance. After incubation, transformants (100 µL) were spread uniformly on LB-agar
plates supplemented IPTG (0.1 mM), ampicillin (100 µg mL-1) and X-gal (80 µg mL-1).
Remaining transformed cell suspension was subjected to centrifugation for 60 seconds at
10,000×g, approximately 800 µL supernatant was decanted off, remaining 100 µL used to
resuspend the cell pellet followed by spread on LB-agar plates and kept these plates at 37°C
for 16-18 hours (overnight). Several colonies were appeared on agar plates, while positive
putative transformants were identified by blue/white screening and further confirmed by
double digestion analysis, colony PCR and DNA sequencing.
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3.2.15. Colony PCR
Colony PCR was performed separately to confirm the positive transformants harboring
recombinant pTZ57R/T–TnbglA and pTZ57R/T–TnbglB plasmids. Prior to colony PCR,
selected ten to sixteen well-isolated white colonies for both cloned genes (TnbglA and
TnbglB), then made short streak or duplically spotted on freshly prepared LB-ampicillin
agar plates (100 µg mL-1) , and kept these plates at 37°C for overnight till the colonies
became 1-2 mm in diameter. Colony suspension of each selected single colonies were
prepared by transferring the colonies aseptically to the sterile individual eppendrof tubes
(1.5 mL) containing sterile water (50 µL), dissolved each colony in their respective tube
and boiled the suspension for 3-5 minutes to lyse putative cloned bacterial cells and
denature the proteins. Subsequently centrifuged (10,000×g) the all tubes for 2 minutes to
collect the condensate, all denature proteins and membranes were sedimented in the bottom
of the tube (pellet). Transferred the upper layer (supernatant) 10 µL from each colony tube
to properly labeled sterile PCR tubes (0.5 mL) and amplification reactions for bglA and
bglB were set up by adding all the components with exactly same concentration as
described above (Table 3.1) except template DNA and using gene-specific primers. Colony
PCR of TnbglA and TnbglB was carried out by applying same conditions as described
earlier. After amplification, the PCR products of all colonies were electrophoresed through
0.8% (w/v) agarose gel to examine the result, presence or absence of insert gene. Positive
bacterial transformants containing recombinant pTZ57R/T–TnbglA and pTZ57R/T–
TnbglB plasmids were maintained in E. coli DH5α with ampicillin as a pressure of
selection, stored at -80°C as glycerol stocks after restriction analysis of recombinant
plasmids.
3.2.16. Mini-preparation of Plasmid DNA
Recombinant pTZ57R/T–TnbglA and pTZ57R/T–TnbglB plasmids were isolated from
cloned DH5α culture using alkaline lysis method (Sambrook and Russell, 2001). PCR
positive colonies were streaked on freshly prepared LB-ampicillin agar plates (100 µg mL-
1), which were further used for making glycerol stocks and recombinant plasmid isolation.
Sterile LB-broth (10 mL) containing ampicillin (100 µg mL-1) was inoculated with a well
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isolated bacterial colony harboring recombinant plasmid in an Erlenmeyer flask. Incubated
the culture flask at 37°C for 16 hour (overnight) in an incubator with vigorous shaking (200
rev min-1). The inoculation and extraction of recombinant plasmid DNA procedure was the
same for all transformants. The overnight recombinant cultures were harvested in sterile
eppendrof tubes (1.5 mL) by centrifugation (4,000×g) for 3-5 minutes at 4°C, discarded
the resultant supernatants and dried the pellets. The pelleted cells were resuspended in ice-
chilled alkaline lysis Solution I (100 µL) by vigorously vortexing. Then, added freshly-
prepared alkaline lysis Solution II (200 µL), mixed the contents by gently vortexing or
inverting the tubes for 3-4 times followed by placed all tubes on ice for 3-5 minutes.
Thereafter ice-chilled Solution III (150 µL) was poured into the tubes and again incubated
on ice for further 10 minutes, then at 4°C centrifuged (10,000×g) the tubes for 10-15
minutes. Pellets were discarded while the resultant supernatants were transferred to the
new sterile eppendrof tubes (1.5 mL) subsequently added equal volume of
phenol:chloroform solution (1:1) to all tubes, and then by vortexing shake well, and
centrifuged (10,000×g, 4°C) for 3-5 minutes. Upper plasmid containing aqueous layer from
all tubes were carefully shifted to fresh sterile tubes, to remove residual phenol (if present)
from the separated aqueous layer, chloroform (equal volume) was poured to each tube and
centrifuged (10,000×g, 4°C) again for 3-5 minutes. Upper layer from all tubes were taken
out in fresh sterile tubes, precipitated the plasmid DNA by adding ice-chilled double
volume of absolute ethanol, kept all tubes on ice for 5 minutes, pelleted by centrifugation
(10,000×g, 4°C) for 5-10 minutes. Washed the precipitated DNA pellets with 70% ethanol
(1 mL), discarded the supernatant cautiously and air dried the pellets. Dissolved the
plasmid pellets in TE buffer (50 µL) containing DNase-free RNase (1 µL) and kept all
these tubes for further 60 minutes at 37°C after that analyzed the plasmids DNA by gel
electrophoresis and stored at -40°C for further use.
3.2.17. Restriction Analysis of pTZ57R/T–TnbglA and pTZ57R/T–TnbglB
Isolated recombinant plasmids (pTZ57R/T–TnbglA and pTZ57R/T–TnbglB) were used to
confirm the presence of insert genes by single and double restriction analysis with specific
molecular scissors, NdeI and HindIII. For restriction analysis of both recombinant
plasmids, reaction (50 µL) was prepared in individual fresh sterile eppendrof tubes (1.5
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66
mL) by mixing plasmid DNA (~15 µg), 10X tango buffer (2X final concentration), NdeI
(~10 units), HindIII (~10 units) and nuclease-free water, to made final volume 50 µL.
Mixed all the restriction reaction components by vortexing and reaction tubes incubated
for overnight (16 hours) at 37°C. The resulting DNA restriction pattern of both reactions
were analyzed under UV-light after electrophoresed on 0.8% agarose gel along with DNA
size marker.
3.2.18. Preparation of Glycerol Stock
After the conformation of positive transformants, all bacterial recombinant colonies were
maintained in the form of glycerol stocks after culturing. To prepare the glycerol stocks,
overnight culture of positive clones were mixed with sterile 30% glycerol (w/v) in a sterile
screw caped cryo tubes, immediately stored at -80°C after mixing properly, for the long
period.
3.2.19. Sequencing of Cloned Genes
To get the plasmid DNA of ‘sequencing-grade’, the recombinant plasmids (pTZ57R/T–
TnbglA and pTZ57R/T–TnbglB) were extracted according to the recommended procedure
of the miniprep kit (Qiagen). The overnight recombinant culture was harvested, and
resuspended the pellet in PI buffer (250 µL) followed by the adding P2 buffer (250 µL),
then gently inverted the tube, poured N3 buffer (350 µL) and again inverted the tube 3–4
times. Centrifuged (10,000×g) for 5 minutes at room temperature, discarded the pellet and
loaded the supernatant to the QIAprep spin column after that centrifuged (10,000×g) again
for 60 seconds. Discarded the flow-through, washed the column with PE buffer (750 µL),
centrifuged (10,000×g) again for 60 seconds, and discarded the flow-through. To eliminate
residues of wash buffer, repeated the centrifugation (10,000×g) step again. To elute bound
DNA, placed the column in a clean sterile tube (1.5 mL), poured TE buffer (50 µL) on to
the column, and allowed to stand for 120 seconds followed by centrifuged (10,000×g) for
60 seconds. Quality and quantity (concentration) of eluted plasmid DNA was scrutinized
by λ260 measurements and electrophoresis. Recombinant plasmids were provided to
Macrogen, Korea for genes sequencing.
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3.2.20. Sequence Analysis and Protein Structure study
By utilizing Blast server at NCBI and ClustalW2 (Clustal omega), nucleotide and amino
acid sequences of both recombinant proteins were analyzed (aligned and compared).
Clustal omega online server was used to find the homology with others related protein
sequences and conserved regions. Theoretical molecular mass, isoelectric point (pI),
positively and negatively charged amino acid residues, aliphatic index and instability index
was calculated for amino acid sequence using ProtParam tool. The deduced protein
sequences of TnBglA and TnBglB were modeled for 3D structure utilizing SWISSMODEL
(Kiefer et al., 2009) and RaptorX sever (Källberg et al., 2012). PDBsum (Laskowski, 2009)
was used to study the secondary structure of TnbglA and TnbglB; and some tools of
ExPASy were also used to study the peptide information, protein structure elucidation
(swissProt) and functional information (Gasteiger et al., 2003).
3.3. Sub-cloning of TnbglA and TnbglB in Expression Vector
To get the adequate level of TnbglA and TnbglB expression, an excellent expression vector
was selected from vector series, amongst which T7 expression system using pET vectors
in E. coli BL21 CodonPlus (DE3)-RIPL prokaryotic host, has proved to be an excellent
over-expression system. An efficiently strong T7 lac promoter has been developed using
pET expression system to regulate the foreign gene of interest expression in E. coli BL21
CodonPlus (DE3). Therefore, pET-21a(+) proficient expression vector was employed to
get the high-level expression of both inserted genes (Figure 3.4). Firstly, constructed the
TnbglA and TnbglB expression plasmids followed by propagated into DH5α and for
expression, BL21 CodonPlus strain of E. coli was used as a mesophilic prokaryotic
expression host, which contains extra copies of the proL, leuW, ileY and argU tRNA
genes.
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Figure 3.4: Map of pET-21a(+) expression vector with cloning and expression sites. Vector contain a
pBR322 plasmid origin of replication (Ori), gene for ampicillin resistance (ampr), T7 lac promoter, which
contain a 25 bp long lac operator sequence. HindIII restriction site for the insertion of genes also shown.
3.3.1. Construction of Recombinant Expression Plasmids
E. coli DH5α competent cells was transformed with the purchased pET-21a(+) expression
vector followed by prepared glycerol stocks of E. coli DH5α containing pET-21a(+) vector
and stored at -80°C. For construction of recombinant expression plasmid, pTZ57R/T–
TnbglA and pTZ57R/T–TnbglB recombinant plasmids as well as pET-21a(+) vector were
isolated from the E. coli DH5α glycerol stocks by using alkaline lysis method (Sambrook
and Russell, 2001). Recombinant plasmids (pTZ57R/T–TnbglA and pTZ57R/T–TnbglB)
and expression vector pET-21a(+) were double cleaved with NdeI and HindIII restriction
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enzymes. Double digestion reactions (50 µL for each) were prepared in individual fresh
sterile eppendrof tubes (1.5 mL) by mixing isolated recombinant plasmid DNA along with
all components with same concentrations for proper reaction, as described earlier. Mixed
all the restriction reaction components by vortexing and incubated at 37°C for overnight
(16 hours). With pET-21a(+) vector, a corresponding double digestion was also carried
out. After incubation, the restricted plasmid reactions were mixed with DNA loading buffer
(6X) followed by electrophoresed on 0.8% agarose gel along with the DNA size marker,
after running the gel plasmid double restriction analysis was evaluated under UV-light.
The digested fragments (bands) of inserted genes (TnbglA and TnbglB) as well as pET-
21a(+) vector were excised from the 0.8% agarose gel, immediately transferred into the
individual sterile tubes (1.5 mL) and removed from gel using QIA quick gel extraction kit
(protocol described earlier). Purification of insert genes and linearized pET-21a(+),
restricted gene fragments (TnbglA and TnbglB) and pET-21a(+) were ligated by using T4
DNA ligase at the corresponding sites. The ligation reaction of bglA and pET was set up
in sterile tubes (1.5 mL) by adding double digested insert TnbglA (500 ng), 10X ligation
buffer (1X final concentration), double restricted pET-21a (100 ng), T4 DNA ligase (5
units) followed by make the final volume with nuclease-free water, while the ligation
reaction of TnbglB had all the components with same concentrations as described above
except the purified insert bglB whose final concentration was 400 ng in the reaction. A
control reaction was also prepared which did not had insert. Mixed all the ligation
component in respective tubes by vortexing and spun down by centrifugation at 4,000×g
for 0.5 minute after that incubated at 16°C for 16 hours (overnight).
3.3.2. Transformation of E. coli DH5α with Constructed Expression Plasmids
Competent cells were prepared chemically using CaCl2, and ligation mixtures of both genes
(TnbglA and TnbglB) were used to transformed E. coli DH5α competent cells individually
by heat shock method (previously described). After transformation, resultant bacterial
transformants cells were plated over uniformly on separate LB-agar plates containing
ampicillin (100 µg mL-1), and then kept at 37°C for 16-18 hours, several white colonies
appeared on agar plates. Positive putative transformants harboring recombinant plasmids
Material & Methods
70
(pET-21a–TnbglA and pET-21a–TnbglB) were screened and separated by colony
polymerase chain reaction and further confirmed by restriction analysis.
3.3.3. Colony PCR and Extraction of Recombinant Expression Plasmids
Putative transformants colonies were used to perform colony PCR separately for both
genes according to the protocol as described earlier. After colony PCR, positive bacterial
transformants harboring recombinant expression plasmids (pET-21a–TnbglA and pET-
21a–TnbglB) were analyzed and confirmed by electrophoresis. Successful clones were
streaked again and kept them on 4°C. Inoculated positive colonies of E. coli DH5α in LB-
broth containing ampicillin (100 µg mL-1) followed by kept in a shaking incubator for
overnight at 37°C, and extracted the recombinant plasmids by alkaline lysis method.
Visualized the quality and quantity of plasmids by agarose electrophoresis.
3.3.4. Restriction Analysis of Recombinant Plasmids
To further confirm the positive transformants, double digestion analysis of isolated
recombinant plasmids pET-21a–TnbglA and pET-21a–TnbglB were carried out in sterile
individual tubes (1.5 mL) using restriction enzymes NdeI and HindIII. Restriction reaction
was prepared separately by adding isolated recombinant plasmid (~15 µg), 10X tango
buffer (2X), NdeI (~10 units), HindIII (~10 units) and water (nuclease-free). Mixed and at
37°C kept the reaction mixture for 16 hours followed by analyzed on 0.8% agarose gel
electrophoresis. Positive clones were stored for long time as glycerol stock at -80°C, after
confirmation of positive recombinants by colony PCR and double restriction analysis.
3.3.5. Transformation of E. coli BL21 CodonPlus with Constructed Expression
Plasmids
Isolated recombinant plasmids (pET-21a–TnbglA and pET-21a–TnbglB) were used to
transform E. coli BL21 CodonPlus competent cells as an expression host by heat shock
method. After transformation, the bacterial transformed cells were plated on LB-agar plates
containing chloramphenicol (50 µg mL-1) and ampicillin (100 µg mL-1) followed by
incubated for overnight (at 37°C), a number of white colonies obtained on plates. To screen
positive transformed colonies, colony PCR was performed again at optimal standard
Material & Methods
71
conditions as described earlier. The positive transformants were stored at -80°C as stocks
for long time according to the same protocol as described earlier and used further for
production and expression analysis.
3.4. Heterologous Expression and Production of TnBglA and TnBglB
The expression analysis of both recombinant proteins were scrutinized by inoculating E.
coli BL21 CodonPlus harboring the pET-21a–TnbglA and pET-21a–TnbglB plasmids were
grown in sterilized LB broth and later on to enhance the production of both cloned proteins
various other modified media were also used, under the pressure of appropriate antibiotics
(ampicillin and chloramphenicol), and induced with isopropyl-β-D-thiogalatopyranoside
(IPTG) or later on with an alternative cheap inducer lactose was used for both recombinant
proteins production and over-expression.
3.4.1. Inoculum preparation
The seed cultures of both clones were prepared by inoculating individual LB sterilized
medium (10 mL) in two Erlenmeyer flasks (100 mL) with a 10 µL of frozen glycerol stocks
of engineered E. coli BL21 (DE3)-RIPL, supplemented with chloramphenicol (50 µg
mL−1) and ampicillin (100 µg mL−1) separately. Followed by both cultures were kept in a
shaking incubator at 200 rev min-1 for overnight (16 hours) at 37°C. These both seed
cultures were used as the standard inoculum to inoculate sterilized LB medium and all
other inducing media.
3.4.2. Production of TnBglA and TnBglB in LB medium
Initially, feasibility of LB-broth as the production medium for E. coli BL21 CodonPlus,
TnBglA and TnBglB expression and enzyme activity were investigated. Used 1% of
overnight both cloned cultures to refresh LB-broth in individual Erlenmeyer flasks
containing both antibiotic solutions with same concentration, kept at 37°C with 200 rev
min-1 till OD600nm reached at 0.6, the preferred phase for heterologous protein TnBglA and
TnBglB expression in our preliminary finding. Then heat shock was given to the bacterial
culture by placing all fermentation experimental flasks in a shaking water bath at 200 rev
min-1 at 42°C for 60 minutes. For TnBglA and TnBglB proteins over-expression,
Material & Methods
72
recombinant cells were induced with different concentration of inducer IPTG (0.1–1.0
mM) or lactose (10–250 mM), immediately after heat shock treatment. After induction, the
culture was allowed to incubate up to 72 hours, at various temperatures (16-42°C) in a
shaker (50-250 rev min-1); bacterial culture samples (fractions) were withdrawn after
regular intervals (2 hours) to assess the cell growth (OD600nm), optimal expression and
activity of heterologous protein TnBglA and TnBglB. By SDS-PAGE analysis,
heterologous proteins expression were scrutinized followed by assayed to determine
recombinant enzyme activity.
3.4.3. Enhanced expression and production using various inducing media
Small-scale expression analysis was set with various inducing media including LB+, ZB,
4×ZB, ZBM, ZYB, 3×ZYB, ZYBM9, 3×ZYBM9, and M9 with some modifications in
their composition were used in this present work (Studier, 2005). Individually, the effect
of ten various cultivation medium on recombinant bacterial growth, expression of
heterologous proteins (TnBglA and TnBglB) and activity were studied after inducing
individually either with IPTG or lactose. All used media with complete composition are
given in table 3.2. To enhance the expression and production of recombinant proteins or to
optimize the fermentation medium, inoculated all respective sterilized broth media (10 mL)
containing both antibiotics with the standard inoculum of recombinant E. coli BL21
CodonPlus (DE3)-RIPL, followed by overnight incubation in a shaker (200 rev min-1) at
37°C. Transferred 1% of all overnight cultures to their respective sterilized media
containing both antibiotics and incubated at 37°C (200 rev min-1), followed by done all
steps including heat treatment to sample fractions withdrawn, as described earlier. Here,
various parameters such as heat shock treatment, IPTG concentration, lactose
concentration, incubation temperature, agitation and time of incubation were studied to
enhance the activity and expression of heterologous proteins (TnBglA and TnBglB).
3.4.4. Analytical methods
Bacterial cell growth was monitored as the described by Basar et al. (2010). During
fermentation, culture broth sample were withdrawn regularly and at 4°C centrifuged
(12,000×g) for 10 minutes, obtained cell pellets were used to find out the cell growth.
Material & Methods
73
Resuspended the cell pellet in NaCl (0.9%, w/v), and OD600nm was measured when optical
density value exceeded 0.9, filtered the cell suspensions using membrane filter, then dried
the retentate in an oven (80°C) for 24 h, to calculate the dry cell weight (DCW).
Relationship of OD and DCW was assessed from many experiments, which showed that
one unit of optical density (OD600nm) was almost equivalent to 0.6 g DCW L-1.
Table 3.2: Compositions of different media.
Medium Composition
LB Yeast Extract (0.5%, w/v), NaCl (1.0%, w/v), Tryptone (1.0 %, w/v)
LB+ Yeast Extract (1.0%, w/v), NaCl (0.5%, w/v), Tryptone (1.6%, w/v).
ZB NaCl (0.5%, w/v), Tryptone (1.0%, w/v)
4×ZB NaCl (2.0%, w/v), Tryptone (4.0%, w/v).
ZBM NaCl (0.5%, w/v), Tryptone (4.0%, w/v)
ZYB Yeast Extract (0.5%, w/v), NaCl (0.5%, w/v), Tryptone (1.0%, w/v)
3×ZYB Yeast Extract (1.5%, w/v), NaCl (1.5%, w/v), Tryptone (3.0%, w/v)
ZYBM9
Yeast Extract (0.5%, w/v), NaCl (0.5%, w/v), Tryptone (1.0%, w/v),
NH4Cl (0.1%, w/v), KH2PO4 (0.3%, w/v), Na2HPO4 (0.6%, w/v),
MgSO4.7H2O (1 mM), and Glucose (0.4%, w/v).
3×ZYBM9
Yeast Extract (1.5%, w/v), NaCl (1.5%, w/v), Tryptone (3.0%, w/v),
NH4Cl (0.1%, w/v), KH2PO4 (0.3%, w/v), Na2HPO4 (0.6%, w/v),
MgSO4.7H2O (1 mM), and Glucose (0.4%, w/v).
M9
NH4Cl (0.1%, w/v), Na2HPO4 (0.6%, w/v), Glucose (0.4%, w/v), KH2PO4
(0.3%, w/v), NaCl (0.5%, w/v), MgSO4. 7H2O (1 mM), and CaCl2
(0.1mM).
3.4.5. Cell fractionation
Regularly, withdrawn bacterial samples were used to scrutinize the optimal expression,
activity assay and solubility of recombinant proteins (TnBglA and TnBglB) in all three
fractions: culture supernatant (extracellular), cell pellet (cell-bound) and cell lysate
Material & Methods
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(intracellular). The recombinant cell culture (5 mL aliquot) collected by centrifugation
(12,000×g) at 4°C for 10 minutes and media supernatant was used as soluble (extracellular)
fraction. The pellet was washed twice and resuspended in Tris-Cl (50 mM) buffer of pH
8.0, disrupted cloned cells by keeping cell suspension on ice during sonication (10×30
seconds bursts with 60 seconds intervals between successive pulses) in a Ultra Sonicator
(UP 400s), then again centrifuged (12,000×g) at 4°C for 10 minutes and the cell lysate
(resultant supernatant) was used as soluble fraction (intracellular), and the pellet of cell
debris was resuspended in Tris-Cl (50 mM) buffer of pH 8.0, which constituted the cell-
bound fraction (insoluble). All soluble and insoluble fractions thus obtained, were
individually assayed to determine the maximum heterologous TnBglA and TnBglB protein
activity and expression.
3.4.6. Protein Estimation
Recombinant Soluble protein (TnBglA and TnBglB) concentration from all fractions were
assessed by either absorbing measurement at λ280 or dye-binding (Bradford) method in
which BSA used as a standard (Bradford, 1976). To estimate the protein in a given sample,
reaction mixture was prepared in well dry test tube by adding appropriately diluted protein
sample (1 mL) followed by Bradford reagent (5 mL), and control reaction had dH2O instead
of protein sample. In Bradford reagent, Coomassie brilliant blue G-250 (binding dye) bind
with basic amino acid residues of protein subsequently blue color develop. Incubated the
reactions at room temperature for 10 minutes prior to measure the absorbance at λ595 using
spectrophotometer after adjusting the control as a blank for auto-zero. A standard curve
was plotted with known concentration of a protein like BSA and applied to compute the
protein concentration in test sample.
3.4.6.1. Standard Curve of BSA
To make the standard curve of BSA for protein estimation, sequentially increased
concentration of BSA dilutions ranging from 02-100 µg were prepared, in properly labelled
individual test tubes by using stock solution of BSA (1 mg mL-1). In each dilution test tube,
Bradford reagent (5 mL) was added and shake well. After 10 minutes incubation at room
temperature, measured the absorbance of all dilution reactions (in duplicate) at λ595 using
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spectrophotometer against a blank reaction (adjusted to auto-zero) which contained
Bradford reagent and dH2O instead of BSA protein dilution. A graph was plotted by taking
absorbance values of all BSA dilutions at ordinate axis and concentration of BSA at
abscissa axis. Protein concentration from given sample was estimated from the slop of this
BSA standard curve (Figure 3.5).
Figure 3.5: Standard curve of Bovine Serum Albumin (BSA).
3.4.7. SDS-PAGE Analysis
The recombinant protein TnBglA and TnBglB expression and molecular weight (MW)
were determined by SDS-PAGE analysis, according to the described method of Laemmli
(1970). 12 % SDS gel was prepared in two phases, initially resolving gel mixture poured
into assemble casting plates followed by stacking gel mixture was poured.
3.4.7.1. Separating/Resolving gel preparation
To prepare 12% resolving gel (10 mL), all required chemical reagents and buffers as
described in table 3.3 except (TEMED) were mixed well in a fresh falcon tube (50 mL).
After mixing, TEMED was added mixed again by gently swirled and immediately poured
the resolving gel mixture between already assemble two plates of Bio-Rad mini-gel
electrophoresis vertical casting apparatus, at the top 0.5 inch (2 cm) space was left for the
stacking gel and poured dH2O (100 µL) at the top of gel to make a flat surface of the gel,
y = 0.0073x + 0.0153R² = 0.9996
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 20 40 60 80 100 120
Ab
sorb
ance
(5
95
nm
)
Concentration µg mL-1
Standard Curve of BSA
Material & Methods
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to prevent dehydration and to remove oxygen, which may inhibit polymerization. Kept the
gel along with caster undisturbed at room temperature for approximately 30 minutes for
gel polymerization followed by completely drained the upper layer of dH2O and prepared
stacking gel was loaded on resolving gel.
Table 3.3: 12% SDS-PAGE resolving gel composition.
Required Chemicals Volume (mL)
dH2O 3.3
1.5 M Tris-Cl (pH 8.8) 2.5
30% (w/v)Acrylamide/Bis-acrylamide 4.0
10% (w/v) SDS 0.1
10% (w/v) APS (freshly prepared) 0.1
TEMED 0.004
3.4.7.2. Stacking gel preparation
After polymerization of resolving gel, the second phase of SDS-gel called stacking gel (3
mL) was prepared by mixing all the chemical components listed in the following table 3.4
except TEMED. After properly mixing, TEMED was added, swirled and poured the gel
mixture at the top of gel assembly. Immediately insert the clean comb into the gel to make
proper wells for loading the samples, kept gel assembly undisturbed at room temperature
for 30 minutes to polymerize the gel.
Table 3.4: SDS-PAGE Stacking gel composition.
Required Chemicals Volume (mL)
dH2O 2.037
0.5 M Tris-Cl (pH 6.8) 0.380
30% (w/v) Acrylamide/Bis-acrylamide 0.5
10% (w/v) SDS 0.03
10% (w/v) APS (freshly prepared) 0.03
0.4% (w/v) Bromophenol blue dye 0.02
TEMED 0.003
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3.4.7.3. Sample preparation
Protein samples before subjected for SDS-PAGE were prepared by mixing protein sample
(10-15 µg) with 5X SDS-gel loading dye in sterile tube (1.5 mL) followed by boiling for
3-5 minutes in a boiling water (100°C) in order to denature sample proteins. Then,
centrifuged for 60 seconds and transferred the sample supernatant in fresh tube and loaded
in well.
3.4.7.4. Electrophoresis
After polymerization, the set gel cassette along with comb was taken out and clamped into
the electrophoresis tank apparatus. Poured Tris-glycine buffer (1X) in the tank and filled
up to the marked level of the tank subsequently the inner region of clamped apparatus was
also filled with the same buffer. Removed gel comb cautiously without disturbing the wells
followed by washed them with glycine buffer by using a syringe. The prepared samples
were loaded along with protein marker (Figure 3.6) into the individual wells carefully by
using a fine tip. Electrophoresis was performed at a constant current (2 mA cm-2) for about
90-120 minutes until from the bottom of the resolving gel bromophenol blue dye came out.
Figure 3.6: Novagen Perfect Protein Marker (10-225 kDa), Cat # 69079-3
Material & Methods
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Turned off power supply immediately and removed the glass plates from the cassette and
separated out the gel carefully from the plates. To visualize the protein bands, stained the
gel with staining solution having Coomassive brilliant blue R-250, and placed the staining
gel tray in a shaker (50 rev min-1) for 30 minutes. After that placed out the gel from staining
solution, washed the gel with dH2O and dipped the gel in destaining solution with constant
agitation. Kept the gel on a shaking plate (50 rev min-1) for overnight to completely
destaining the gel, background became transparent and bands of protein were visible in the
gel.
3.4.8. Native-PAGE Analysis
The recombinant protein TnBglA and TnBglB were analyzed by Native-PAGE also. Native
gel was also prepared in two phases, initially resolving (separating) gel mixture poured into
assemble casting plates followed by stacking gel mixture was poured.
3.4.8.1. Separating gel preparation
To prepare 12% resolving gel (10 mL), all required chemical reagents and buffers as
described in table 3.5 except TEMED were mixed well in a fresh falcon tube (50 mL).
After mixing, TEMED was added mixed again by gently swirled and immediately poured
the resolving gel mixture between already assemble two plates of Bio-Rad mini-gel
electrophoresis apparatus. Kept the gel undisturbed at room temperature for
polymerization.
Table 3.5: 12% Native resolving/separating gel composition
Required Chemicals Volume (mL)
dH2O 3.396
1.5 M Tris-Cl (pH 8.8) 2.5
30% (w/v)Acrylamide/Bis-acrylamide 4.0
10% (w/v) APS (freshly prepared) 0.1
TEMED 0.004
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3.4.8.2. Stacking gel preparation
After polymerization of resolving gel, the second phase of native gel called stacking gel (3
mL) was prepared by mixing all the chemical components listed in the following table 3.6
except TEMED. After properly mixing, TEMED was mixed and poured the gel mixture at
the top of gel assembly. Immediately insert the clean comb into the gel to make proper
wells for loading the samples, kept gel assembly undisturbed for polymerization.
Table 3.6: Native stacking gel composition
Required Chemicals Volume (mL)
dH2O 2.1
0.5 M Tris-Cl (pH 6.8) 0.380
30% (w/v) Acrylamide/Bis-acrylamide 0.5
10% (w/v) APS (freshly prepared) 0.03
0.4% (w/v) Bromophenol blue dye 0.02
TEMED 0.003
3.4.8.4. Sample preparation
Protein samples before subjected for native PAGE were prepared by mixing protein sample
(~10 µg) with 2X native-gel loading dye in sterile tube (1.5 mL) and loaded in well.
Samples were not heated.
3.4.8.5. Electrophoresis
After polymerization, the set gel cassette along with comb was taken out and clamped into
the electrophoresis tank apparatus. Poured native Tris-glycine buffer (1X) in the tank and
filled up to the marked level of the tank subsequently the inner region of clamped apparatus
was also filled with the same buffer. Removed gel comb cautiously without disturbing the
wells followed by washed them with native glycine buffer by using a syringe. Loaded the
sample and run the gel with same method as described for SDS-PAGE.
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3.4.9. Determination of recombinant enzymes activity
The recombinant TnBglA and TnBglB enzyme activity assay were performed individually
in separate heat resistant caped tubes, using pNPG as a substrate and measured the amount
of pNP released from pNPG. A reaction mixture, contained 0.2 mL of pNPG (4 mg mL-1,
prepared in McIlvaine buffer of pH 7.0) and 0.4 mL of appropriately diluted enzyme (100
ng of BglA or BglB in 10 µL and 390 µL of pH 7.0 McIlvaine buffer) with 600 µL of final
volume. A single test was carried out in triplicate along with a control reaction, which had
all components with same concentration except recombinant enzyme (TnBglA or TnBglB),
under the assay conditions at 70°C for 10 minutes. All reactions were stopped by adding 3
mL of 1 M Na2CO3, followed by addition of diluted enzyme in control reaction tube and
to determine the released amount of pNP, measure the absorbance at 405 nm. A standard
curved of pNP was used to estimate the released amount of pNP and finally know about
the enzyme catalytic efficiency in term of units.
3.4.9.1 Standard Curve of para-Nitrophenol (pNP)
A standard curve of pNP was used to measure the enzyme activity of TnBglA and TnBglB.
This standard curve was drawn to convert the absorbance values into µM of pNP. Firstly,
prepared 1 mM p-nitrophenol of stock solution. Then, diluted the pNP serially ranging
from 20-500 µM in well dried label test tubes using stock solution, and made final volume
600 µL. A control was also run parallel by replacing pNP with dH2O, followed by adding
3 mL of 1 M Na2CO3 in each reaction tube and using spectrophotometer measured the
absorbance at 405 nm. A standard curve of pNP was plotted by taking absorbance along
Y-axis and various concentration of pNP along X-axis (Figure 3.7).
3.4.9.2. Enzyme Unit
“One unit of recombinant enzyme (TnBglA or TnBglB) activity corresponds to the amount
of enzyme necessary to release 1 μmoL (unit) of pNP per minute under the described
standard assay conditions”. Enzyme activity in term of units was calculated by applying
the following formula:
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81
Figure 3.7: Standard curve of para-nitrophenol (pNP)
3.4.9.3. Specific activity
Specific activity of TnBglA and TnBglB enzymes were described as enzyme units per mg
of the recombinant protein, and calculated by using the following formula:
3.5. Purification of Recombinant TnBglA and TnBglB
Various strategies were employed for the purification of recombinant β-1,4-glucosidases
enzyme after large scale (up to 3 L) production. After recombinant proteins production,
cultures were subjected to heat treatment, followed by ammonium sulphate NH4)2SO4
precipitation, dialysis and chromatography techniques were applied to purify cloned
TnBglA and TnBglB enzymes from the E. coli BL21 CodonPlus host proteins.
y = 0.0073x + 0.0153R² = 0.9996
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 50 100 150 200 250 300 350 400 450 500
Ab
sorb
an
ce (
40
5 n
m)
Concentration µM
Standard Curve of pNP
Conc. from graph (µM) × Dil. factor × 1000
Incubation time (min) × Total reaction volume (µL) Enzyme activity (U mL-1 min-1) =
U mL-1 min-1 of enzyme
mg of protein mL-1 Specific activity (U mg-1) =
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82
3.5.1. Batch production
Individually, the recombinant TnBglA and TnBglB enzymes produced on large scale at
optimal conditions which were found by small flask scale production. Initially, sterilized
LB broth medium was inoculated with 16 hours old overnight recombinant cultures
containing chloramphenicol and ampicillin with 50 µg mL-1 and 100 µg mL-1 as final
concentration, followed by left at 37°C in a shaking incubator for attaining the OD600nm of
culture 0.6. At 42°C, heat treatment was given for 60 minutes after that induced the cultures
with IPTG (0.5 mM) and kept flasks at 22°C with other optimal conditions for production
up to 72 hours. The bacterial culture was collected at optimal crude activity and expression
of recombinant TnBglA and TnBglB enzyme. Both enzymes were soluble, extracellular
and highly active, therefore crude supernatants were stockpiled after 72 hours and
concentrated twenty folds by using freeze drier or lyophilizer (Alpha 1-4 LO).
3.5.2. Heat Treatment
Lyophilized powder of both recombinant enzymes TnBglA and TnBglB from T.
naphthophila were resuspended in dH2O followed by subjected to thermal incubation
because both enzymes was isolated from hyperthermostable bacterium. Therefore, it was
observed that both enzymes must have thermal inactivation. To purify the recombinant
TnBglA and TnBglB, incubated the crude lyophilized enzymes in a pre-heated water bath
at 70°C for 60 minutes, after thermal precipitation the enriched enzyme samples were
immediately cooled at 4°C in an ice bath for 0.5 h, after that centrifuged (12,000×g) to
remove all denatured host heat labile proteins, for 20 minutes at 4°C.
3.5.3. Fractional Ammonium sulfate precipitation
Heat treated recombinant protein samples were subjected to ammonium sulfate
precipitation with 55–60% saturation according to the method described by De-Moraes et
al. (1999). Precipitation was performed at 4°C by slowly pouring the salt to the heat treated
partially purified enzyme samples with step wise increment and constant gentle stirring for
2-3 hours at 4°C, to attain various level of saturation. After each saturation stage,
centrifuged the samples at 12,000×g for 10 minutes at 4°C, to pellet down the precipitated
Material & Methods
83
recombinant proteins. Transferred the supernatant to another falcon and dissolved the pellet
in Tris-Cl buffer (50 mM) having pH 8.0, to ensure the precipitation of enzymes, activity
assay and protein contents were observed in each fractions. Stockpiled the fractional pellets
and dialyzed the resultant suspension to remove the salt from the precipitated protein.
3.5.4. Dialysis
Dialysis was carried out in order to remove the salt molecules from the precipitated protein
by using regeberated cellulose tubular membrane (Celluosep T4 #1430-33, membrane
filtration product Inc. USA) having nominal molecular weight cut off (MWCO) 12,000-
14,000. The dialyzing membrane was washed with buffer twice, and specific closure was
applied to close membrane tube one end. Followed by pouring the precipitated protein
suspension into tube with the help of micropipette, and also closed the other end of tube.
The dialyzing bag having sample suspended in pH 8.0 buffer of Tris-Cl (50 mM) of in a
beaker, placed the beaker on magnetic stir plate with continued stirring, regularly changed
the outer Tris–Cl buffer after 3–4 hours, process was continued at 4°C (approximately up
to 24 hours) till the suspension became clear and salt was completely removed. After
dialysis volume of the sample was increased, protein estimation and activity assay was
performed.
3.5.5. Anion Exchange Column (AEC) Chromatography
Followed by heat treatment, ammonium sulfate precipitation and dialysis to remove salt
both recombinant TnBglA and TnBglB were subjected to a Resource Q anion exchange
column (Amersham Pharmacia Biotech, Sweden) for anion exchange chromatography.
After studying the isoelectric point (pI) of cloned TnBglA and TnBglB enzymes using the
Protparam ExPASy tool, an appropriate choice was Tris-Cl buffer (50 mM, pH 8.0) as a
binding buffer (buffer A) and as an elution buffer or buffer B, 1 M NaCl salt mixed in 50
mM Tris-Cl buffer having pH 8.0 was prepared, both buffers kept separately after
sterilization at 4°C.
Initially, the column preparation was performed by washing Resource Q pre-packed
column with five column volume (5 CV) of double filtered (0.22 µm membrane millipore
Material & Methods
84
filter) deionized water at the rate of 1 mL min-1, followed by equilibrated with ten CV of
buffer A for approximately 10 minutes. The dialyzed sample was passed through a
millipore membrane filter (0.45 µm) before loading to avoid the blockage of column, and
the resulting enzyme fraction was applied to a Resource Q ion exchange column at a flow
rate of 3 mL min-1 and equilibrated with five column volumes (5V) of binding buffer (pH
8.0). At all steps, flow-through was collected, and during equilibration three unbound
fractions were collected. After washing, bound protein fraction was eluted from the column
with a 0-1 M NaCl linear increasing gradient at a flow rate of 1 mL min-1 and 0.6 psi
pressure. The elution profile was examined at 280 nm throughout the process. According
to elution peak, all collected fractions were analyzed separately by SDS-PAGE and activity
assay followed by active fractions pooled were together.
3.5.6. Hydrophobic Interaction Column (HIC) Chromatography
Resource ISO pre-packed column (Amersham Pharmacia Biotech, Sweden)
chromatography was selected for further purification. Resource ISO column was washed
with 3 column volume (3CV) of Tris-Cl 50 mM buffer of pH 8.0 containing 1 M (NH4)2SO4
(binding buffer A) and equilibrated with this buffer prior to applied active pooled fractions.
Before applying to the column, pooled active fractions solution was adjusted to 1.5 M
(NH4)2SO4 by adding salt, since the appropriate adsorption was occurred at high salt
concentration. Properly dissolved the salt in the sample followed by membrane filtration
down. After the application of sample (active fractions), the column was washed with the
same equilibration buffer till the UV absorption returned to near baseline at a flow rate of
1 mL min-1. The bound enzyme fraction was eluted with a 1.5-0 M (NH4)2SO4 decreasing
gradient. Flow rate was maintained at 2 mL min-1 during protein elution and measured the
absorption by online UV monitor. Across the major peak, various fractions were collected
and evaluated by SDS-PAGE analysis. Active enzyme was obtained as unbound fractions,
pooled together all active fractions of recombinant enzymes TnBglA and TnBglB. Purified
protein (TnBglA and TnBglB) concentration was determined by Bradford method and
thereafter purified recombinant enzymes were either lyophilized or directly (in liquid form)
stored at -80°C for further analytical studies.
Material & Methods
85
3.6. Characterization of Recombinant TnBglA and TnBglB
Characterization of recombinant TnBglA and TnBglB enzymes from T. naphthophila were
performed to determine molecular weight, optimal pH and temperature, thermal
inactivation, pH stability, substrate specificity, kinetics parameters, thermodynamics study
of substrate hydrolysis and enzymes inactivation, shelf life of enzymes, effect of chemical
inhibitors, metallic cation, glucose and xylose on the catalytic efficiency of cloned
enzymes.
3.6.1. Determination of Molecular weight
Purity and apparent molecular weight of recombinant TnBglA and TnBglB proteins were
analyzed by SDS-PAGE. Individually the purified TnBglA and TnBglB enzymes were
loaded on 12% SDS-gel along with control protein sample from both induced and
uninduced cells of E. coli BL21 CodonPlus harboring pET-21a(+) without insert for the
comparison of expressions and to determine the molecular weight of recombinant enzymes.
3.6.2. Optimal pH of TnBglA and TnBglB
To determine the optimal pH of purified recombinant TnBglA and TnBglB enzymes,
various buffers were used in the present study. For example, McIlvaine buffer (for pH 2.2-
8.0), sodium acetate buffer (for pH 3.7-5.6), Tris-Cl buffer (for pH 7.0-9.0), MES buffer
(for pH 5.6-6.6), Sorensen’s phosphate buffer (for pH 5.8-8.0), HEPES buffer (for pH 6.8–
8.2), MOP buffer (for pH 7.0-7.8), and CAPS buffer (for pH 9.0-11.0). To scrutinize the
optimum pH and buffer for TnBglA and TnBglB catalytic activity, the enzymes were
diluted suitably in buffers from pH 3.0-11.0, in increments of 0.5 unit of pH, under the
assay conditions as described earlier. The results were compiled as the percentage of
TnBglA and TnBglB activities.
3.6.3. Optimal Temperature of TnBglA and TnBglB
The optimal temperature of cloned TnBglA and TnBglB were measured over a broad
temperature ranged from 30-100°C, under standard assay conditions using already
investigated optimal pH buffer for both enzyme that was McIlvaine buffer having pH 7.0
Material & Methods
86
for TnBglA and pH 5.0 for TnBglB. Both enzymes were incubated for 10 minutes at
different temperatures (30-100°C), with the increments of 1 to 5 degree C followed by stop
the reactions with the addition of 3 mL 1 M Na2CO3. All results related to temperature
profile was expressed as percentage of TnBglA and TnBglB activities.
3.6.4. Optimal Incubation Time
Time course profile of recombinant enzymes (TnBglA and TnBglB) for substrate
hydrolysis were monitored to find the optimum incubation time for reaction under the
optimal assay conditions of TnBglA and TnBglB incubated at 95°C and 85°C using pH 7.0
and 5.0 buffer, respectively. Therefore, standard reaction mixtures of both enzymes were
incubated from 01 to 15 minutes subsequently computed the product released per minutes.
3.6.5. pH Stability
To evaluating the effect of H+ ions concentration on recombinant enzyme stability, pre-
incubated the purified enzymes (TnBglA and TnBglB) for 60 minutes in different buffers
ranging from pH 3.0-11.0 at 70°C, followed by diluted each sample by using McIlvaine
buffer (pH 7.0 for TnBglA and pH 5.0 for TnBglB) at 1:50 of dilution ratio and immediately
determined the residual activity of enzymes under optimal assay conditions for TnBglA
and TnBglB at 95°C (in pH 7.0 buffer) and 85°C (in pH 5.0 buffer), respectively. Results
were expressed as percentage of obtained residual activity, and enzyme activity defined as
100% which was without pre-incubation.
3.6.6. Thermal stability at different pH
Thermostability of recombinant purified TnBglA and TnBglB were investigated by
measuring the residual activity of pre-incubated aliquots at temperature ranging from 30-
100°C for different time intervals (up to 12 hours) in different buffer solutions ranging
from pH 4.0-7.5, in the absence of substrate. Aliquots were taken out followed by incubated
on ice for 30 minutes. Activity assay of TnBglA (95°C, pH 7.0) and TnBglB (85°C, pH
5.0) enzymes were performed under the standard assay conditions. Activity of TnBglA and
TnBglB without pre-incubation was considered as 100%.
Material & Methods
87
3.6.7. Effect of metallic ions and different inhibitors
To find out the inhibitory influence of various metallic cations on the activity of purified
TnBglA and TnBglB, enzymes were incubated in various metal cations (Mn2+, Ca2+, Zn2+,
Ni2+, Pb2+, Mg2+, Hg2+, Cu2+, Co2+, Cd2+ and Fe3+) at final concentration of 10 mM. The
effect of various chemical reagents and detergents (1% of β-mercaptoethanol, SDS, Triton-
X-100, Tween-80, and Urea) on the activity of recombinant enzymes were also
investigated. Chelating agent (10 mM EDTA) as well as different % (v/v) of short chain
alcohols (methanol, ethanol, n-butanol, and isopropanol) and organic solvents like ethyl
acetate, acetonitrile, acetone, dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF)
were also used as chemical additives. The TnBglA and TnBglB residual activity were
measured using pNPG, under standard assay conditions. The activity of TnBglA and
TnBglB without any inhibitor, metallic ions and modifying agents was defined as 100%.
3.6.8. Effect of glucose and xylose
The effect of glucose and xylose on purified TnBglA and TnBglB enzyme activities were
investigated. The Ki value, representing the amount of glucose and xylose required to
inhibit activity of TnBglA and TnBglB up to 50% (Pei et al., 2012), was found by
incubating the purified recombinant enzyme in different concentrations of glucose and
xylose (20-1500 mM) and the residual activity of both purified enzymes were studied under
the standard assay conditions. Activity of enzymes without the addition of glucose and
xylose were considered as 100%.
3.6.9. Shelf life of recombinant TnBglA and TnBglB
The storage stability of recombinant TnBglA and TnBglB crude supernatant, crude after
heat treatment (for 1 h, at 70°C) and purified enzymes were monitored at room temperature
(30-35°C) and under refrigeration (4°C) conditions. Recombinant enzymes residual
activity were measured intermittently for a period of one year using standard assay
conditions as describe earlier.
Material & Methods
88
3.6.10. Substrate specificity
Substrate specificity of purified TnBglA and TnBglB were determined by using various
para- and ortho-nitrophenyl substrates such as pNP-β-D-galactopyranoside (pNPGal),
oNP-β-D-galactopyranoside (oNPGal), pNP-β-D-fucopyranoside (pNPF), oNP-β-D-
fucopyranoside (oNPF), pNP-β-D-xylopyranoside (pNPX), oNP-β-D-glucopyranoside
(oNPG), pNP-β-D-cellobioside (pNPC), pNP-α-D-glucopyranoside (pNPαG), pNP-α-L-
arabinofuranoside (pNPαA), pNP-β-D-mannoside (pNPM), pNP-β-D-glucuronide
(pNPGn) pNP-α-D-cellobioside (pNPαC) in the same way as with pNP-β-D-
glucopyranoside (pNPG, 4 mg mL-1) under the optimal assay conditions of TnBglA and
TnBglB.
When 1% cellobiose was used as substrate, the released amount of glucose was determined
using a glucose assay kit GAGO 20 (Sigma, Aldrich) according to the manufacturer’s
protocol. Glucose standard curve was prepared under the same assay conditions and was
used to find out the glucose concentration. Various polysaccharides substrates (4% CMC,
2% birchwood xylan, 2% beechwood xylan, 1% avicel, 1% sucrose and 0.5% laminarin)
were used in a 1 mL reaction mixture, under described assay conditions as in following
section.
3.6.11. Activity assay using polysaccharides
Various polysaccharides substrates (4% CMC, 2% birchwood xylan, 2% beechwood xylan,
avicel and laminarin) were used to scrutinize the catalytic activity, assays performed in seal
caped heat resistant tubes. Reaction mixture (1 mL) contained respective substrate (0.5
mL) in McIlvaine buffer (pH 7.0 and pH 5.0 accordingly) and diluted TnBglA (100 ng of
enzyme in 10 µL and 490 µL buffer, pH 7.0) or TnBglB (100 ng of enzyme in 10 µL and
490 µL buffer, pH 5.0). A single substrate assay with both enzymes were carried out in
triplicate along with a control reaction, followed by incubation at 95°C for TnBglA and at
85°C for TnBglB. After 10 minutes incubation, reaction was stopped using 3,5-
dinitrosalicylic acid (3 mL DNS) reagent, and mixture was incubated in a boiling water
bath for 10 minutes, measured absorbance against blank at 540 nm, and the concentration
Material & Methods
89
of liberated reducing sugars were quantified using DNS method described by Miller
(1959). The released reducing sugar was measured by using standard curve of glucose.
3.6.11.1. Standard Curve of Glucose and Xylose
To make the standard curve of glucose and xylose, stock solutions of glucose (50 mg mL-
1) and xylose (50 mg mL-1) were prepared in dH2O. Both glucose and xylose were diluted
serially ranging from 0.1-1.2 mg mL-1 in well dried label test tubes by using respective
stock solutions, and made final volume 1000 µL of the reaction. A control was also run
parallel by replacing glucose/xylose with dH2O, followed by incubation in boiling water
for 10 minutes, subsequently adding 3 mL of DNS in each serially diluted reaction tubes
and measured the absorbance at 540 nm using spectrophotometer. A standard curve of
glucose (Figure 3.8) and xylose were plotted by taking absorbance along Y-axis and
various concentration of glucose along X-axis (Figure 3.9).
Figure 3.8: Standard curve of Glucose.
y = 0.0073x + 0.0153R² = 0.9996
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Ab
sorb
ance
(54
0 n
m)
Concentration mg mL-1
Standard Curve of Glucose
Material & Methods
90
Figure 3.9: Standard curve of Xylose.
3.6.11.2. Enzyme Unit
“One unit of recombinant enzyme activity is defined as the amount of enzyme that releases
1 µmol of reducing sugar per minute under the standard assay conditions.” Enzyme activity
in term of units was computed by applying the following formula:
3.6.11.3. Specific activity
Specific activity of TnBglA and TnBglB enzymes were described as enzyme units per mg
of the recombinant protein, and calculated by using the following formula:
3.6.12. Kinetic studies
To calculate Michaelis constant (Km), maximum velocity (Vmax) and enzyme turnover or
catalytic constants (kcat) Lineweaver-Burk plot was applied, various concentrations of
y = 0.0073x + 0.0153R² = 0.9996
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Ab
sorb
an
ce (
54
0 n
m)
Concentration mg mL-1
Standard Curve of Xylose
Conc. from graph (mg mL-1) × Dil. factor × 1000
Incubation time (min) × MW of glucose/xylose Enzyme activity (U mL-1 min-1) =
U mL-1 min-1 of enzyme
mg of protein mL-1 Specific activity (U mg-1) =
Material & Methods
91
pNPG, pNPF, pNPGal, pNPX, pNPC and cellobiose substrates used with fixed amount of
recombinant enzymes. Rate of substrates hydrolysis for various concentration were
measured using McIlvaine buffer having enzyme optimal pH. Graphs were plotted by
taking 1/V along Y-axis and 1/[S] along X-axis, the values of Km and Vmax for all substrate
were calculated through Lineweaver-Burk plots and the value of kcat for each substrate was
computed by the following equation.
kcat = Vmax / [e]
Where, [e] is the concentration of enzyme.
3.6.13. Thermodynamics of substrate hydrolysis
Thermodynamics of pNPG hydrolysis were calculated as elucidated by Haq et al. (2012).
Activation energy (Ea) was calculated from the plot of ln Vmax against 1/T and
thermodynamic data (∆H*, ∆S* and ∆G*) were calculated by using the following
equations:
Ea (activation energy) = -slope × R
∆H* (enthalpy of activation) = Ea-RT
∆G* (free energy of activation) = -RT ln (kcath / kBT)
∆S* (entropy of inactivation) = (∆H* – ∆G*) / T
Where, ‘kB’ is the Boltzmann’s constant, kB is equal to the 1.3806488 × 10-23 J K-1, ‘T’ is
the absolute temperature in kelvin, ‘R’ is the gas constant which is equal to 8.3144621 J
K−1, and ‘h’ is the Planck’s constant which is equal to 6.62606957 x 10-34 J s or 11.04 x 10-
36 J. min.
3.6.14. Thermodynamics of TnBglA and TnBglB inactivation
Thermodynamic parameters for irreversible thermal denaturation of recombinant enzymes
were estimated by incubating purified enzyme at different temperatures for a specific time
period followed by determined the residual activity of enzymes. Thermal denaturation (Kd)
Material & Methods
92
constant was calculated from a logarithmic plot of percent of remaining activity versus
time to determine the thermodynamic parameters for irreversible thermal denaturation of
recombinant TnBglA and TnBglB. EaD was calculated from plot of ln Kd against 1/T and
ΔH*D, ΔS*D and ΔG*D parameters were calculated using above mentioned equations after
replacing kcat and Ea with Kd and EaD, respectively as calculated by Haq et al. (2012).
Apparent half-lives (t1/2) of TnBglA and TnBglB were calculated using following equation.
t1/2 = In 2 / Kd
3.6.15. Statistical Analysis
All experimental reactions were conducted in CRD (completely randomized design) with
three replicates, data collection was statistically analyzed using student’s t-test analysis and
one-way ANOVA, with the significance results (p≤0.05) of ANOVA, Duncan’s multiple
range test was applied to analyze the comparison of means using SPSS software.
3.6.16. Docking Studies of Enzyme-Substrate
The minimized energy model structure of TnBglA and TnBglB were used for the docking
studies by PatchDock (Schneidman-Duhovny et al., 2005). Different substrates utilized in
the present study to test both enzymes activities, were also utilized for forecasting the
possible details of molecular enzyme substrate actions. The substrates docked with the both
β-glucosidases (TnBglA and TnBglB) included, cellobiose and p-nitrophenyl substrates.
All PatchDock results of docking were analyzed for evaluating the accuracy of docked
model and predicting the molecular details of both proteins in terms of non-covalent
interactions among enzyme (TnBglA and TnBglB) and substrates by using PDBsum
(Laskowski, 2009).
CHAPTER-IV
RESULTS
Results
93
4. RESULTS
The quest for thermotolerant and efficient cellulases led the way towards thermophiles.
The members of genus Thermotoga are believed to be an excellent source of highly active,
acidic-alkali resistant and highly heat-stable glycoside hydrolases (GHs) especially
cellulases. They are strictly anaerobes, rod-shaped, gram-negative bacteria which
optimally grow at 80-90°C. Therefore, in the present research work a marine
hyperthermophilic bacterium T. naphthophila RKU-10T (ATCC BAA-489/DSM
13996/JCM 10882) was selected for the isolation of novel cellulolytic genes because this
strain contains a large number of putative cellulolytic genes. Among these, two cellulolytic
genes were selected for molecular gene cloning and overexpression in mesophilic host E.
coli BL21 (DE3) followed by characterization and thermodynamic studies of both purified
enzymes were conducted.
All results related to the present study are discussed in the following section.
4.1. β-1,4-glucosidase genes from T. naphthophila
T. naphthophila RKU-10T complete genomic sequence was available on NCBI server
under GenBank accession number CP001839.1 and its genomic DNA has 1809823 base
pairs with several cellulolytic genes. Using the online NCBI database two putative
cellulolytic (β-1,4-glucosidase) genes fragments of 1,341 and 2,166 bp with locus tag
Tnap_0602 and Tnap_0656 were selected for the present research work and designated as
TnbglA and TnbglB. Gene bglA from T. naphthophila (TnbglA) encoding 446 amino acid
residues of a protein (TnBglA) and second gene bglB from T. naphthophila (TnbglB)
encoding 721 amino acid residues of a protein (TnBglB). Proteins TnBglA and TnBglB
have molecular weight (MW) of 51.50 kDa and 81.14 kDa, respectively.
All characteristics detail of both genes along with protein _ids from online NCBI database
software are presented in the following table 4.1.
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94
Table 4.1: Characteristics details of TnbglA and TnbglB genes of T. naphthophila.
Property TnbglA TnbglB
Source Organism T. naphthophila RKU-10T T. naphthophila RKU-10T
Culture Collection ID DSM 13996, JCM 10882 DSM 13996, JCM 10882
Gene Size 1,341 bp 2,166 bp
Gene symbol TnbglA TnbglB
GenBank Accession no. CP001839.1 CP001839.1
Gene ID 281373136 281373190
Gene region 615833-617173 675125-677291
Locus tag Tnap_0602 Tnap_0656
Glycoside Hydrolase (GH)
family
GH family 1 (GH1) GH family 3 (GH3)
Protein_id ADA66698.1 ADA66752.1
4.2. Gene Sequence of TnbglA
The complete gene sequence of the putative cellulolytic gene belonged to GH family 1
was available on NCBI. Primers (forward and reverse) were designed using the obtained
gene sequences and the underline bold base pairs showed the NdeI restriction site
upstream (underlined) which was introduced to digest the gene with NdeI restriction
enzyme.
4.2.1. FASTA Sequence
“CATATGAACGTGAAAAAGTTCCCTGAAGGATTCCTCTGGGGTGTTGCAACAGCTTCCT
ACCAGATCGAGGGTTCTCCCCTCGCAGACGGAGCTGGTATGTCTATCTGGCACACCTTC
TCCCATACTCCTGGAAATGTAAAGAACGGTGACACGGGAGATGTGGCCTGCGACCACTA
CAACAGATGGAAAGAGGACATTGAAATCATAGAGAAACTCGGAGTAAAGGCTTACAGAT
TTTCAATCAGCTGGCCAAGAATACTTCCGGAAGGAACAGGAAGGGTGAATCAGAAAGGA
CTGGATTTTTACAACAGGATCATAGACACCCTGCTGGAAAAAGGTATCACACCCTTTGT
GACCATCTATCACTGGGATCTTCCCTTCGCTCTTCAGTTGAAAGGAGGATGGGCGAACA
GAGAAATAGCGGATTGGTTCGCAGAATACTCAAGGGTTCTCTTTGAAAATTTCGGCGAC
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95
CGTGTGAAGAACTGGATCACCTTGAACGAACCGTGGGTTGTTGCCATAGTGGGGCATCT
GTACGGAGTCCACGCTCCTGGAATGAGAGATATTTACGTGGCTTTCCGAGCTGTTCACA
ATCTCTTGAGGGCACACGCCAAAGCGGTGAAAGTGTTCAGGGAAACTGTGAAAGATGGA
AAGATCGGAATAGTTTTCAACAATGGATATTTCGAACCTGCGAGTGAAAAAGAGGAGGA
CATCAGAGCGGCGAGATTCATGCATCAGTTCAACAACTATCCTCTCTTTCTCAATCCGA
TCTACAGAGGAGATTATCCGGAGCTCGTTCTGGAATTTGCCAGAGAGTATCTACCGGAG
AATTACAAAGATGACATGTCCGAGATACAGGAAAAGATCGACTTTGTTGGATTGAACTA
TTACTCCGGTCATTTGGTGAAGTTCGATCCAGATGCACCAGCTAAGGTCTCTTTCGTTG
AAAGGGATCTTCCAAAAACAGCCATGGGATGGGAGATCGTTCCAGAAGGAATCTACTGG
ATCCTGAAGAAGGTGAAAGAAGAATACAACCCACCAGAGGTTTACATCACAGAGAATGG
GGCTGCTTTTGACGACGTAGTTAGTGAAGATGGAAGAGTTCACGATCAAAACAGAATCG
ATTATTTGAAGGCCCACATTGGTCAGGCATGGAAGGCCATACAGGAGGGAGTGCCGCTT
AAAGGTTACTTCGTCTGGTCGCTCCTCGACAATTTCGAATGGGCAGAGGGATACTCTAA
GAGATTTGGTATTGTGTACGTGGACTACAGTACTCAAAAACGCATCATAAAAGACAGTG
GTTACTGGTACTCGAACGTGGTCAAAAGCAACAGTCTGGAAGATTGA”
4.2.2. Amino acid sequence of TnBglA
"MNVKKFPEGFLWGVATASYQIEGSPLADGAGMSIWHTFSHTPGNVKNGDTGDVACDHY
NRWKEDIEIIEKLGVKAYRFSISWPRILPEGTGRVNQKGLDFYNRIIDTLLEKGITPFV
TIYHWDLPFALQLKGGWANREIADWFAEYSRVLFENFGDRVKNWITLNEPWVVAIVGHL
YGVHAPGMRDIYVAFRAVHNLLRAHAKAVKVFRETVKDGKIGIVFNNGYFEPASEKEED
IRAARFMHQFNNYPLFLNPIYRGDYPELVLEFAREYLPENYKDDMSEIQEKIDFVGLNY
YSGHLVKFDPDAPAKVSFVERDLPKTAMGWEIVPEGIYWILKKVKEEYNPPEVYITENG
AAFDDVVSEDGRVHDQNRIDYLKAHIGQAWKAIQEGVPLKGYFVWSLLDNFEWAEGYSK
RFGIVYVDYSTQKRIIKDSGYWYSNVVKSNSLED”
4.3. Gene Sequence of TnbglB
The complete gene sequence of putative cellulolytic gene belonged to GH family 3 was
available on NCBI. Primers (forward and reverse) were designed using the obtained gene
sequences and the underline bold base pairs showed the Nde I restriction site upstream
(underlined) which was introduced to digest the gene with NdeI restriction enzyme.
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96
4.3.1. FASTA Sequence
“CATATGGAAAGGATCGATGAAATCCTCTCTCAGTTAACTACAGAGGAAAAGGTGAAGC
TCGTTGTGGGGGTCGGTCTTCCAGGACTTTTTGGAAACCCACATTCCAGAGTGGCGGGT
GCGGCTGGAGAAACACATCCCATTCCAAGACTTGGAATTCCTGCGTTTGTCCTGGCAGA
TGGTCCCGCAGGACTCAGAATAAATCCCACAAGGGAAAACGATGAAAACACTTACTACA
CGACGGCATTTCCCGTTGAAATCATGCTTGCTTCTACCTGGAACAGAGACCTTCTGGAA
GAAGTGGGAAAAGCCATGGGAGAAGAAGTTAGGGAATACGGTGTCGATGTGCTTCTTGC
ACCTGCGATGAACATTCACAGAAACCCTCTTTGTGGAAGGAATTTCGAGTACTACTCAG
AAGATCCTGTCCTTTCCGGTGAAATGGCTTCAGCCTTTGTCAAGGGAGTTCAATCTCAA
GGGGTGGGAGCCTGCATAAAACACTTTGTCGCGAACAACCAGGAAACGAACAGGATGGT
AGTGGACACGATCGTGTCCGAGCGAGCCCTCAGAGAAATATATCTGAAAGGTTTTGAAA
TTGCCGTCAAGAAAGCAAGACCCTGGACCGTGATGAGCGCTTACAACAAACTGAATGGA
AAATACTGTTCACAGAACGAATGGCTTTTGAAGAAGGTTCTCAGGGAAGAATGGGGATT
TGACGGTTTCGTGATGAGCGACTGGTACGCGGGAGACAACCCTGTAGAACAGCTCAAGG
CCGGAAACGATATGATCATGCCTGGAAAAGCGTATCAGGTGAACACGGAAAGAAGAGAT
GAAATAGAAGAAATCATGGAGGCGTTGAAGGAGGGAAAGTTGAGTGAGGAGGTTCTCGA
TGAGTGTGTGAGAAACATTCTCAAAGTTCTTGTGAACGCGCCTTCCTTCAAAGGGTACA
GGTACTCAAACAAACCGGATCTCGAATCTCACGCGGAAGTCGCCTACAAAGCAGGTGCG
GAGGGTGTTGTCCTTCTTGAGAACAACGGTGTTCTTCCGTTCGATGAAAATACCCATGT
CGCCGTCTTTGGCACCGGTCAAATCGAAACAATAAAGGGAGGAACGGGAAGTGGAGACA
CCCATCCGAGATACACGATCTCTATCCTTGAAGGCATAAAAGAAAGAAACATGAAGTTC
GACGAAGAACTCGCTTCCACTTATGAGGAGTACATAAAAAAGATGAGAGAAACAGAGGA
ATATAAACCCAGAACCGACTCCTGGGGAACGGCCATAAAACCGAAACTTCCAGAGAACT
TCCTCCCAGAAAAAGAGATAAAGAAAGCTGCAAAGAAAAACGATGTTGCAGTTGTTGTG
ATCAGCAGGATCTCCGGTGAGGGATACGACAGAAAGCCGGTGAAAGGTGATTTCTACCT
CTCCGATGACGAGCTGGAACTCATAAAAACCGTCTCGAAAGAATTCCACGATCAGGGTA
AGAAAGTTGTGGTTCTTCTGAACATCGGAAGTCCCATCGAAGTCGCAAGCTGGAGAGAC
CTTGTGGATGGAATTCTTCTTGTCTGGCAGGCGGGACAGGAGATGGGAAGAATAGTGGC
CGATGTTCTTGTGGGAAAGATTAATCCCTCCGGAAAACTTCCAACGACCTTCCCGAAGG
ATTACTCGGACGTTCCATCCTGGACGTTCCCAGGAGAGCCAAAGGACAATCCGCAAAGA
GTGGTGTACGAGGAAGACATCTACGTGGGATACAGGTACTACGACACCTTTGGTGTGGA
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97
ACCTGCCTACGAGTTCGGCTACGGCCTCTCTTACACAAAGTTTGAATACAAAGATTTAA
AGATCGCTATCGACGGAGATATACTCAGAGTGTCGTACACGATCACAAACACCGGGGAC
AGAGCTGGAAAGGAAGTCTCACAGGTTTATGTCAAAGCTCCAAAAGGGAAAATAGACAA
ACCCTTCCAGGAGCTGAAAGCGTTCCACAAAACAAAACTTTTGAACCCGGGTGAATCCG
AAAAGATCTTTCTGGAAATTCCTCTTAGAGATCTTGCGAGTTTCGATGGGAAAGAATGG
GTTGTCGAGTCAGGAGAATACGAGGTCAGGGTCGGTGCATCTTCGAGGGATATAAGGTT
GAAAGATGTTTTCTTGGTTGAAGAAGAAAAGAGATTCAAACCATGA”
4.3.2. Amino acid sequence of TnBglB
“MERIDEILSQLTTEEKVKLVVGVGLPGLFGNPHSRVAGAAGETHPIPRLGIPAFVLAD
GPAGLRINPTRENDENTYYTTAFPVEIMLASTWNRDLLEEVGKAMGEEVREYGVDVLLA
PAMNIHRNPLCGRNFEYYSEDPVLSGEMASAFVKGVQSQGVGACIKHFVANNQETNRMV
VDTIVSERALREIYLKGFEIAVKKARPWTVMSAYNKLNGKYCSQNEWLLKKVLREEWGF
DGFVMSDWYAGDNPVEQLKAGNDMIMPGKAYQVNTERRDEIEEIMEALKEGKLSEEVLD
ECVRNILKVLVNAPSFKGYRYSNKPDLESHAEVAYKAGAEGVVLLENNGVLPFDENTHV
AVFGTGQIETIKGGTGSGDTHPRYTISILEGIKERNMKFDEELASTYEEYIKKMRETEE
YKPRTDSWGTAIKPKLPENFLPEKEIKKAAKKNDVAVVVISRISGEGYDRKPVKGDFYL
SDDELELIKTVSKEFHDQGKKVVVLLNIGSPIEVASWRDLVDGILLVWQAGQEMGRIVA
DVLVGKINPSGKLPTTFPKDYSDVPSWTFPGEPKDNPQRVVYEEDIYVGYRYYDTFGVE
PAYEFGYGLSYTKFEYKDLKIAIDGDILRVSYTITNTGDRAGKEVSQVYVKAPKGKIDK
PFQELKAFHKTKLLNPGESEKIFLEIPLRDLASFDGKEWVVESGEYEVRVGASSRDIRL
KDVFLVEEEKRFKP”
4.4. Cloning and Sequencing of TnbglA and TnbglB Genes
Two cellulolytic (β-1,4-glucosidases) TnbglA and TnbglB genes fragment of 1,341 bp
(Tnap_0602) and 2,166 bp (Tnap_0656) from T. naphthophila RKU-10T, encoding 446
and 721 amino acid residues, respectively. Genomic DNA of T. naphthophila used as a
template for the amplification of β-1,4-glucosidase TnbglA and TnbglB genes, followed by
initially cloned in pTZ57R/T vector and after that sequencing were performed. All results
related to cloning in pTZ57R/T vector and sequencing analysis are described as below.
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98
4.4.1. Determination of Non-cutter restriction enzymes
To identify all the non-cutter (zero-cutter) restriction enzymes for the TnbglA and TnbglB,
an online NEBcutter V2.0 software was used in order to introduce restriction site in the
forward primer. The software showed a linear sequence of target genes along with non-
cutter restriction enzymes.
4.4.2. Non-cutter enzymes for TnbglA
After uploading the FASTA nucleotide sequence, all restriction enzymes were displayed.
TnbglA contained 46% GC and 54% AT contents and encoding 446 amino acid residues.
Following figure 4.1a shows the single cutter of TnbglA gene and list of zero-cutters are
displays in the table 4.2a.
Figure 4.1 (a): Linear sequence of TnbglA gene with NEB single cutter endonucleases.
4.4.3. Non-cutter enzymes for TnbglB
TnbglB contained 47% GC and 53% AT contents and encoding 721 amino acid residues.
Following figure 4.1b shows the single cutter of TnbglB gene and list of zero-cutters are
displays in the table 4.2b.
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99
Figure 4.1 (b): Linear sequence of TnbglB gene with NEB single cutter endonucleases.
Table 4.2 (a): List of Non-cutter enzymes for TnbglA.
AatII, Acc65I, AccI, AclI, AfeI, AflII, AgeI, AhdI, AleI, AlwNI, ApaI, ApaLI, AscI, AseI, AsiSI, AvaI, AvaII,
AvrII,
BaeI, BanI, BbsI, BbvCI, BceAI, BclI, BfaI, BglI, BglII, BlpI, BmgBI, BmtI, BpmI, BsaBI, BsaHI, BseYI,
BsgI, BsiWI, BsmBI, BsmFI, BsmI, BsoBI, BspCNI, BspHI, BsrDI, BsrFI, BsrGI, BssHII, BssSI, BstAPI,
BstEII, BstUI, BstZ17I, Bsu36I, BtgZI, BtsI,
CspCI,
DraI, DraIII, DrdI,
EagI, EciI, EcoO109I, EcoRI, EcoRV,
FauI, FseI, FspI,
HaeII, HgaI, HhaI, HinP1I, HincII, HindIII, HpaI,
KasI, KpnI,
MfeI, MluI, MslI,
NaeI, NarI, NciI, NdeI, NgoMIV, NheI, NmeAIII, NotI, NruI,
PacI, PaeR7I, PflFI, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspOMI, PspXI, PstI, PvuI,
RsrII,
SacII, SalI, SbfI, SexAI, SfiI, SfoI, SgrAI, SmaI, SnaBI, SpeI, SphI, SrfI, SspI, StuI, SwaI
TspMI, Tth111I,
XbaI, XcmI, XhoI, XmaI,
ZraI
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100
Table 4.2 (b): List of Non-cutter enzymes for TnbglB
AatII, Acc65I, AccI, AclI, AfeI, AflII, AgeI, AhdI, AleI, AlwNI, ApaI, ApaLI, AscI, AseI, AsiSI, AvaI, AvaII,
AvrII,
BaeI, BanI, BbsI, BbvCI, BceAI, BclI, BfaI, BglI, BglII, BlpI, BmgBI, BpmI, BsaBI, BsaHI, BseYI, BsgI,
BsiWI, BsmBI, BsmFI, BsmI, BsoBI, BspCNI, BspHI, BsrDI, BsrFI, BsrGI, BssHII, BssSI, BstAPI, BstEII,
BstUI, Bsu36I
CspCI,
DraI, DraIII, DrdI,
EagI, EciI, EcoO109I, EcoRI, EcoRV,
FauI, FseI, FspI,
HaeII, HgaI, HhaI, HinP1I, HincII, HindIII, HpaI,
KasI, KpnI,
MfeI, MluI, MslI,
NaeI, NarI, NciI, NdeI, NgoMIV, NheI, NmeAIII, NotI, NruI,
PacI, PaeR7I, PflFI, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspOMI, PspXI, PstI, PvuI,
SacII, SalI, SbfI, SexAI, SfiI, SfoI, SgrAI, SmaI, SnaBI, SpeI, SphI, SrfI, SspI, StuI, SwaI,
TspMI, Tth111I,
XbaI, XcmI, XhoI, XmaI,
ZraI
4.4.4. Primers Designing
To amplify the coding regions of TnbglA (ADA66698.1) and TnbglB (ADA66752.1), two
sets of primers were designed using Vector NTI Advance TM 10.3 software (Invitrogen
Corporation, California) and OligoCalc software (Kibbe, 2007). NdeI restriction site
(CATATG) upstream (underlined) at 5' end of the both forward primers of TnbglA and
TnbglB genes were introduced. No restriction site was introduced in reverse primers
because in multiple cloning site of pTZ57R/T (cloning vector) has HindIII site (second
restriction endonuclease used for restriction digestion). The primers were used to amplify
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101
TnbglA and TnbglB genes size of 1,341 and 2,166 bp, all thermodynamic properties and
sequences of designed oligomers (primers) are briefly described in the following Table 4.3.
Table 4.3: Oligonucleotides for PCR amplification of TnbglA and TnbglB genes.
Gene
Name Primer Sequence 5' 3'
R*
Site
Primer
length
(nt)
product
size
(bp)
MW Tm
TnbglA
F-CATATGAACGTGAAAAAGTTCCCTGAAGG
R-TCAATCTTCCAGACTGTTGCTTTTGACC
NdeI
-
29
28
1,341
8,959
8,481
61.2
61.0
TnbglB
F-CATATGGAAAGGATCGATGAAATCCTCTCTCA
R-TCATGGTTTGAATCTCTTTTCTTCTTCAACCAAG
NdeI
-
32
34
2,166
9,816
10,323
61.8
61.3
R* represented the restriction site, F (Forward), R (Reverse), MW (molecular weight), nt (nucleotides), bp
(base pairs), and Tm (melting temperature). TnbglA represented bglA gene and TnbglB represented bglB
gene from T. naphthophila.
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4.4.5. Extraction of Genomic DNA
T. naphthophila was grown at 80°C for 166 hours in an anaerobic conditions followed by
the genomic DNA extracted from the bacterial culture using standard protocol. Total
extracted DNA was analyzed after agarose gel electrophoresis under UV light (Figure 4.2).
Concentration was determined by absorption measurement at λ260, DNA quantity was
estimated to be approximately 120-140 ng µL-1.
Figure 4.2: Agarose gel of isolated genomic DNA from Thermotoga naphthophila, 6 µg DNA was loaded
in Lanes 1-3 along with DNA Marker (M) #SM0333.
4.4.6. TnbglA and TnbglB Genes Amplification
Target β-1,4-glucosidases (TnbglA and TnbglB) genes fragments of 1.341 kb and 2.166 kb
from T. naphthophila RKU-10T were amplified using gene specific primers in a
thermocycler. PCR often generates point mutations, therefore all reactions were performed
at least in triplicate. After the completion of PCR, amplicons were analyzed using 0.8%
agarose electrophoresis, gene fragment of 1.341 kb and 2.166 kb were obtained (Figure
4.3a and 4.3b).
M 1 2 3
10
Genomic DNA
3
1
0.5
kb
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103
Figure 4.3 (a): Analysis of TnbglA amplified product resolved on 0.8% agarose gel. Lane M, DNA ladder
# SM0333; Lane 1 and 2 showed PCR-amplified product of 1.341 kb.
Figure 4.3 (b): Analysis of TnbglB amplified product after agarose gel electrophoresis. Lane M, DNA
ladder # SM0333; Lane 1 and 2 showed PCR-amplified gene product of 2.166 kb length.
M 1 2
kb
10 8.0 6.0 5.0 4.03.0 2.52.0 1.5 1.2 1.00.90.8 0.7 0.6 0.5 0.4 0.30.20.1
1.341 kb
M 1 2 kb 10 8.0 6.0 5.0 4.03.0 2.52.0 1.5 1.2 1.00.90.8 0.7 0.6 0.5 0.4 0.3
2.166 kb
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104
4.4.7. Purification of Amplicons
PCR amplicons of TnbglA and TnbglB yielded a single product of their required sizes,
excised the bands of both amplified DNA and subsequently extract from agarose gel using
QIA quick gel extraction kit. Gene clean products of PCR were electrophoresed and results
are shown in figure 4.4.
Figure 4.4: Analysis of TnbglA and TnbglB purified PCR product of 1.341 kb and 2.166 kb, respectively on
0.8% agarose gel after DNA gel extraction (gene clean) using Qiagen Quick gel extraction kit. Lane M, DNA
ladder # SM0333; Lane 1, displayed TnBglA purified product (1.341 kb); and Lane 2, showed purified PCR
TnbglB gene product (2.166 kb).
4.4.8. Cloning of genes in pTZ57R/T
Purified TnbglA (1.341 kb) and TnbglB (2.166 kb) genes product were cloned in linear
pTZ57R/T vector using TA cloning strategy as elucidated in materials and methods. After
the incubation of ligation reaction, recombinant pTZ57R/T–TnbglA and pTZ57R/T–
TnbglB thus obtained, which were used to transform competent cells of DH5α strain
followed by spread on individual LB-agar plates supplemented with X-gal, IPTG, and
ampicillin, kept all plates at 37°C for 16-18 hours. Since the cloning vector pTZ57R/T is
genetically marked with LacZ, bacterial transformants could be selected by blue/white
screening. High efficiency of transformation was observed because a fairly good number
of white (putative positive transformants) colonies appeared on both plates after overnight
incubation (Figure 4.5).
M 1 2
3.0
2.0 2.5
1.5
1.0
kb
1.341 kb
2.166 kb
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105
Figure 4.5: After transformation of E. coli strain DH5α (competent cells) with ligation product of
pTZ57R/T–TnbglA and pTZ57R/T–TnbglB, several white and blue colonies were appeared on LB-agar
plates containging X-gal/IPTG/ampicilline. Putative recombinant (positive) well isolated white colonies were
selected by using blue-white sereening method.
4.4.9. Spotting of selected white colonies
Ten to sixteen well isolated putative recombinant white colonies harboring pTZ57R/T–
TnbglA and pTZ57R/T–TnbglB were selected (marked), duplically spotted or streak shortly
on freshly prepared LB-ampicillin agar plates and after that kept at 37°C incubator for
overnight, results were shown in the Figure 4.6.
Figure 4.6: Randamly selected well-isolated white colonies from master plates (TnbglA and TnbglB) and
made short streak or duplically spotted on freshly prepared LB-ampicillin agar plates.
TnbglA TnbglB
TnbglA TnbglB
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106
4.4.10. Colony PCR
By using gene-specific primers, the presence of insert genes (TnbglA and TnbglB) were
confirmed by colony PCR. Ten putative white colonies were screened for TnbglA and
thirteen colonies for TnbglB from spotted plates, and performed colony PCR followed by
gel electrophoresis to visualized the result as the described method (Figure 4.7a and 4.7b).
Figure 4.7 (a): Screening of positive transformants of E. coli DH5α harboring pTZ57R/T–TnbglA plasmids
by colony-PCR. Lane M, DNA ladder; Lane 1-10, amplified products of randomly selected white colonies;
Lane C, control reaction product. All selected white colonies were positive for TnbglA gene, arrow indicated
the position of 1.341 kb amplicon.
Figure 4.7 (b): Screening of positive transformants of E. coli DH5α harboring pTZ57R/T–TnbglB by colony-
PCR. Lane M, DNA size marker; Lane 1-13, amplified products of different selected colonies (PCR
reactions); Lane C, control reaction. All selected colonies were positive except colony no. 11 which was
negative for TnbglB insert. The arrow specified the position of 2.166 kb amplicon.
1.5
2.0
0.5
1.0
1.2
3.0
4.0
5.0
M 1 2 3 4 5 6 7 8 9 10 C
1.341 kb
kb
2.5 2.0
M 1 2 3 4 5 6 7 8 9 10 11 12 13 C
kb
8.0 3.0
2.166 kb
1.0
0.2 0.5
Results
107
4.4.11. Extraction of Recombinant Plasmids
After analyzing the results of colony PCR of both genes, recombinant plasmids
(pTZ57R/T–TnbglA and pTZ57R/T–TnbglB) were extracted from four positive bacterial
transformants (encircled) by alkaline lysis method and treated with RNase (Figure 4.8a and
4.8b) for further restriction analysis and sequencing.
Figure 4.8: Isolated recombinant plasmids pTZ57R/T–TnbglA and pTZ57R/T–TnbglB from four to five
selected positive transformants of E. coli DH5α after the screening by colony-PCR of both genes. (a) Lane
M, DNA ladder; Lane 1-5, recombinant uncut pTZ57R/T–TnbglA plasmids (circular) from five different
colonies after RNase treatment. (b) Lane M, DNA ladder; Lane 1-4, recombinant pTZ57R/T–TnbglB
plasmids (circular/uncut) from four different colonies after RNase treatment.
4.4.12. Single Restriction Analysis of Recombinant Plasmids
Plasmids (pTZ57R/T–TnbglA and pTZ57R/T–TnbglB) were used to confirm the presence
of insert genes by single restriction analysis with specific restriction enzymes, NdeI or
HindIII. Restriction analysis of both inserts were performed individually. DNA restriction
pattern of both recombinant plasmids were analyzed under UV-light after electrophoresed
on 0.8% agarose gel along with DNA size marker (Figure 4.9a and 4.9b).
kb
M 1 2 3 4 5 M M 1 2 3 4
(a) (b)
6.0 3.0 1.0
Uncut
Plasmids
Results
108
Figure 4.9 (a): Restriction analysis of pTZ57R/T–TnbglA plasmid using single restriction enzyme. Lane M,
DNA ladder; Lane 1-4, restricted plasmids with HindIII from four different selected colonies and arrow
specified resultant bands of ~4.227 kb (2.886 kb pTZ+1.341 kb insert) for linearized pTZ57R/T–TnbglA.
Figure 4.9 (b): Restriction analysis of pTZ57R/T–TnbglB plasmid using single restriction enzyme. Lane M,
DNA ladder; Lane 1-4, restricted plasmids with HindIII from four selected colonies and resultant bands of
~5.052 kb (2.886 kb pTZ+2.166 kb insert) showed linear pTZ57R/T–TnbglB plasmid.
4.4.13. Sequence Analysis of TnbglA and TnbglB
Isolated recombinant plasmids (pTZ57R/T–TnbglA and pTZ57R/T–TnbglB) from four
selected positive transformants (encircled) were subjected to sequence analysis, both in
forward and reverse direction. The complete TnbglA and TnbglB sequences were identical
for all cloned. Nucleotide and amino acid sequences were analyzed (aligned) to the
kb
~ 4.227kb 4.0
8.0
3.0
1.5
1.0
0.5
M 1 2 3 4
M 1 2 3 4
kb
~ 5.052 kb
6.0
3.0
5.0
4.0
Results
109
sequences available in GenBank database by utilizing Blast server at NCBI and Clustal
Omega.
4.4.14. Nucleotide Sequences Comparison of TnbglA gene
The nucleotide query sequence of TnbglA gene was blasted to identify and compare the
gene sequence with highly similar sequences in the NCBI database. Query produced
significant alignments after run and majority aligned sequences were β-glucosidases of
hyperthermophilic bacteria especially belonging to the genus Thermotoga and other
thermophiles. The query sequence of TnbglA showed 100% identity to the sequence of
Thermotoga naphthophila RKU-10T (CP001839.1) with 100% query coverage (Figure
4.10a). Further, the query sequence had high similarity of 99%, 99%, 99%, 99%, 98%,
98%, 82% and 82% to β-glucosidases from Thermotoga sp. RQ2, Thermotoga sp. Cell2,
Thermotoga sp. 2812B, T. petrophila RKU-1, T. maritima strain Tma100, T. maritima
MSB8, T. neapolitana DSM 4359 and Thermotoga sp. RQ7, respectively with 100% query
coverage. However, TnbglA nucleotide sequence showed 68%, 68%, 68%, 68% and 67%
similarity to β-glucosidases from Petrotoga mobilis SJ95, Clostridium thermocellum,
Ruminiclostridium thermocellum DSM 2360, Fervidobacterium pennivorans strain DYC
and Defluviitoga tunisiensis, respectively. Genomic sequence analysis revealed that β-1,4-
glucosidase TnbglA belongs to GH family 1. The eight significant aligned sequences are
presented in table 4.4a.
BLAST Result
Score Expect Identities Gaps Strand
2477 bits(1341) 0.0 1341/1341(100%) 0/1341(0%) Plus/Minus
Results
110
Results
111
Figure 4.10 (a): Sequence alignment between cloned TnbglA and NCBI retrieved sequence. Query = Cloned
TnbglA sequence, Subject = retrieved sequence from NCBI.
Table 4.4 (a): Nucleotide sequence BLAST result of TnbglA.
Description Accession
No.
Genomic
length
Query
Cover Score
E
value Gaps
Max.
Identity
Thermotoga sp. RQ2 CP000969.1 1877693 100% 2444 bits
(1323) 0.0
0/1341
(0%)
1335/134
(99%)
T. petrophila RKU-1 CP000702.1 1823511 100% 2466 bits
(1335) 0.0
0/1341
(0%)
1339/134
(99%)
Thermotoga sp. Cell2 CP003409.1 1749904 100% 2438 bits
(1320) 0.0
0/1341
(0%)
1334/134
(99%)
Thermotoga sp. 2812B CP003408.1 1843731 100% 2372 bits
(1284) 0.0
0/1341
(0%)
1322/134
(99%)
T. maritima MSB8 CP011107.1 1869610 100% 2361 bits
(1278) 0.0
0/1341
(0%)
1320/134
(98%)
T. neapolitana DSM 4359 CP000916.1 1884562 100% 1344 bits
(1490) 0.0
4/1343
(0%)
1105/134
3 (82%)
Defluviitoga tunisiensis LN824141.1 2053097 96% 356 bits
(394) 8e-94
34/131
5 (2%)
877/1315
(67%)
Fervidobacterium
pennivorans CP003260.1 2166381 53%
109 bits
(120) 2e-19
5/368
(1%)
248/368
(67%)
Results
112
4.4.15. Nucleotide Sequences Comparison of TnbglB gene
The recombinant pTZR/T57–TnbglB gene was sequenced and FASTA nucleotide query
sequence of TnbglB gene was blasted to identify and compare the gene sequence with
highly similar sequences in the NCBI database. The query sequence of TnbglB showed
100% identity to the sequence of Thermotoga naphthophila RKU-10 (CP001839.1) with
100% query coverage (Figure 4.10b).Query produced significant alignments after run and
majority aligned sequences were beta-glucosidases of hyperthermophilic bacteria
especially belonged to the genus Thermotoga and other thermophiles. The query sequence
of TnbglB had high similarity of 99%, 99%, 98%, 98%, 95%, 80%, 79%, 73% and 67% to
β-glucosidases of GH family 3 from Thermotoga sp. RQ2, Thermotoga sp. Cell2,
Thermotoga sp. 2812B, T. maritima MSB8, T. petrophila RKU-1, Thermotoga sp. RQ7, T.
neapolitana DSM 4359, T. hypogea DSM 11164 and Fervidobacterium islandicum,
respectively. Genomic sequence analysis revealed that β-1,4-glucosidase TnbglB belongs
to GH family 3. The nine significant aligned sequences are presented in table 4.4b.
BLAST Result
Score Expect Identities Gaps Strand
4000 bits(2166) 0.0 2166/2166 (100%) 0/2166 (0%) Plus/Minus
Results
113
Results
114
Figure 4.10 (b): Sequence alignment between cloned TnbglB and NCBI retrieved sequence. Query =
Cloned TnbglA sequence, Subject = retrieved sequence from NCBI.
Results
115
Table 4.4 (b): Nucleotide sequence BLAST result of TnbglB.
Description Accession
No.
Genomic
length
Query
Cover Score
E
value Gaps
Max.
Identity
Thermotoga sp. RQ2 CP000969.1 1877693 100%
3956 bits
(2142) 0.0
0/2166
(0%)
2158/2166
(99%)
Thermotoga sp. Cell2 CP003409.1 1749904 100%
3921 bits
(2123) 0.0
1/2166
(0%)
2152/2166
(99%)
Thermotoga sp. 2812B CP003408.1 1843731 100%
3757 bits
(2034) 0.0
0/2166
(0%)
2122/2166
(98%)
T. maritima MSB8 CP004077.1 1869612 100%
3746 bits
(2028) 0.0
0/2166
(0%)
2120/2166
(98%)
T. petrophila RKU-1 CP000702.1 1823511 99%
3374 bits
(1827) 0.0
1/2164
(0%)
2052/2164
(95%)
T. neapolitana DSM
4359 CP000916.1 1884562 97%
1576 bits
(853) 0.0
22/2127
(1%)
1706/212
(79%)
T. hypogea DSM
11164 CP007141.1 2165416 97%
1196 bits
(1326) 0.0
22/2122
(1%)
1548/2122
(73%)
T. caldifontis AZM44 AP014509.1 2014912 96%
1135 bits
(1258) 0.0
28/2115
(1%)
1529/2115
(72%)
Fervidobacterium
islandicum CP014334.1 2237377 94%
627 bits
(694) 5e-175
43/2056
(2%)
1386/2056
(67%)
4.4.16. Multiple sequence alignment of TnBglA and TnBglB
4.4.16.1. Alignment of TnBglA
Clustal omega program is used for the multiple sequence alignment of TnBglA to the others
members of GH1 from Genbank database, which exhibited high amino acid sequence
homology with β-glucosidases of genus Thermotoga especially with T. petrophila RKU-1
and T. maritima with 99% and 98% similarity, respectively. According to the homology
and structure modeling study, TnBglA has conserved structural folds and catalytic cleft for
substrate, with two or at the highest three conserved acidic amino acids residues (Glu166,
Glu351 and Glu405) present in the catalytic cleft or active site, one Glu residue in N-terminal
Results
116
and other in middle or C-terminal position, play role in hydrolysis (Figure 4.11a), which
presented the low evolutionary pressure on these catalytic regions.
Alignment of β-glucosidase A from T. naphthophila (TnBglA)
T. naphthophila -MNVKKFPEGFLWGVATASYQIEGSPLADGAGMSIWHTFSHTPGNVKNGDTGDVACDHY 58 T. maritima ---MKKFPEGFLWGVATASYQIEGSPLADGAGMSIWHTFSHTPGNVKNGDTGDVACDHY 56 T. pseudethanolicus ----MIKFPKDFLWGTATSSYQIEGAVNEDGRTPSIWDTFSKTEGKTYNGHTGDVACDHY 56 T. ethanolicus ---MEVKFPKDFLWGTATSSYQIEGAVNEDGRTPSIWDTFSKTEGKTYNGHTGDVACDHY 57 C. staminisolvens --MSKINFPKDFIWGSATAAYQIEGAYNEDGKGESIWDRFSHTPGNIENGHNGDIACDHY 58 S. griseoaurantiacus MTIDLGAFPRDFLWGTATAAYQIEGAAAEDGRSPSIWDTYSHTPGKVAGGDHGDVACDHY 60 N. alba -MSKPDDFPEDFLWGAATASFQIEGATTADGRGRSIWDTFAETPGKVLNGDTGDPADDHY 59 T. naphthophila NRWKEDIEIIEKLGVKAYRFSISWPRILPEGTGRVNQKGLDFYNRIIDTLLEKGITPFVT 118 T. maritima NRWKEDIEIIEKLGVKAYRFSISWPRILPEGTGRVNQKGLDFYNRIIDTLLEKGITPFVT 116 T. pseudethanolicus HRYKEDVEILKEIGVKAYRFSIAWPRIFPE-EGKYNPKGMDFYKRLIDELLKKDIMPTAT 115 T. ethanolicus HRYKEDVEILKEIGVKAYRFSIAWPRIFPE-EGKYNSKGMDFYKRLVDELLKKDIMPTAT 116 C. staminisolvens HRYEEDIKIMKEIGLKSYRFSISWPRIFPEGTGQLNQKGLDFYKRLTNMLLENGIMPAIT 118 S. griseoaurantiacus HRWEEDIELMRRLGTNAYRLSVAWPRVVPGGTGEVNAKGLDFYDRLIDALLAAGITPSVT 120 N. alba HRYAEDIGLMRALNLGAYRFSIAWPRILPEGEGKVNQAGLDFYDRLVDALLEAGIRPWAT 119 * T. naphthophila IYHWDLPFALQLKGG-WANREIADWFAEYSRVLFENFGDRVKNWITLNEPWVVAIVGHLY 177 T. maritima IYHWDLPFALQLKGG-WANREIADWFAEYSRVLFENFGDRVKNWITLNEPWVVAIVGHLY 175 T. pseudethanolicus IYHWDLPQWAYDKGGGWLNRDSVKWYVEYATKLFEELGDVIPLWITHNEPWCSSILSYGI 175 T. ethanolicus IYHWDLPQWAYDKGGGWLNRDSVKWYVEYATKLFEELGDVIPLWITHNEPWCASILSYGI 176 C. staminisolvens LYHWDLPQKLQDKGG-WKNRDTTDYFTEYCEAIFKSLGDIVPMWITHNEPRVVALLGHFL 177 S. griseoaurantiacus LYHWDLPQVLQDRGG-WPERDTALAFASYAGVVAERLGDRVKMWTTLNEPLCSAWIGHLE 179 N. alba LYHWDLPQPLEDRGG-WPERDTALRFADYATVVAEALGDRVGDWMTINEPWCSAFLGYDN 178 T. naphthophila GVHAPGMRDIYVAFRAVHNLLRAHAKAVKVFRETVKD-GKIGIVFNNGYFEPASEKEEDI 236 T. maritima GVHAPGMRDIYVAFRAVHNLLRAHARAVKVFRETVKD-GKIGIVFNNGYFEPASEKEEDI 234 T. pseudethanolicus GEHAPGHKNYREALIAAHHILLSHGEAVKAFREMNIKGSKIGITLNLTPAYPASEKEEDK 235 T. ethanolicus GEHAPGHKNYREALIAAHHILLSHGEAVKAFREMNIKGSKIGITLNLTPAYPASEKEEDK 236 C. staminisolvens GIHAPGIKDLRTSLEVSHNILLSHGKTVKLFREMNID-AQIGIALNLSHHYPVSEKPEDI 236 S. griseoaurantiacus GLMAPGLTDLTAAVRASYHLLLGHGLAVRAVRAAVPD-AKVGIVNNLAWVEPATDRPEDV 238 N. alba GHHAPGRRDTAAALAATHHLLLGHGLAVEAIRSTGHP-ARVGLAHNQAVIRANGAHAADV 237 T. naphthophila RAARFMHQFNNYPLFLNPIYRGDYPELVLEFAREYLPEN---YKDDMSEIQEKIDFVGLN 293 T. maritima RAVRFMHQFNNYPLFLNPIYRGDYPELVLEFAREYLPEN---YKDDMSEIQEKIDFVGLN 291 T. pseudethanolicus LAAQYADGFANR-WFLDPIFKGNYPEDMMELYSKIIGEFDFIKEGDLETISVPIDFLGVN 294 T. ethanolicus LAAQYADGFANR-WFLDPIFKGNYPEDMMELYSKIIGEFDFIKEGDLKTISVPIDFLGVN 295 C. staminisolvens AAAELSFSIAGR-WYLDPLFKGCYPEDALEYYKKKGIELSFPQD-DLKLISQPIDFLAFN 294 S. griseoaurantiacus AAARRMDGHSNR-WWLDPVHGRGFPED-MREVYG-VELP--ERAGDLAAMAEPLDWLGLN 293 N. alba RAARRADGVRNR-IFTDPLFKGSYPADVLKDIAH-ISDFSFVREGDLATISAPIDFLGVN 295 T. naphthophila YYSGHLVKFD----------------PDAPAKVSFVERDLPKTAMGWEIVPEGIYWILKK 337 T. maritima YYSGHLVKFD----------------PDAPAKVSFVERDLPKTAMGWEIVPEGIYWILKK 335 T. pseudethanolicus YYTRSIVKYN----------------EDSMLKAENVPGPGKRTEMGWEISPESLYDLLKR 338 T. ethanolicus YYTRSIVKYN----------------EDSMLKAENVPGPGKRTEMGWEISPESLYDLLKR 339 C. staminisolvens NYSSDIIQYDPSD-------------ESGFSFAESILDKFEKTDMGWIIYPEGLYDLLML 341 S. griseoaurantiacus YYFPSVVED-----DPAG--------PAPRARAVRR-PGLPRTGMDWEVDAHGIERLLLR 339 N. alba YYTPEFVAGSDRGLDPAGVDGGGGAWPGAEPEEVHVSQGLPVTQMGWEIDPRGLYDVLQR 355 * T. naphthophila VKEEYNPPEVYITENGAAFDDVVSEDGRVHDQNRIDYLKAHIGQAWKAIQEGVPLKGYFV 397 T. maritima VKEEYNPPEVYITENGAAFDDVVSEDGRVHDQNRIDYLKAHIGQAWKAIQEGVPLKGYFV 395 T. pseudethanolicus LDREYTKLPMYITENGAAFKDEVTEDGRVHDDERIEYIKEHLKAAAKFIGEGGNLKGYFV 398 T. ethanolicus LDREYTKLPMYITENGAAFKDEVTEDGRVHDDERIEYIKEHLKAAAKFIGERGNLKGYFV 399 C. staminisolvens LDRDYGKPNLIISENGAAFKDEISSSGRIEDTKRIQYLKSYLTQAHRAIQDGVNLKGYYL 401 S. griseoaurantiacus LTEDYGARRLYITENGSAYPDVVGPDGTVDDPERTAYLEEHLAACASAVRKGVPLAGYFA 399 N. alba LAGESGGIDLYVTENGCAFEDRVSEDGAVHDPERTDYYEAHLRAAREAVHAGIPLRGYFA 415
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* T. naphthophila WSLLDNFEWAEGYSKRFGIVYVDYSTQKRIIKDSGYWYSNVVKSNSLED- 446 T. maritima WSLLDNFEWAEGYSKRFGIVYVDYSTQKRIVKDSGYWYSNVVKNNGLED- 444 T. pseudethanolicus WSLMDNFEWAHGYSKRFGIVYVDYETQKRILKDSALWYKEVIQKNSIE— 446 T. ethanolicus WSLMDNFEWAHGYSKRFGIVYVDYETQKRILKDSALWYKEVIQKNSMS— 447 C. staminisolvens WSLMDNFEWSFGYNKRFGIVHVNFETQERKIKDSGYWYKEVIKNNGF--- 448 S. griseoaurantiacus WSLLDNFEWAYGYEKRFGLVHVDYATQRRTIKGSGHRYAELIRAHREPKA 449 N. alba WSLLDNFEWAWGYSRRFGIVYVDYETQERVVKDTGHWYAELAGTGRFPER 465
Figure 4.11 (a): Multi-alignment of β-glucosidase from T. naphthophila (TnBglA) with some other GH1
family β-glucosidases. Highly conserved amino acid residues are indicated by blocks. The catalytic sites are
represented by asterisk (*) on the top of alignment, the conserved catalytic Glu residues which act as proton
donor (E166) and nucleophile active site (E351 and E405). Thermotoga naphthophila RKU-10 (ADA66698.1),
Thermotoga maritima (WP_004082398.1), Clostridium straminisolvens (WP_038286797.1),
Thermoanaerobacter pseudethanolicus ATCC 33223 (ABY95558.1), Thermoanaerobacter ethanolicus
(WP_003869870.1), Streptomyces griseoaurantiacus (WP_006139294.1), Nocardiopsis alba
(WP_014910366.1).
4.4.16.2. Alignment of TnBglB
Clustal omega (ClustalW2) program is used for the multiple sequence alignment of TnBglB
amino acid sequence to the others members of thermostable GH3 from Genbank database,
which exhibited high amino acid sequence homology with β-glucosidases of genus
Thermotoga especially with T. petrophila RKU-1 and T. maritima with 99% and 98%
similarity, respectively. According to the homology and structure modeling study, TnBglB
has conserved structural folds and catalytic cleft for substrate, with two conserved acidic
amino acids residues (Asp242 and Glu458) present in conserved regions of amino acid
residues and formed the catalytic cleft or active site, which played hydrolytic role (Figure
4.11b). The conserved regions represented the low evolutionary pressure on these regions.
Alignment of β-glucosidase B from T. naphthophila (TnBglB)
Thermotoga naphthophila -MERIDEILSQLTTEEKVKLVVGVGLPGLFGNPHSRVAGAAGETHPIPRL 49 Thermotoga petrophila MMGKIDEILSQLTIEEKVKLVVGVGLPGLFGNPHSRVAGAAGETHPVPRL 50 Thermosipho africanus ----IEKIISQMTVEEKLKLLVGVGLPGMFGNKSSRVPGAAGETHQIERL 46 Fervidobacterium nodosum -MLDIEKVISQMTLEEKIHFVVGVGVLGIEDNPKARVSGAAGETFEIPRL 49 Thermotoga maritima -MERIDEILSQLTTEEKVKLVVGVGLPGLFGNPHSRVAGAAGETHPVPRL 49 Thermotoga neapolitana -MEKVNEILSQLTLEEKVKLVVGVGLPGLFGNPHSRVAGAAGETHPVPRV 49 Thermofilum sp. ----IEELLSKLSIEEKVKILVGIG--DTTSSQLARVPGAAGQTHPIERL 44 Thermofilum pendens -----------LSLEEKASLVVGWG------S-SRRLPGAAGETRPVR-- 30 Caldivirga maquilingensis --------VEDLSLEERVQLLVGAS------WRLRRIHGTAGETRPVRG- 35
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Thermotoga naphthophila GIPAFVLADGPAGLRINPTRENDENTYYTTAFPVEIMLASTWNRDLLEEV 99 Thermotoga petrophila GIPSFVLADGPAGLRINPTRENDENTYYTTAFPVEIMLASTWNKDLLEEV 100 Thermosipho africanus KIPSTVHADGPAGLRIDPERENDSNKYHATAFPIASMLASTWNKEILFEV 96 Fervidobacterium nodosum GIPRTVYADGPAGLRIDPERESDERKYHATAFPVETMLASTWNKNILKKV 99 Thermotoga maritima GIPAFVLADGPAGLRINPTRENDENTYYTTAFPVEIMLASTWNRDLLEEV 99 Thermotoga neapolitana GLPAFVLADGPAGLRINPTRENDENTYYTTAFPVEIMLASTWNRELLEEV 99 Thermofilum sp. NIPGFVLADGPAGVRIEN------PACRATAFPVEVMLASTWNPELVEQV 88 Thermofilum pendens -VPSIVTADGPSGLRVEP---SGGRRWFATAFPVPTMLAATWNPEVVERV 76 Caldivirga maquilingensis -LPMVAMADGPSGIRIEP---NPIRRWPATAFPVPTMLASTWNPELVEAV 81 Thermotoga naphthophila GKAMGEEVREYGVDVLLAPAMNIHRNPLCGRNFEYYSEDPVLSGEMASAF 149 Thermotoga petrophila GKAMGEEVREYGVDVLLAPAMNIHRNPLCGRNFEYYSEDPVLSGEMASAF 150 Thermosipho africanus GKAMGNEAKEYGVDFLLAPAINIHRNPLCGRNFEYYSEDPILTGELASAF 146 Fervidobacterium nodosum GEAMGEEVREYGVDVLLAPAINIHRNPLCGRNFEYYSEDPLLTGELAASF 149 Thermotoga maritima GKAMGEEVREYGVDVLLAPAMNIHRNPLCGRNFEYYSEDPVLSGEMASAF 149 Thermotoga neapolitana GKAMGEEVREYGVDVLLAPAMNIHRNPLCGRNFEYYSEDPVLSGEMASSF 149 Thermofilum sp. GKAMGEEARSCGVDVLLAPALNIHRHPLGGRNFEYFSEDPLLSGKIASAY 138 Thermofilum pendens GRAIGEECRAYGVDVLLAPGVNMHRHPLCGRNFEYFSEDPLLSGEMAAAY 126 Caldivirga maquilingensis GRAMGEEARDYGIGVFLAPGINIHRHPLCGRNFEYFSEDPLLAGKIASAY 131 Thermotoga naphthophila VKGVQSQGVGACIKHFVANNQETNRMVVDTIVSERALREIYLKGFEIAVK 199 Thermotoga petrophila VKGVQSQGVGACIKHFVANNQETNRMVVDTIVSERALREIYLKGFEIAVK 200 Thermosipho africanus VEGVQSEGIGTSLKHFAANNQETNRMKIDTIVSERALREIYLKGFEIVVK 196 Fervidobacterium nodosum VEGVQSQGVGACLKHFVVNEQETNRMTVDTIVSERALREIYLKPFEIAIK 199 Thermotoga maritima VKGVQSQGVGACIKHFVANNQETNRMVVDTIVSERALREIYLKGFEIAVK 199 Thermotoga neapolitana VKGVQSQGVGACIKHFVANNQETNRMVVDTIVSERALREIYLRGFEIAVK 199 Thermofilum sp. VRGVQSAGIGACIKHFVGNEQETGRWGLDTFVSERALREIYLKPFEIAVK 188 Thermofilum pendens VRGVQSVGVGATLKHFAANDQETNRTVIDTVVSERALREIYLKPFEIAVK 176 Caldivirga maquilingensis VKGVQSVGVAATPKHFAANEQETNRTTVDTIVDERTLREIYLKPFEIVVK 181
* Thermotoga naphthophila KARPWTVMSAYNKLNGKYCSQNEWLLKKVLREEWGFDGFVMSDWYAGDNP 249 Thermotoga petrophila KARPWTVMSAYNKLNGKYCSQNEWLLKKVLREEWGFDGFVMSDWYAGDNP 250 Thermosipho africanus KAKPWTVMSAYNKLNGKYCSQNKWLLTKVLREEWGFEGFVMSDWFAGDNP 246 Fervidobacterium nodosum KSKPWTVMSSYNKLNGYYTSQNSWLLTKVLRYEWQFDGFVMTDWFAGDDG 249 Thermotoga maritima KARPWTVMSAYNKLNGKYCSQNEWLLKKVLREEWGFDGFVMSDWYAGDNP 249 Thermotoga neapolitana KSKPWSVMSAYNKLNGKYCSQNEWLLKKVLREEWGFEGFVMSDWYAGDNP 249 Thermofilum sp. EAKPWSVMSAYNKLNGVHCSENEWLLTTVLREEWGFDGFVMTDWGAGEDV 238 Thermofilum pendens KAKPWCVMSSYNKLNGKYSSQNEWLLTRVLREEWGFDGVVMTDWGAGDDS 226 Caldivirga maquilingensis EAKPWAIMSSYNKLNGKYASQNEWLLTKVLREEWGFDGIVMSDWGAGDNP 231 Thermotoga naphthophila VEQLKAGNDMIMPGKAYQVNTERRDEIEEIMEALKEGKLSEEVLDECVRN 299 Thermotoga petrophila VEQLKAGNDMIMPGKAYQVNTERRDEIEEIMEALKEGRLSEEVLNECVRN 300 Thermosipho africanus VEQIKAGNDLIMPGKTYNVFKDRKDEIKELKQAYEKGEITDDIINERVRT 296 Fervidobacterium nodosum AKQMAAGNDLIMPGKSHQVLKHRRNEIDDIRKAIENGELTEEVLNERIRN 299 Thermotoga maritima VEQLKAGNDMIMPGKAYQVNTERRDEIEEIMEALKEGKLSEEVLDECVRN 299 Thermotoga neapolitana VEQLKAGNDLIMPGKAYQVNTERRDEIEEIMEALKEGKLSEEVLDECVRN 299 Thermofilum sp. VRQINAGNDVIMPG--------GQDKLEQVLKAVREGKIPIETIDRAVTR 280 Thermofilum pendens VEQVNAGNDLIMPG--------SDEAVEKLLEAARSGRLRLEALEASAER 268 Caldivirga maquilingensis IEQVKAGNDLIMPG--------SDEIVSRLIDAVKRGELSEDYVNRSAAR 273 Thermotoga naphthophila ILKVLVNAPSFKGYRYSNKPDLESHAEVAYKAGAEGVVLLENNGVLPFDE 349 Thermotoga petrophila ILKVLVNAPSFKGYRYSNKPDLESHAKVAYEAGVEGVVLLENNGVLPFDE 350 Thermosipho africanus ILNILMKTPSFKGYKYSNAPDFEKHARISYNAASEGVVLLKNNDVLPFSA 346 Fervidobacterium nodosum ILRVLVKMPSFKGYNYSNNPDLEKNAKISYEAGCEGVVLLKNNEVLPISK 349 Thermotoga maritima ILKVLVNAPSFKGYRYSNKPDLESHAEVAYEAGAEGVVLLENNGVLPFDE 349 Thermotoga neapolitana ILKVLVNAPSFKNYRYSNKPDLEKHAKVAYEAGAEGVVLLRNEEALPLSE 349 Thermofilum sp. VLSKLLGSAGYK--STPGKPDLEAHAKIAYEAAVEGMVLLKNEGALPIDR 328 Thermofilum pendens VLRLVRKSLTYRGYRPRGAPDLDGHARVAYEAASEGVVLLKNEGALPLGP 318 Caldivirga maquilingensis VLEFIKRTLAYKGYKPTNSPNLKEHAKLAYEAAAEGVILLKNNDALPLNA 323
Results
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Thermotoga naphthophila NTHVAVFGTGQIETIKGGTGSGDTHPRYTISILEGIKERNMKFDEELAST 399 Thermotoga petrophila SIHVAVFGTGQIETIKGGTGSGDTHPRYTISILEGIKERNMKFDEELTSI 400 Thermosipho africanus DAKVSIFGTGQIETVKGGFGSGDTHPRYTISIFEGFKEKGVKVDEKIGNF 396 Fervidobacterium nodosum DTTIALFGTGQIETIRGGTGSGETHPMYTINFLDGVVERGLNFDKELAQF 399 Thermotoga maritima NTHVAVFGTGQIETIKGGTGSGDTHPRYTISILEGIKERNMKFDEELAST 399 Thermotoga neapolitana NSKIALFGTGQIETIKGGTGSGDTHPRYAISILEGIKERGLNFDEELAKT 399 Thermofilum sp. GKKIAFFGVGQILTVKGGMGSGHTHPPYVSTILDAARERSLAIDEELAKK 378 Thermofilum pendens GARVALFGTGQVETLKGGMGSGHTHPRYVVTVLEGLKSGGLLVDEELSSI 368 Caldivirga maquilingensis NARIALFGTGQVETNRGGLGSGHTHPRYFINILDGLRSRGLRIDEELSSI 373 Thermotoga naphthophila YEEYIKKMRETEEYKPRTDSWGTAIKPKLPENFLPEKEIKKAAKKNDVAV 449 Thermotoga petrophila YEDYIKKMRETEEYKPRTDSWGTVIKPKLPENFLSEKEIKKAAKKNDAAV 450 Thermosipho africanus YKEKVFELRNGNYRPNYVNEFNLKIPPKLPEDILDESMIDEASETNDLAI 446 Fervidobacterium nodosum YRAKIEELRNGEYRITRG-QWNEEIKPKLPENLFIVSQLEDIAKRNDVAI 448 Thermotoga maritima YEEYIKKMRETEEYKPRTDSWGTVIKPKLPENFLSEKEIKKAAKKNDVAV 449 Thermotoga neapolitana YEDYIKKMRETEEYKPRRDSWGTIIKPKLPENFLSEKEIHKLAKKNDVAV 449 Thermofilum sp. YAEAVSTYKEELEIFYRD----EPDKPSISEDVVGEADIYATAQRNDLAV 424 Thermofilum pendens YERYVREARGEEFLEKLY--LDEVYADPLPQDIVSEGDAARFAERNDAAV 416 Caldivirga maquilingensis YVNYVKENRGEDYLCALY--YEEAYSEPLPQDIISEEQVRKYAERNDAAI 421
* Thermotoga naphthophila VVISRISGEGYDRKPVKGDFYLSDDELELIKTVSKEFHDQGKKVVVLLNI 499 Thermotoga petrophila VVISRISGEGYDRKPVKGDFYLSDDELELIKTVSREFHEQGKKVVVLLNI 500 Thermosipho africanus IVISRISGEFVDRRAVKGDYYLSDDEQKLIENVSKKFHSKGKKVIVLLNI 496 Fervidobacterium nodosum IFITRISGEGYDRRAEKGDYYLTDDEYELIKNVSETFHKYGKKSLVVLNI 498 Thermotoga maritima VVISRISGEGYDRKPVKGDFYLSDDELELIKTVSKEFHDQGKKVVVLLNI 499 Thermotoga neapolitana IVISRISGEGYDRKPVKGDFYLSDDETDLIKTVSREFHEQGKKVIVLLNI 499 Thermofilum sp. IVVTRVSGEGWDLAP--EDFYLRDDEKWLIDTVSEAYRKIGKPVVAILNI 472 Thermofilum pendens VVLYRVSGEGWDRRPVRGDFYLTESEERLLRLVSREFRGRGKKVVVVLNV 466 Caldivirga maquilingensis VVISRNSGEGWDRRAVKGDYYLTDSERRLIEIVSRQFHALGKKVTVLLNI 471 Thermotoga naphthophila GSPIEVASWRDLVDGILLVWQAGQEMGRIVADVLVGKINPSGKLPTTFPK 549 Thermotoga petrophila GSPIEVASWRDLVDGILLVWQAGQEMGRIVADVLVGRVNPSGKLPTTFPK 550 Thermosipho africanus GGPIEIASWIELVDGLLLIWQPGQEAGRVVADVCLGTVNPSGKLPTTFPK 546 Fervidobacterium nodosum GSPIEVESWKELTDGILLVWQPGQEAGRIFADVISGKVNPSGKLATTFPK 548 Thermotoga maritima GSPIEVASWRDLVDGILLVWQAGQEMGRIVADVLVGKINPSGKLPTTFPK 549 Thermotoga neapolitana GSPVEVVSWRDLVDGILLVWQAGQETGRIVADVLTGRINPSGKLPTTFPR 549 Thermofilum sp. GTPIDVASWREKVDAILLAWQPGQEAGRAIIDTLLGLVNPSGKLPTTFPK 522 Thermofilum pendens CGPIEVASWRDLVDAILVVWLPGQEAGRVVADVLAGRVNPSGKLPMTWPR 516 Caldivirga maquilingensis PAPIEVASWRDLVDAILLVWLPGQEAGRVIADALIGVVNPSGKLPVTFPK 521 Thermotoga naphthophila DYSDVPSWT----FPGEPKDNPQRVVYEEDIYVGYRYYDTFGVEPAYEFG 595 Thermotoga petrophila DYSDVPSWT----FPGEPKDNPQRVVYEEDIYVGYRYYDTFGVEPAYEFG 596 Thermosipho africanus DYQDIPSKS----FPGKPVENPLEVVYEEDIYVGYRYYDTFQIDPLFEFG 592 Fervidobacterium nodosum DYKDVPSRS----FPGEPKENPTSVTYDEGIYVGYRYYDTFRVEPSFEFG 594 Thermotoga maritima DYSDVPSWT----FPGEPKDNPQRVVYEEDIYVGYRYYDTFGVEPAYEFG 595 Thermotoga neapolitana DYSDVPSWT----FPGEPKDNPQKVVYEEDIYVGYRYYDTFGVEPAYEFG 595 Thermofilum sp. TLGDVPSWT----FGGEPAGSPKSVTYEEDIYVGYRYYDTFGKEPAYEFG 568 Thermofilum pendens DWTDVPAAKAPECYPGLPVEDPRRVVYCEGVYVGYRYYDTFGVEPAYEFG 566 Caldivirga maquilingensis DWGDVPTSKSPECYPGIPKENPGAVRYCEGIYVGYRYYDKYSVEPAYEFG 571 Thermotoga naphthophila YGLSYTKFEYKDLKIAIDGDILRVSYTITNTGDRAGKEVSQVYVKAPKGK 645 Thermotoga petrophila YGLSYTKFEYKDLKIAIDGDILRVSYTITNTGDRAGKEVSQVYVKAPKGK 646 Thermosipho africanus YGLSYTKFDYKNLIVKHDDENIEICFEIENSGNVEGKEIAQVYVKAPKAR 642 Fervidobacterium nodosum FGLSYTKFEYSNLNLYKDGSSIVVCFDVKNVGNVPGKEITQLYVRAPRVK 644 Thermotoga maritima YGLSYTKFEYKDLKIAIDGETLRVSYTITNTGDRAGKEVSQVYIKAPKGK 645 Thermotoga neapolitana YGLSYTTFEYSDLNVSFDGETLRVQYRIENTGGRAGKEVSQVYIKAPKGK 645 Thermofilum sp. YGLSYTTFSYSDLSIEQDSEQVKVKFRVTNTGKMPGKEVAQVYVKPPKGK 618 Thermofilum pendens YGLSYTKFEYRGLRVALSRGALKVSFEVVNAGSRPGKEVAQVYVRAPRGR 616 Caldivirga maquilingensis YGLSYTRFEYRGLSVVKDGELIKVSFDVVNVGKHPGKEVAQVYVKAPQGK 621
Results
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Thermotoga naphthophila IDKPFQELKAFHKTKLLNPGESEKIFLEIPLRDLASFDGKE--WVVESGE 693 Thermotoga petrophila IDKPFQELKAFHKTKLLNPGESEKIFLEIPLRDLASFDGKE--WVVESGE 694 Thermosipho africanus LDKPFQELKGFYKTKMLKPGEKEEISIKIALRDLTSFCKDK--WVLEEGE 690 Fervidobacterium nodosum IDKPYQELKGFFKTDTLAPGEMQKVKITVQISDLASFDGEK--WVVEEGI 692 Thermotoga maritima IDKPFQELKAFHKTKLLNPGESEEISLEIPLRDLASFDGKE--WVVESGE 693 Thermotoga neapolitana IDKPFQELKAFHKTRLLNPGESEEVVLEIPVRDLASFNGEE--WVVEAGE 693 Thermofilum sp. IDKPQKELKAFKKTRLLQPGEHEDIVLTIGYKDMASFNGKE--WVLEKGT 666 Thermofilum pendens IDKPFQELKAFRKTRLLEPGEAERIKLRVSLRDLASFDEREKVWVVEPGE 666 Caldivirga maquilingensis LDKPVQELKAFKKTRLLNPGELEHVELTINVRDLASFDESRGMWIIDDGE 671 Thermotoga naphthophila YEVRVGASSRDIRLKDVFLVEEEKRFKP 721 Thermotoga petrophila YEVRVGASSRDIRLRDIFLVEGEKRFKP 722 Thermosipho africanus YRIRVGASSRDIRLEEKVFLKE------ 712 Fervidobacterium nodosum YEFRVGASSRDIRLK------------- 707 Thermotoga maritima YEVRVGASSRDIRLRDIFLVEGEKRFKP 721 Thermotoga neapolitana YEVRVGASSRNIKLKGTFSVGEERRFKP 721 Thermofilum sp. YKIMVGSSSRRIHLEGEFKIHEEKTFR- 693 Thermofilum pendens YEVRVGSSSRDIRLTEHFEVKQELRFAP 694 Caldivirga maquilingensis YEVRVGASSRDIRLTGKFTVSGVIEFKP 699
Figure 4.11 (b): Multi-alignment of β-glucosidase from T. naphthophila RKU-10T (TnBglB) with some
other GH3 family β-glucosidases. Highly conserved amino acid residues are indicated by blocks. The
catalytic sites are represented by asterisk (*) on the top of alignment, the conserved catalytic Aspartic acid
residue act as nucleophile active site (D242) and conserved Glutamic acid act as catalytic proton donor (E458)
and. Thermotoga naphthophila (ADA66752.1), Thermosipho africanus (WP_012580493.1),
Fervidobacterium nodosum (WP_011994742.1), Thermotoga maritima (WP_004082478.1), Thermotoga
petrophila (ABQ46915.1), Thermotoga neapolitana (WP_015919165.1), Thermofilum sp.
(WP_020961743.1), Thermofilum pendens (WP_011753156.1), Caldivirga maquilingensis
(WP_012186357.1).
4.4.17.1. TnBglA Prediction of Signal Peptide
By using online SignalP4.1 prediction server (predicts the presence and location of signal
peptide sequences), determined the presence of signal peptide sequence in the TnBglA
protein. After studied the translation sequence, it was confirmed that TnBglA protein has
no putative signal peptide sequence (Figure 4.12a). SignalP 4.1 neutral network produced
three scores in the form of output for each regions (position) in the sequence of TnBglA
amino acid input. In result, C-score (raw cleavage site score) represented the signal peptide
cleavage sites (immediately after the cleavage site showed high at the position). Signal
peptide score (S-score), output from the SignalP networks are used to discriminate
positions within signal peptides from mature part (position) of the proteins and from
without signal peptides proteins. Whereas, combined cleavage site score (Y-score) is an
average of the slope of S-score and C-score which predicted the cleavage site in a better
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way as compare to the raw C-score alone. The maximum C and S-score for position 24
were 0.141 and 0.148, respectively (Table 4.5a). The SignalP results demonstrated that
there was no signal peptide probability.
Figure 4.12 (a): Signal peptide prediction graph of TnBglA
Table 4.5 (a): Output data of SignalP 4.1 for TnBglA Signal peptide prediction.
Measure Position Value Cutoff signal peptide
Max. C-score 24 0.141
0.450 NO
Max. Y-score 24 0.148
Max. S-score 10 0.217
Mean S 1-23 0.157
D-score 1-23 0.153
C-score= Raw cleavage site score, Y-score= Combined cleavage site score, S-score= Signal peptide
score, D= Discrimination.
4.4.17.2. TnBglB Prediction of Signal Peptide
The amino acid sequence of TnBglB was analyzed with the help of online SignalP4.1 server
and determined the presence or absence of signal peptide sequence. After using SignalP4.1
prediction server, it was confirmed that TnBglB protein has no putative signal peptide
Results
122
sequence (Figure 4.12b). The maximum C-score, Y-score and S-score were 0.122 for
position 31, 0.109 for position 49 and 0.146 for position 36, respectively. All output results
of TnBglB for signal peptide prediction was mention in the following table 4.5b.
Figure 4.12 (b): Signal peptide prediction graph of TnBglB
Table 4.5 (b): Output data of SignalP 4.1 for TnBglB Signal peptide prediction.
Measure Position Value Cutoff Signal Peptide
(SP)
Max. C-score 31 0.122
0.450 NO
Max. Y-score 49 0.109
Max. S-score 36 0.146
Mean S 1–48 0.099
D-score 1–48 0.104
C-score= Raw cleavage site score, Y-score= Combined cleavage site score, S-score= Signal peptide
score, D= Discrimination.
Results
123
4.4.18. Theoretical Properties of TnBglA and TnBglB Proteins
Many Physical and chemical properties of a given protein sequence was computed by using
ProtParam (ExPASy–Bioinformatics) tool. The amino acid sequences of TnBglA and
TnBglB were submitted to compute all parameters including amino acid composition,
aliphatic index, instability index, extinction coefficient, theoretical pI value, atomic
composition and GRAVY, which are listed in the table 4.6. According to the study of
ProtParam tool, the instability index of TnBglA and TnBglB are computed to be 31.23 and
30.83, respectively which classifies both proteins as stable.
Table 4.6: Theoretical Properties of TnBglA and TnBglB Protein using ProtParam tool.
Properties TnBglA TnBglB
Number of amino acids 446 721
Molecular weight 51.509 kDa 81.142 kDa
Positively charged residues (Arg+Lys) 51 94
Negatively charged residues (Asp+Glu) 63 116
Theoretical pI 5.56 5.19
Extinction coefficients (M-1 cm-1) 121240 102930
Instability index 31.23 30.83
Aliphatic index 82.60 84.17
Formula C2376H3537N611O664S7 C3648H5702N958O1099S18
GRAVY -0.373 -0.427
Total number of atoms 7195 11425
GRAVY = Grand average of hydropathicity, Asp = Aspartic acid, Arg = Arginine, Lys = Lysine,
and Glu = Glutamic acid.
Results
124
4.5. Sub-cloning of TnbglA and TnbglB in Expression Vector
To regulate the adequate level of genes expression, an excellent vector pET-21a(+) was
selected for the construction of recombinant expression plasmids. TnbglA and TnbglB
genes were sub-cloned in pET-21a(+) vector and overexpressed under the control of T7/lac
promoter system in host cells. Following are the steps of sub-cloning of genes in expression
plasmids.
4.5.1. Double Restriction of Recombinant Plasmids
Recombinant pTZ57R/T–TnbglA and pTZ57R/T–TnbglB plasmids were double restricted
with NdeI and HindIII in separate individual sterile tubes followed by electrophoresed on
0.8% agarose gel and evaluated the resultant fragment bands under UV-light. Double
cleaved recombinant pTZ57R/T–TnbglA plasmids from selected positive transformants
gave three prominent bands of ~4.227 kb, 2.886 kb and 1.341 kb bands on gel that
represented the uncut recombinant plasmid, pTZ57R/T vector and lastly insert TnbglA
fragment, respectively (Figure 4.13a).
Figure 4.13 (a): Analysis of recombinant pTZ57R/T–TnbglA plasmid after double digestion using two
restriction endonucleases. Lane M, DNA ladder; Lane 1-8, restricted plasmids with NdeI and HindIII and
three resultant bands of approximately 4.227 kb (uncut plasmid), 2.886 kb (pTZ57R/T) and 1.341 kb (insert
TnbglA gene) were observed.
kb
~ 4.227 kb
2.886 kb
1.341 kb
0.5
8.0
5.0
3.0
1.0
1.5
M 1 2 3 4 5 6 7 8
Results
125
After double digestion of recombinant plasmids pTZ57R/T–TnbglB isolated from selected
positive transformants colonies, three bands of ~5.052 kb (uncut recombinant plasmid),
2.886 kb (linerized pTZ57R/T) and 2.166 kb (insert TnbglB) bands were observed on
agarose gel along with DNA ladder (Figure 4.13b).
Figure 4.13 (b): Analysis of recombinant pTZ57R/T–TnbglB plasmid after double digestion using two
restriction endonucleases. Lane M, DNA size ladder; Lane 1-6, double restricted plasmids with NdeI and
HindIII enzymes, and three resultant bands of approximately 5.052 kb (uncut plasmid), 2.886 kb (pTZ57R/T)
and 2.166 kb (insert TnbglB gene) were observed.
4.5.2. Isolation and Double Restriction of pET-21a(+)
Glycerol stock of E. coli DH5α harboring expression vector pET-21a(+) was used to streak
freshly prepared LB-ampicillin plate followed by kept for overnight at 37°C. Next day, a
well isolated single colony selected (Figure 4.14) and inoculated in fresh sterile LB-
ampicillin broth for mini-preparation. After plasmid pET-21a(+) extraction, treated with
RNase to purify the pET from RNA remains (Figure 4.15a). After that purified pET-21a(+)
was subjected to restriction with NdeI and HindIII subsequently analyzed on 0.8% agarose
gel, a prominent band of 5.443 kb was obtained (Figure 4.15b).
M 1 2 3 4 5 6
2.886 kb
kb
10
5.0
3.0
2.0 2.166 kb
1.0
~5.052 kb
Results
126
Figure 4.14: Streak plate of E. coli DH5α harboring pET-21a(+)
Figure 4.15 (a): Isolation of plasmid pET-21a(+), Lane M, DNA size marker; Lane 1 and 2, uncut plasmid
treated with RNase. (b) Double restricted pET-21a(+) using NdeI and HindIII restriction endonucleases, M,
DNA marker; Lane 1-4, bands of 5.443 kb linearized pET-21a(+) after double digestion.
4.5.3. Purification of Double Digested pET–21a(+), TnbglA and TnbglB
Double restricted fragments of pET-21a (5.443 kb), TnbglA (1.341 kb) and TnbglB (2.166
kb) were excised from gel using extraction kit. To evaluate the purity of gene clean double
digested DNA fragments, a small amount (2 µL) of purified products were loaded in the
wells of 0.8% agarose gel and purified products of double digested genes (TnbglA and
TnbglB) fragments and pET were observed under UV-light after electrophoresed (Figure
4.16a and 4.16b).
M 1 2 3 4 M 1 2
Uncut
pET-21a kb
3.0
1.0
0.5
5.443 kb
kb
4.0
5.0
3.0
1.0
0.5
(a) (b)
Results
127
Figure 4.16 (a): Purified double restricted pET-21a(+), Lane M, DNA size marker; Lane 1 and 2, linearized
double cleaved pET-21a(+) after gene clean (5.443 kb). (b) Purified double digested DNA fragments (TnbglA
and TnbglB) after gene clean. Lane M, DNA marker; Lane 1 and 2, purified TnbglB (2.166 kb); Lane 3 and
4 purified TnbglA (1.341 kb).
4.5.4. Ligation and Transformation of E. coli DH5α with Constructed Plasmids
Purified double digested expression vector pET-21a(+) and genes fragments (TnbglA and
TnbglB) were ligated together using T4 DNA ligase followed by incubated at 16°C for 16-
18 hours. Ligation products of both genes (TnbglA and TnbglB) were used to transformed
competent cells of strain DH5α followed by spread transformants of both genes separately
on freshly prepared LB-agar plates containing ampicillin (100 µg mL-1) and kept for
overnight at 37°C incubator. Many white colonies were appeared on both (TnbglA and
TnbglB) LB-agar plates (Figure 4.17). Positive putative transformants were screened by
colony PCR and confirmed by double restriction analysis.
4.5.5. Colony PCR
Selected well isolated white colonies from both plates (TnbglA and TnbglB) and analyzed
the presence of insert gene in pET-21a(+) by colony PCR. After PCR process, all reactions
were loaded on 0.8% agarose gel separately and screened out the positive recombinant
colonies for both genes (Figure 4.18a and 4.18b).
M 1 2 M 1 2 M 3 4
(a) (b)
kb
8.0
kb
2.166 kb
1.341 kb 2.0
10 8.0 6.0 5.0 4.03.0 2.0 1.0 0.5 0.4 0.3 0.2
3.0
5.443 kb
1.2
0.5
Results
128
Figure 4.17: Several white bacterial colonies were appeared on LB-agar plates supplement with ampicillin
after transformation of E. coli DH5α with constructed expression plasmids. (a) Control plate has shown
transformants colonies harboring circular pET-21a(+). (b) Positive putative transformants harboring
recombinant plasmids pET-21a–TnbglA. (c) Positive putative transformants colonies harboring recombinant
plasmids pET-21a–TnbglB.
Figure 4.18: Colony PCR for the screening of positive transformants of E. coli DH5α harboring constructed
recombinant plasmids. (a) Screening of positive transformants harboring pET-21a–TnbglA plasmids by
colony-PCR. Lane M, DNA marker; Lane 1-7, represent 1.341 kb TnbglA amplicons of different colony-
PCR tubes and all selected colonies are positive. (b) Lane M, DNA ladder; Lane 1-6, show 2.166 kb TnbglB
amplicons of different selected colony-PCR and all colonies are positive.
(a) Control Plate (b) pET-21a–TnbglA Plate (c) pET-21a–TnbglB Plate
M 1 2 3 4 5 6 7
(a) (b)
M 1 2 3 4 5 6
10 8.0 6.0 5.0 4.03.0 2.0 1.5 1.00.80.5 0.4 0.3 0.2
kb
1.34 kb
2.166 kb
Results
129
4.5.6. Extraction of Recombinant Expression Plasmids
After colony PCR, positive bacterial transformants harboring recombinant expression
plasmids (pET-21a–TnbglA and pET-21a–TnbglB) were further confirmed by double
restriction analysis. Therefore, recombinant plasmids were isolated from particular positive
cloned E. coli bacterial colonies, and extracted plasmids after RNase treatment were
visualized and analyzed by agarose gel electrophoresis (Figure 4.19a and 4.19b).
Figure 4.19: Isolated recombinant plasmids treated with RNase (a) M, DNA marker; Lane 1-3, uncut circular
recombinant pET-21a–TnbglA plasmids. (b) M, DNA marker; Lane 1-4, uncut circular recombinant pET-
21a–TnbglB plasmids from selected positive transformants.
4.5.7. Restriction Analysis of Recombinant Plasmids
To confirm the positive transformants, double digestion analysis of isolated recombinant
plasmids pET-21a–TnbglA and pET-21a–TnbglB were carried out in sterile individual
tubes with NdeI and HindIII restriction endonucleases followed by analyzed on 0.8%
agarose gel electrophoresis (Figure 4.20a and 4.20b).
4.5.8. Transformation of E. coli BL21 CodonPlus (DE3) with Constructed Expression
Plasmids
After the confirmation of insert genes by restriction analysis, the isolated recombinant
plasmids (pET-21a–TnbglA and pET-21a–TnbglB) were used to transform E. coli
CodonPlus competent cells as a mesophilic expression host. Bacterial transformed spread
10 8.0 6.0 5.0 4.03.0 2.0 1.5 1.00.70.5 0.4 0.3 0.2 0.1
kb
1 2 3 M M 1 2 3 4
(a) (b)
Uncut
Plasmid
Results
130
on LB-agar plates containing ampicillin and chloramphenicol (100 µg mL-1 and 50 µg mL-
1, respectively) followed by incubated for overnight at 37°C, a number of white colonies
obtained on plates (Figure 4.21a and 4.21b).
Figure 4.20: Analysis of recombinant constructed expression plasmids after double digestion using
restriction endonucleases. (a) Lane M, DNA ladder; Lane 1-4, double restricted pET-21a–TnbglA plasmids
with NdeI and HindIII enzymes and resultant three bands of ~ 6.784 kb (uncut plasmid), 5.443 kb (linearized
pET-21a) and 1.341 kb (insert TnbglA gene) were observed. (b) Lane M, DNA size ladder; Lane 1-4, double
restricted pET-21a–TnbglB plasmids with NdeI and HindIII, and three bands were observed, in which first
one was ~7.609 kb (uncut plasmid), second 5.443 kb (pET-21a) and third 2.166 kb (insert TnbglB gene)
fragments.
Figure 4.21: Several white bacterial colonies were appeared on LB-agar plates supplement with ampicillin
and chloramphenicol after transformation of E. coli BL21 CodonPlus (DE3)-RIPL with recombinant circular
plasmids. (a) Plate represent transformants colonies harboring recombinant plasmids pET-21a–TnbglA (b)
Transformants colonies harboring recombinant plasmids pET-21a–TnbglB.
10 8.0 6.0 5.0 4.03.0 2.0 1.5 1.00.5
~6.784 kb ~7.609 kb
5.443 kb
2.166 kb
5.443 kb
1.341 kb
(a) (b)
M 1 2 3 4 M 1 2 3 4 kb
(a) pET-21a–TnbglA in BL21 (b) pET-21a–TnbglB in BL21
Results
131
4.5.9. Colony PCR
To screen the positive transformed colonies of pET-21a–TnbglA and pET-21a–TnbglB,
colony PCR was performed individually using specific primers at optimal standard
conditions of PCR as described earlier. The result of colony PCR of both recombinant
colonies were analyzed after electrophoresis and observed that all selected colonies were
positive for both cloned genes (Figure 4.22a and 4.22b).
Figure 4.22: Colony PCR for the screening of positive transformants of E. coli BL21 CodonPlus harboring
constructed recombinant plasmids. (a) Transformants harboring pET-21a–TnbglA plasmids confirmed by
colony-PCR. Lane M, DNA marker; Lane 1-6, represent 1.341 kb TnbglA amplicons after colony-PCR and
all randomly selected colonies are positive. (b) Lane M, DNA ladder; Lane 1-6, show 2.166 kb TnbglB
amplicons after colony-PCR and all selected colonies are positive.
4.6. Expression Analysis of Recombinant Proteins (TnBglA and TnBglB)
The positive bacterial transformants of E. coli CodonPlus harboring pET-21a–TnbglA and
pET-21a–TnbglB plasmids were grown separately in LB-broth medium supplemented with
ampicillin and chloramphenicol (100 µg mL−1 and 50 µg mL−1, respectively), induced
initially with 0.5 mM IPTG and kept in a shaking incubator (200 rev min-1) at 37°C for the
production of recombinant enzymes. Initially, heterologous crude proteins (TnbglA and
TnbglB) expression in supernatant were analyzed after 24 h by 12% SDS-PAGE along with
2.166
kb
kb kb
1.341
kb
10 8.0 6.0 5.0 4.03.5 3.0 2.5 2.0 1.51.2 1.0 0.9 0.8 0.6 0.5
M 1 2 3 4 5 6 M 1 2 3 4 5 6
(a) (b) 10 8.0 6.0 5.0 4.03.5 3.0 2.5 2.0 1.51.2 1.0 0.9 0.8 0.6 0.5
Results
132
crude control supernatant of E. coli BL21 CodonPlus harboring pET-21a (+) with and
without induction (Figure 4.23a and 4.23b). Recombinant protein TnBglA and TnBglB
displayed a prominent band of 51.5 kDa and 81 kDa molecular weight, respectively on
SDS-PAGE; no such bands were observed in the control samples.
Figure 4.23: SDS-PAGE analysis of recombinant proteins expression in E. coli BL 21 CodonPlus DE3-
(RIPL). (a) Heterologous expression of TnBglA by 12% SDS-PAGE. Lane M, Protein marker (cat # 69079-
3); Lane 1, Crude control supernatant harboring pET-21a (+) after induction with 0.5 mM IPTG; Lane 2,
Crude control supernatant harboring pET-21a(+) without induction; Lane 3, Crude supernatant harboring
pET-21a–TnBglA after induction with 0.5 mM IPTG. (b) Heterologous expression of TnBglB. Lane M,
Protein marker; Lane 1, Crude control supernatant harboring pET-21a(+) after induction with 0.5 mM IPTG;
Lane 2, Crude control supernatant harboring pET-21a(+) without induction; Lane 3, Crude supernatant
harboring pET-21a–TnBglB after induction with 0.5 mM IPTG.
4.7. Preliminary Characterization of TnBglA and TnBglB
Initially, both cloned cells were cultivated in individual sterilized LB-medium, to evaluate
the preliminary characterization of both recombinant enzymes. IPTG (0.5 mM) used to
induce the cultures after that they kept for 96 hours at 37°C and were withdrawn the sample
at regular intervals. After centrifugation, the supernatant (extracellular), cell-free lysate
225 150
100
kDa
(a) (b)
51.5 kDa
81 kDa
35
25
15
10
50
75
25
35
50
75
100
150
225
kDa
Results
133
(intracellular) and cell pellet (cell-bound) fractions of culture were analyzed by SDS-PAGE
and assayed to determine recombinant enzyme activity. Preliminary, optimal temperature
and pH were evaluated to perform activity assay for both recombinant enzymes.
4.7.1 Optimal buffer system for TnBglA and TnBglB
Optimal pH and most suitable buffer for the recombinant TnBglA and TnBglB enzymes
were evaluated by using various buffering system such as, McIlvaine buffer (for pH 2.2-
8.0), sodium acetate buffer (for pH 3.7-5.6), Tris-Cl buffer (for pH 7.0-9.0), MES buffer
(for pH 5.6-6.6), Sorensen’s phosphate buffer (for pH 5.8-8.0), HEPES buffer (for pH 6.8-
8.2), MOP buffer (for pH 7.0-7.8), and CAPS buffer (for pH 9.0-11.0). pH profile was
determined at 70°C for 10 min incubation against pNPG (4 mg mL-1) as substrate, at
different pH values ranging from 3.0-11.0 using various buffer system. Low activity was
observed in MES buffer and MOP buffer. Finally, McIlvaine buffer system was found
optimal buffering system for both cloned enzymes and maximum activity was found at pH
7.0 and pH 5.0 for crude TnBglA and TnBglB, respectively (Figure 4.24a and 4.24b).
4.7.2. Optimal Temperature for TnBglA and TnBglB
The optimal temperature of cloned TnBglA and TnBglB were measured over a broad
temperature ranged from 30-100°C (increments of 5°C), using pNPG (4 mg mL-1) as
substrate, in optimal pH buffer system for both enzymes that was McIlvaine buffer having
pH 7.0 for TnBglA and pH 5.0 for TnBglB. Maximum TnBglA and TnBglB activities were
observed at 95°C and 85°C temperature, respectively (Figure 4.25). All results related to
temperature profile was expressed as percentage of relative activity.
4.7.3. Incubation Time for Enzyme Assay
To find out the optimal incubation time for the hydrolysis of pNPG (4 mg mL-1) substrate,
activity assays were performed for both recombinant enzymes (TnBglA and TnBglB).
Enzyme-substrate reaction mixtures for TnBglA and TnBglB were incubated for 1–15
minutes at their respective optimal conditions, individually. Reactions were withdrawn
periodically and scrutinized the optimum time for the substrate hydrolysis. Maximum
Results
134
activity of TnBglA and TnBglB were observed after 10 minutes incubation at 95°C and
85°C temperature, respectively (Figure 4.26).
Figure 4.24: Effect of pH and various buffer system on recombinant enzymes. Sodium acetate buffer (closed
diamond, pH 4.0-5.5), McIlvaine buffer (closed square, pH 5.0-7.5), Tris-Cl buffer (open triangle, pH 7.0-
9.0), HEPES buffer (closed circle, pH 7.0-8.0), phosphate buffer (opened circle, for pH 6.0-8.0), and CAPS
buffer (dash, pH 9.0-11.0) buffers. (a) The maximum TnBglA activity was observed at pH 7.0 with McIlvaine
buffer. (b) TnBglB showed maximum activity at pH 5.0 with McIlvaine buffer, both are defined as 100%
activity to calculate the relative activity. Reported data is the average of at least three independent
experiments with the standard deviation (+ SD) presented as error bars, which differ significantly at p≤0.05.
0
20
40
60
80
100
120
4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11
Rel
ati
ve
act
ivit
y %
pH
(a) Effect of various buffer on TnBglA activity
0
20
40
60
80
100
120
4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11
Rel
ativ
e ac
tivi
ty %
pH
(b) Effect of various buffer on TnBglB activity
Results
135
Figure 4.25: Effect of temperature on TnBglA and TnBglB. TnBglA displayed optimal activity at 95°C while
TnBglB showed optimal catalytic activity at 85°C, both are defined as 100% activity to calculate the relative
activity. Y-error bars show the standard deviation (±SD) of parallel three experimental reactions. Probability
of all the variables is < 5% significance level, which entail that elected variables are significant at 95%
confidence level.
Figure 4.26: Effect of time course profile of recombinant enzymes. TnBglA and TnBglB displayed optimal
catalytic activity after 10 minutes of incubation at 95°C and 85°C, respectively. Both are defined as 100%
activity to calculate the relative activity. Reported values in results are the average of three independent
experiments, Y-error bars show the standard deviation (±SD) among three reactions, which differ
significantly at p≤0.05.
0
20
40
60
80
100
120
45 50 55 60 65 70 75 80 85 90 95 100
Rel
ati
ve
act
ivit
y %
Temperature oC
Effect of Temperature on TnBglA and TnBglB activity
TnBglA TnBglB
0
20
40
60
80
100
120
0 5 10 15
Rel
ativ
e ac
tivi
ty %
Time (minute)
Effect of Incubation Time for Activity Assay
TnBglA TnBglB
Results
136
4.7.4. Cell fractionation
Heterologous expression and activity of proteins (TnBglA and TnBglB) were analyzed in
all three fractions: extracellular, intracellular and cell-bound fractions. All fractions have
exhibited expression and enzymes activity while highest TnBglA and TnBglB activities
were observed in soluble extracellular fraction (culture supernatant) followed by the
intracellular and the cell-bound fractions (Figure 4.27a and 4.27b). For both enzymes,
maximum protein concentration was found in cell lysate fraction but optimal enzyme
activity was observed in culture supernatant (soluble extracellular fraction). As analysis
was conducted by SDS-PAGE, less expression of recombinant TnBglA and TnBglB
observed in cell lysate as compared to soluble fraction supernatant (Figure 4.28a and
4.28b).
Figure 4.27: Cell fractionation analysis for cloned TnBglA and TnBglB. (a) TnBglA represented optimal
activity (at 95°C using pH 7.0 buffer) and expression in soluble extracellular fraction. (b) TnBglB showed
peak catalytic activity (at 85°C, pH 5.0 buffer) and expression in soluble extracellular fraction, both are
defined as 100% activity to calculate the relative activity. All values shown in results are the average of
triplicate experiments. Y-error bars show the standard deviation (±SD) among three reactions, and probability
of all the variables is < 5% significance level, which entail that elected variables are significant at 95%
confidence level.
0
1
2
3
4
0
20
40
60
80
100
120
Cell lysate Mediasupernatant
Cell bound
Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ati
ve
acti
vit
y %
Fractions
(a) Cell fractionation analysis of TnBglA
Enzyme Activity Protein Conc.
0
1
2
3
4
0
20
40
60
80
100
120
Cell lysate Mediasupernatant
Cell bound
Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ati
ve
act
ivit
y %
Fractions
(b) Cell fractionation analysis of TnBglB
Enzyme Activity Protein Conc.
Results
137
Figure 4.28: SDS-PAGE analysis of cell lysate (intracellular) and supernatant (extracellular) fractions of
recombinant proteins expressed in E. coli BL 21 CodonPlus DE3-(RIPL). (a) Expression of Intra- and
extracellular fractions of TnBglA by 12% SDS-PAGE. Lane M, Protein molecular weight marker; Lane 1,
Crude control cell lysate of E. coli harboring pET-21a (+) after induction with 0.5 mM IPTG; Lane 2, Crude
cell lysate fraction of host after induction with 0.5 mM IPTG; Lane 3, Crude extracellular supernatant fraction
of TnBglA after induction with 0.5 mM IPTG. (b) Heterologous expression of TnBglB by applying both
fractions. Lane M, Protein marker; Lane 1, Crude control cell lysate harboring pET-21a (+) after induction
with 0.5 mM IPTG; Lane 2, Crude supernatant harboring pET-21a (+) after induction with 0.5 mM IPTG;
Lane 3, Crude supernatant harboring pET-21a–TnBglB after induction with 0.5 mM IPTG; Lane 4, Crude
cell lysate of host cells harboring pET-21a–TnBglB after induction with 0.5 mM IPTG.
4.8. Production of Recombinant TnBglA and TnBglB
Engineered E. coli CodonPlus cells harboring pET-21a–TnbglA and pET-21a–TnbglB
plasmids, were used for the production of cloned enzymes, in shake flasks. Various
cultivation conditions and parameters including pre-induction optical density or induction
stage (influence the yield and reproducibility of expressed proteins), medium pH, heat
shock treatment, agitation, inducer concentrations, inducement temperature and time, were
optimized generally in LB medium to achieve maximum yield of heterologous proteins,
prior to preparative-scale experiments.
81 kDa 150
225
25
35
50
75
kDa
51.5 kDa
(a) (b)
Results
138
4.8.1. Pre-induction optimal cell density
Optimum culture density of the E. coli BL21 CodonPlus (DE3) containing recombinant
plasmids (pET-21a–TnbglA and pET-21a–TnbglB) at the time of induction was determined
by using LB-broth medium supplied with both antibiotics (with same concentration as
described earlier), in the individual flasks. Recombinant genes were induced with IPTG
(0.5 mM) at different optical density of culture (OD600nm) 0.5 to 0.9; the optimal enzyme
activity and expression was observed when both recombinant bacterial cultures were
induced at 0.6 optical density (OD600nm) of E. coli cells (Figure 4.29a and 4.29b).
Figure 4.29: Effect of Pre-induction optimal cell density of cloned bacterial on recombinant enzymes
expression (a) Represents the optimal TnBglA activity (at 95°C using pH 7.0 buffer) and expression at 0.6
OD600nm. (b) TnBglB showed optimal catalytic activity (at 85°C, pH 5.0 buffer) and expression at 0.6 OD600nm,
both are defined as 100% activity to calculate the relative activity. Y-error bars show the standard deviation
(±SD) of parallel three experimental reactions. Probability of all the variables is < 5% significance level,
which entail that elected variables are significant at 95% confidence level.
0
0.5
1
1.5
2
2.5
3
0
20
40
60
80
100
120
0.5 0.6 0.7 0.8 0.9
Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ativ
e ac
tivi
ty %
OD600 nm
(a) Optimization of Cell density on TnBglA
Enzyme Activity Protein Conc.
00.511.522.53
020406080
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0.4 0.5 0.6 0.7 0.8 0.9
Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
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act
ivit
y %
OD600nm
(b) Optimization of Cell Density on TnBglB
Enzyme Activity Protein Conc.
Results
139
4.8.2. Effect of Heat Shock
To investigate the effect of heat shock treatment, host cells were incubated at 42°C (1 h)
before and after induction with IPTG, simultaneously a flask was run parallel in which
culture did not treat with heat shock. Direct induction of recombinant cells at OD600nm 0.6,
resulted in comparatively lower enzymes activity and expression as compared to when
bacterial culture was given a heat shock in a shaking water bath (200 rev min-1) after that
culture was induced with IPTG (0.5 mM). Both enzymes activities and expression were
also lower when heat shock was given after induction (Figure 4.30a and 4.30b).
Figure 4.30: Effect of heat shock treatment on recombinant TnBglA and TnBglB expression. (a) Represents
optimal TnBglA activity when culture gave heat shock before induction (HSBI) at 0.6 OD600nm. (b) Optimal
TnBglB expression was obtained when culture gave heat shock before induction (HSBI) at 0.6 OD600nm, both
are defined as 100% activity to calculate the relative activity. Y-error bars show the standard deviation (±SD)
of parallel three reactions. Probability of all the variables is < 5% significance level, which entail that elected
variables are significant at 95% confidence level. WHSI represented, without heat shock induction and HSAI,
heat shock after induction.
0
0.5
1
1.5
2
2.5
3
0
20
40
60
80
100
120
WHSI HSBI HSAI Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ativ
e a
ctiv
ity
%
Heat Shock Treatment
(a) Effect of Heat Shock on TnBglA
Enzyme Activity Protein Conc.
00.511.522.53
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WHSI HSBI HSAI
Pro
tein
Co
nc.
(m
g m
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)
Rel
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Heat Shock Treatment
(b) Effect of Heat Shock on TnBglB
Enzyme Activity Protein Conc.
Results
140
4.8.3. Optimal IPTG concentration
TnbglA and TnbglB genes ligated into pET-21a (+), propagated and expressed in host
under the control of T7 RNA polymerase promoter. Therefore, to over-express
heterologous protein, induced the culture cells with different concentration of IPTG (0.1-
0.9 mM), which revealed that optimal level of recombinant TnBglA and TnBglB activity,
expression, cell density and culture proteins were observed with 0.5 mM concentration,
beyond 0.5 mM, no increase in expression level was observed on SDS-PAGE analysis
(Figure 4.31a and 4.31b). Moreover, further increment in IPTG concentration (0.9 mM)
retarded the growth of cloned bacterial cells as well as lowered the TnBglA and TnBglB
activities (Figure 4.32a and 4.32b).
Figure 4.31: Analysis of different concentration of IPTG as inducer on TnBglA and TnBglB expression. (a)
Expression of extracellular fractions of TnBglA induced with different concentration of IPTG. Lane C, Crude
control supernatant of E. coli harboring pET-21a (+) without induction; Lane 1, Crude extracellular
supernatant of recombinant bacterial culture after induction with 0.2 mM IPTG; Lane 2, Crude supernatant
after induction with 0.3 mM IPTG; Lane 3, Crude supernatant after induction with 0.4 mM IPTG; Lane 4,
Crude supernatant after induction with 0.5 mM IPTG; Lane 5, Crude supernatant after induction with 0.6
mM IPTG. (b) Heterologous expression of TnBglB, induced with different concentration of IPTG. Lane M,
Protein marker; Lane 1, Crude control supernatant harboring pET-21a (+) without induction; Lane 2, Crude
supernatant after induction with 0.2 mM IPTG; Lane 3, Crude supernatant after induction with 0.3 mM IPTG;
Lane 4, Crude supernatant after induction with 0.4 mM IPTG; Lane 5, Crude supernatant after induction with
0.5 mM IPTG; Lane 6, Crude supernatant after induction with 0.6 mM IPTG.
(a) (b)
C 1 2 3 4 5
51.5 kDa
81 kDa
Results
141
Figure 4.32: Effect of different concentration of IPTG on recombinant enzymes expression (a) Optimal
TnBglA activity and expression was observed at 0.5 mM IPTG. (b) TnBglB showed optimal activity and
expression at 0.5 mM IPTG, both are defined as 100% activity to calculate the relative activity. Y-error bars
show the standard deviation (±SD) of parallel three experimental reactions, which significant at probability
5%.
4.8.4. Optimal induction temperature
Immediately after heat shock and induction, the bacterial culture was incubated at 16-42°C
temperatures in a shaking incubator at 200 rev min-1. Samples were withdrawn periodically
0
0.5
1
1.5
2
2.5
3
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g m
L-1
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Rel
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ve
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ivit
y %
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(a) Optimization of IPTG Conc. on TnBglA
Enzyme Activity Protein Conc.
0
0.5
1
1.5
2
2.5
3
3.5
0
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tein
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c. (
mg
mL
-1)
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ty %
IPTG Concentration (mM)
(b) Optimization of IPTG Conc. for TnBglB
Enzyme Activity Protein Conc.
Results
142
from each incubated flask for the analysis of TnBglA and TnBglB enzyme expression and
total cell protein. The optimal incubation temperature for maximum cell growth and
expression in terms enzymes activities was observed at 22°C for both cloned enzymes
(Figure 4.33a and 4.33b).
Figure 4.33: Effect of different induction temperature on recombinant TnBglA and TnBglB expression (a)
Optimal TnBglA expression was found when culture was induced at 22°C with 0.5 mM IPTG induction. (b)
TnBglB showed optimal at 22°C with 0.5 mM IPTG induction, both are defined as 100% activity to calculate
the relative activity. Y-error bars display the standard deviation (±SD) of parallel three experimental
reactions, which significant at probability 5%.
0
0.5
1
1.5
2
2.5
3
0
20
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60
80
100
120
16 22 26 30 37 40 42 Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ati
ve
acti
vity
%
Temperature (oC)
(a) Optimization of Induction Temperature of TnBglA
Enzyme Activity Protein Conc.
0
0.5
1
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2
2.5
3
0
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120
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g m
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ivit
y %
Temperature (oC)
(b) Optimization of Induction Temperature of TnBglB
Enzyme Activity Protein Conc.
Results
143
4.8.5. Optimal agitation speed
Incubated the both recombinant bacterial culture after induction with 0.5 mM IPTG at
different agitation speed (50, 100, 150, 200 and 250 rev min-1) at 22°C. Best host culture
production, TnBglA and TnBglB activity were determined at an agitation speed of 200 rev
min-1 (Figure 4.34a and 4.34b).
Figure 4.34: Effect of agitation speed on culture growth and recombinant enzymes expression (a) Optimal
TnBglA expression was found when culture was incubated at 200 rev min-1 and 22°C temperature with 0.5
mM IPTG induction. (b) TnBglB optimal expression was obtained when host culture was incubated at 200
rev min-1 and 22°C with 0.5 mM IPTG induction, both are defined as 100% activity to calculate the relative
activity. Y-error bars show the standard deviation (±SD) of parallel three experimental reactions. Probability
of all the variables is < 5% significance level which entail that elected variables are significant at 95%
confidence interval.
0
0.5
1
1.5
2
2.5
3
0
20
40
60
80
100
120
50 100 150 200 250 Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ati
ve
act
ivit
y %
Revolution minute-1
(a) Effect of Agitation on TnBglA Expression
Enzyme Activity Protein Conc.
0
0.5
1
1.5
2
2.5
3
3.5
0
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50 100 150 200 250 Pro
tein
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nc.
(m
g m
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Rel
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e ac
tivi
ty %
Revolution minute-1
(b) Effect of Agitation on TnBglB Expression
Enzyme Activity Protein Conc.
Results
144
4.8.6. Optimal Medium pH
To determine the optimum pH of medium for TnBglA and TnBglB production, proper
protein folding and for better growth of engineered bacterial cultures. Recombinant E. coli
culture was inoculated individually in different pH media ranging from 5.5-8.0 and
incubated at optimal conditions after induction with IPTG (0.5 mM). The highest yield of
both recombinant proteins TnBglA and TnBglB were observed at pH 7.0 (Figure 4.35a and
4.35b). Between pH 6.0-7.5, growth rate of cloned bacterial cells and 0.6 optimal density
(OD600nm) remained unaffected, while the heterologous protein secretion and overall
production of total cell protein (TCP) were affected remarkably, most probably due to
protease activity in fermentation culture broth. Bacterial transformants, therefore, grown
well in neutral pH medium at 22°C induction temperature.
00.511.522.533.544.5
0
20
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5 5.5 6 6.5 7 7.5 8P
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in C
on
c. (
mg
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-1)
Rel
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y %
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(a) Optimization of Medium pH for TnBglA
Enzyme Activity Protein Conc.
Results
145
Figure 4.35: Effect of medium pH on the expression of recombinant proteins (a) Optimally TnBglA
expression was observed at pH 7.0. (b) TnBglB optimal expression was obtained at neutral pH, both are
defined as 100% activity to calculate the relative activity. Y-error bars show the standard deviation (±SD) of
parallel three experimental reactions. Probability of all the variables is < 5% significance level which entail
that elected variables are significant at 95% confidence interval.
4.8.7. Optimal incubation time after induction
After induction, the incubation time period at which cloned host cell growth, thermophilic
enzymes TnBglA and TnBglB showed maximum expression and activities were optimized.
Recombinant enzyme activity and host culture production was monitored up to 96 hours
after regular intervals. Both recombinant enzymes TnBglA and TnBglB activity increased
up to 72 h post induction beyond which further increment in culture cell growth and
activities were not detected in any of the culture flasks under study, it remained constant.
Therefore, maximum activity was observed after 72 h of induction at 22°C in LB-medium,
TnBglA and TnBglB expression was analyzed on SDS-PAGE (Figure 4.36a and 4.36b).
The maximum expression was found after 72 h induction period and after that slightly
reduced the activities and expression, completely demonstrated in figure 4.37a and 4.37b.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0
20
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60
80
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5 5.5 6 6.5 7 7.5 8
Pro
tein
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(m
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)
Rel
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pH
(b) Optimization of Medium pH for TnBglB
Enzyme Activity Protein Conc.
Results
146
Figure 4.36: Analysis of TnBglA and TnBglB expression after induction at different time interval. (a)
Heterologous expression of TnBglA after different time intervals, induced with 0.5 mM IPTG. Lane M,
Protein marker; Lane IC, Crude control supernatant harboring pET-21a (+) after induction with 0.5 mM
IPTG; Lane CL, Crude cell lysate after 24 h induction; Lane S1, Crude supernatant after 24 h induction; Lane
S2, Crude supernatant after 48 h induction; Lane S3, Crude supernatant after 72 h induction; Lane S4, Crude
supernatant after 96 h induction. (b) Expression of extracellular fractions of TnBglB sample withdrawn after
24 h, 48 h and 72 h incubation time, induced with 0.5 mM IPTG. Lane M, Protein marker; Lane 1, Crude
control supernatant harboring pET-21a (+) induced with IPTG; Lane 2, Crude supernatant after 24 h
induction; Lane 3, Crude supernatant after 48 h induction; Lane 4, Crude supernatant after 72 h induction;
Lane 5, Crude supernatant after 96 h induction.
51.5 kDa
81 kDa
25
35
50
(b)
kDa
kDa 225
150
75
50
35
25
100
75
150
(a)
Results
147
Figure 4.37: Effect of incubation time on recombinant enzymes expression (a) After 72 h of induction
maximum TnBglA expression was observed at 22°C with 0.5 mM IPTG induction. (b) Similarly, optimal
expression of TnBglB was found after 72 h of induction at 22°C temperature (0.5 mM IPTG induction), both
are defined as 100% activity to calculate the relative activity. Y-error bars show the standard deviation (±SD)
of parallel three independent experimental reactions. Probability of all the variables is < 5% significance
level which entail that elected variables are significant at 95% confidence level.
0
1
2
3
4
5
0
20
40
60
80
100
120
6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96
Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ati
ve
acti
vit
y %
Time (h)
(a) Optimization of Incubation Time for TnBglA Expression
Enzyme Activity Protein Conc.
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0
20
40
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6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96
Pro
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Rel
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Time (h)
(b) Optimization of Induction Time for TnBglB
Enzyme Activity Protein Conc.
Results
148
4.9. Purification of Recombinant TnBglA and TnBglB Proteins
For the purification of soluble extracellular unrefined recombinant TnBglA and TnBglB
proteins, positive bacterial transformants harboring the pET-21a–TnbglA and pET-21a–
TnbglB construct plasmids, were grown individually in fresh sterilized LB-broth medium
(5 L flasks) supplemented with both antibiotic (100 µg mL−1 ampicillin and 50 µg mL-1
chloramphenicol). The recombinant enzymes were grown at optimal cultivation conditions
such as production in neutral pH media, induced culture at 0.6 OD600nm with reduced
concentration of IPTG (0.5 mM) after heat shock treatment at 42°C for 60 minutes followed
by incubated at 22°C for 72 h in a shaking incubator (200 rev min-1). Unrefined cloned
enzymes TnBglA and TnBglB were purified by employing ion exchange (IEX) and
hydrophobic interaction (HIC) chromatography techniques after heat precipitation,
ammonium sulfate fractionation and dialysis. Both recombinant TnBglA and TnBglB
enzymes were efficaciously purified to homogeneity with single bands on 12% SDS-
PAGE. However, activities of both enzymes were monitored at each step of the purification
process, 7.28 fold (40.8% yield) and 7.39 fold purification (39.5% yield) were achieved,
respectively.
All results of purification steps were discussed in the following section.
4.9.1. Heat Treatment
Transformed host cells of E. coli BL21 CodonPlus were harvested from the culture after
72 h induction period, TnBglA and TnBglB expressions and catalytic activities were
detected in all fractions, and the maximum activities were observed with the extracellular
fraction. Therefore, stockpiled the both culture supernatants and concentrated twenty folds
on a lyophilizer or freeze dryer. After that enzyme samples was subjected to water bath at
70°C for 60 minutes, and enriched samples centrifuged (10,000×g) to remove all denatured
host heat labile proteins. After heat treatment, both TnBglA and TnBglB enzymes samples
were run on the SDS-PAGE to analyze the cloned protein purity, most of the mesophilic
host proteins were eliminated which proved it was an effective way to purified
thermostable recombinant protein (Figure 4.38a and 4.38b).
Results
149
4.9.2. Ammonium sulfate precipitation and Dialysis
Followed by heat treatment, the enzyme samples were subjected to precipitation with 55–
60% ammonium sulfate saturation to remove denatured host proteins, collected all
fractional pellets followed by dialysis the resultant suspension to remove (NH4)2SO4 salt
from the precipitated protein samples.
4.9.3. Anion Exchange Chromatography (AEC)
Both recombinant enzymes (TnBglA and TnBglB) were subjected to anion exchange
chromatography, individually. The sample aliquot was loaded to a Resource Q anion
exchange column (Amersham Pharmacia Biotech, Sweden) after dialysis, and active bound
protein fractions were eluted efficiently with a 0-1 M NaCl gradient. According to peak,
collected fractions were analyzed separately, by activity assay and SDS-PAGE after that
active fractions pooled were together. After anion exchange column (AEC)
chromatography, both TnBglA and TnBglB were almost purified but to make desired
protein samples completely purified, the pooled fractions sample was loaded to Resource
ISO pre-packed high performance hydrophobic interaction column (Amersham Pharmacia
Biotech, Sweden).
4.9.4. Hydrophobic Interaction Chromatography (HIC)
For further purification, pooled active fraction sample was loaded on hydrophobic
interaction column (HIC) chromatography individually for both recombinant enzymes. The
bound enzyme fractions were eluted with a 1.5-0 M (NH4)2SO4 decreasing gradient;
various fractions were collected across the major peak. Active enzyme was obtained as
unbound fractions, pooled all active fractions of recombinant enzymes. The final active
fractions of recombinant TnBglA and TnBglB proteins were subjected to SDS-PAGE
analysis, a single prominent band revealed that the target heterologous TnBglA and TnBglB
protein was successfully purified (Figure 4.38a and 4.38b). Recombinant enzymes
activities were monitored at each step of the purification process, 7.28 fold with a recovery
of 40.8% yield and 7.39 fold purification with a recovery of 39.5% yield were achieved,
respectively (Table 4.7a and 4.7b).
Results
150
Figure 4.38 (a): SDS-PAGE analysis of TnBglA from pET-21a–TnbglA in E. coli BL21 CodonPlus (DE3)-
RIPL. Lane M, Novagen Protein marker (Cat # 69079 Lane -3); Lane 1: Crude control supernatant of E. coli
BL harboring pET-21a(+) induced with IPTG (0.5 mM); Lane 2, Crude control supernatant of E. coli
harboring pET-21a(+) without IPTG induction; Lane 3, Crude TnBglA supernatant of E. coli harboring pET-
21a–TnbglA without induction; Lane 4, Crude TnBglA supernatant of E. coli harboring pET-21a(+)–TnbglA
induced with IPTG (0.5 mM); Lane 5, Crude supernatant of TnBglA after heat treatment (at 70°C for 1 h);
Lane 6, purified TnBglA after AEC and HIC (3 µg).
Table 4.7 (a): Purification summary of recombinant β-1,4-glucosidase from T. naphthophila (TnBglA).
Purification steps
Total
protein
(mg)
Total
activity
(kU)
Specific
activity
(kU mg-1)
Yield
(%)
Purification
(fold)
Crude extract 16.4 574 35 100 1.0
Heat treatment and (NH4)2SO4
precipitation 3.16 339.7 107.5 59.2 3.07
Ion-exchange and hydrophobic
interaction chromatography 0.92 234.6 255 40.8 7.28
Each value represent the average of triplicate measurements and varies from the mean by not more than 10%.
kDa
50
35
25
75
100
150
225
51.5 kDa
Results
151
Figure 4.38 (b): SDS-PAGE analysis of TnBglB from pET-21a–TnbglB in E. coli BL21 CodonPlus (DE3)-
RIPL. Lane M, Novagen Protein marker (Cat # 69079-3); Lane 1: Crude control supernatant of E. coli
harboring pET-21a(+) induced with IPTG (0.5 mM); Lane 2, Crude control supernatant of E. coli harboring
pET-21a(+) without induction; Lane 3, Crude TnBglB supernatant of E. coli harboring pET-21a– TnbglB
without induction; Lane 4, Crude TnBglB supernatant of E. coli harboring pET-21a(+)–TnbglB induced with
IPTG (0.5 mM); Lane 5, Crude supernatant of TnBglB after heat treatment (at 70°C for 1 h); Lane 6, purified
TnBglB after AEC and HIC (3 µg).
Table 4.7 (b): Purification summary of recombinant β-1,4-glucosidase from T. naphthophila (TnBglB)
Purification steps
Total
protein
(mg)
Total
activity
(kU)
Specific
activity
(kU mg-1)
Yield
(%)
Purification
(fold)
Crude extract 16.8 295.68 17.6 100 1.0
Heat treatment and (NH4)2SO4
precipitation 3.15 176.4 56 59.6 3.18
Ion-exchange and hydrophobic
interaction chromatography 0.90 117 130 39.5 7.38
Each value represent the average of triplicate measurements and varies from the mean by not more than 10%.
kDa
225 150
100
25
35
50
75
81 kDa
Results
152
4.9.5. Native-PAGE analysis
The purified TnBglA and TnBglB subjected to native-PAGE analysis under non-reducing
conditions, with and without boiling for 10 minutes. Recombinant purified TnBglA
remained as a monomeric form even after boiling for 10 minutes; similarly, TnBglB
remained as a trimeric form even after boiling for 10 minutes which demonstrated that
TnBglB was a homotrimer, with a monomeric molecular weight of 81.1 kDa (Figure 4.39a
and 4.39b).
Figure 4.39: Native PAGE analysis of TnBglA and TnBglB. (a) Lane 1, monomeric recombinant TnBglA (3
µg) without boiling, represented a single prominent band; Lane 2, monomeric TnBglA (3 µg) form displayed
a single band even after 10 minutes boiling at 100°C. (b) Lane 1, homotrimeric recombinant TnBglB (3 µg)
with a single band without boiling; Lane 2, homotrimeric form with a single band of TnBglB (3 µg) even
after 10 minutes boiling.
(a) (b)
Results
153
4.10. Characterization of Purified TnBglA and TnBglB
The complete characterization of recombinant purified TnBglA and TnBglB enzymes from
T. naphthophila were done and determined their molecular weight, optimal pH and
temperature, thermal and pH stability, substrate specificity, kinetics parameters,
thermodynamics study of substrate hydrolysis and enzymes inactivation, shelf life of
enzymes, effect of chemical inhibitors, metallic cation, glucose and xylose on the catalytic
efficiency of cloned thermostable enzymes.
4.10.1. Determination of Molecular weight
The final active fractions of recombinant proteins TnBglA and TnBglB exhibited a single
noticeable band of 51.50 kDa and 81 kDa molecular weight (MW) by 12% SDS-PAGE
analysis (Figure 4.38a and 4.38b). Theoretical molecular weight of cloned enzymes were
50.51 kDa and 81.14 kDa, deduced from amino acid sequence of protein, which revealed
the target that heterologous TnBglA and TnBglB enzymes was successfully purified.
4.10.2. Buffer system and pH profile of TnBglA and TnBglB
Various buffering systems were used to evaluate the optimal buffer for the purified
recombinants TnBglA and TnBglB enzymes, the results were quite similar as experienced
with crude recombinant enzymes and the best buffer system was McIlvaine buffer (for pH
3.0-7.0), HEPES buffer (for pH 8.0) and CAPS buffer (for pH 9.0-11.0), while relatively
reduced hydrolytic efficiency was observed in Tris-Cl buffer, MES buffer, Sorensen’s
phosphate buffer, and MOP buffer. Purified TnBglA and TnBglB enzymes exhibited
maximum activity at pH 7.0 and pH 5.0 McIlvaine buffer, respectively. TnBglA retained
more than 90% of optimal activity over a broad pH range of 6.0-9.0 (Figure 4.40a), whereas
activity was completely repressed at pH 3.0-4.0 and at pH 10.0-11.0. TnBglB exhibited
90% catalytic activity at acidic pH 4.0, retained more than 70% efficiency in McIlvaine
buffer at pH 6.5 and completely repressed in alkaline aqueous condition (Figure 4.40b).
Results
154
Figure 4.40: Effect of pH on the activity of purified TnBglA and TnBglB from T. naphthophila. pH profile
was determined at 70°C for 10 min incubation against pNPG (4 mg mL-1) as substrate, at different pH values
ranging from 3.0-11.0 using McIlvaine buffer (closed square, pH 3.0-7.0), HEPES (closed triangle, pH 8.0)
and CAPS (closed circle, pH 9.0-11.0) buffers. (a) The maximum TnBglA activity observed at pH 7.0, while
(b) TnBglB exhibited maximum activity at pH 5.0, both are defined as 100% activity to calculate the relative
activity. All values shown in results are the average of triplicate experiments. Y-error bars indicate standard
deviation (+SD) among the triplicate experimental reactions, which differ significantly at p≤0.05.
0
20
40
60
80
100
120
3 4 5 6 7 8 9 10 11
Rel
ati
ve
act
ivit
y %
pH
(a) Effect of pH on TnBglA activity
0
20
40
60
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120
3 4 5 6 7 8 9 10 11
Rel
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ty %
pH
(b) Effect of pH on TnBglB activity
Results
155
4.10.3. Temperature profile
Optimal temperature of purified enzymes were scrutinized by incubating assay for 10 min
at different temperature (30-100°), pNPG (4 mg mL-1) used as a substrate. TnBglA and
TnBglB displayed peak catalytic activity at 95°C (pH 7.0) and 85°C (pH 5.0), respectively.
TnBglA retained more than 80% of maximum activity over a range of temperature at 80-
100°C, while TnBglB showed more than 75% activity at 70-95°C (Figure 4.41a and 4.41b).
Figure 4.41: Effect of temperature on the activity of purified TnBglA and TnBglB. Temperature profile was
determined, at various temperature range from 30-100°C using pNPG (4 mg mL-1) as substrate, by incubating
TnBglA and TnBglB enzymes for 10 minutes in McIlvaine buffer pH 7.0 and pH 5.0, respectively. (a) TnBglA
displayed optimal activity at 95°C while (b) TnBglB showed optimal catalytic activity at 85°C, both are
defined as 100% activity to calculate the relative activity. Reported data is the average of at least three
independent experiments with the standard deviation (+ SD) presented as error bars, which differ
significantly at p≤0.05.
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105
Rel
ativ
e ac
tiv
ity
%
Temperature °C
(a) Effect of Temperature on TnBglA activity
0
20
40
60
80
100
120
30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105
Rel
ativ
e ac
tivi
ty %
Temperature °C
(b) Effect of Temperature on TnBglB activity
Results
156
4.10.4. Optimal Incubation Time
Time course profile of recombinant TnBglA and TnBglB enzymes for the hydrolysis of
pNPG substrate were monitored, and the optimal incubation time for TnBglA at 95°C
(using buffer pH 7.0) and TnBglB at 85°C (using buffer pH 5.0) was 10 minutes. Both
enzymes slowly increased the catalytic activity and after 10 minute 100% activity was
observed (Figure 4.42).
Figure 4.42: Effect of time course profile of recombinant enzymes. TnBglA and TnBglB displayed optimal
catalytic activity after 10 minutes of incubation at 95°C and 85°C, respectively. Both are defined as 100%
activity to calculate the relative activity. Reported values in results are the average of three independent
experiments, Y-error bars show the standard deviation (±SD) among three reactions, which differ
significantly at p≤0.05.
4.10.5. Thermal stability
Thermal stability of purified TnBglA and TnBglB revealed that both recombinant enzymes
showed remarkable thermotolerant behavior, at pH neutral or near neutral conditions. Both
purified enzymes were incubated in various pH buffers ranging from pH 4.0-7.5 at different
temperature (30-100°C), the enzyme sample was withdrawn at regular interval followed
by activity assay. Purified TnBglA and TnBglB enzymes showed great thermal stability
ranging from 30-85°C temperature at pH 6.5 to 7.5, whereas in acidic conditions (pH 4.0-
5.5) both recombinant enzymes were less stable at 30-85°C as compared to neutral
environment. TnBglA was completely stable after 720 minutes (12 h) incubation at 85°C
in pH 6.5 and 7.5 buffering conditions, while purified TnBglB enzyme showed 100%
0
20
40
60
80
100
120
0 5 10 15
Rel
ativ
e ac
tivi
ty %
Time (minute)
Effect of Incubation Time for Activity Assay
TnBglA TnBglB
Results
157
stability at 85°C after 480 minutes (8 h) incubation in neutral aqueous environment.
TnBglA lost its 50% original activity after 120 incubation at 100°C (Figure 4.43a) whereas,
TnBglB lost its 45% original (optimal) hydrolytic efficiency after 30 minutes incubation at
100°C (Figure 4.43b).
Figure 4.43: Thermal stability of TnBglA and TnBglB at neutral aqueous environment. (a) Thermostability
of TnBglA was determined by pre-incubating purified TnBglA enzyme at temperature ranging from 85-100°C
for different time intervals. (b) Thermostability of TnBglB was determined by pre-incubating purified
TnBglB enzyme at temperature ranging from 80-100°C for different time intervals. Reported values in results
are the average of three independent experiments, Y-error bars show the standard deviation (±SD) among
three reactions, which differ significantly at p≤0.05.
0
20
40
60
80
100
120
0 60 120 180 240 300 360 420 480 540 600 660 720
Rel
ativ
e a
ctiv
ity
%
Time (min)
(a) Thermostability of TnBglA
85°C 90°C 95°C 100°C
0
20
40
60
80
100
120
0 60 120 180 240 300 360 420 480 540 600 660 720
Rel
ativ
e ac
tivi
ty %
Time (min)
(b) Thermostability of TnBglB
85°C 90°C 95°C 100°C
Results
158
4.10.6. pH Stability
Aqueous environment with different pH, greatly affect the enzyme stability, TnBglA and
TnBglB exhibited high pH stability when incubated purified enzymes at 70°C in different
pH buffers ranging from pH 3.0-11.0 for 60 minutes. TnBglA retained more than 90%
residual activity from pH 6.0-9.0 (Figure 4.44a). While, TnBglB retained 100% of its
original activity at pH 6.5-7.5 and more than 50% of its original activity at was observed
pH 4.0-5.5 (Figure 4.44b). Hence, both enzymes were more stable at neutral pH condition
as compared to the acidic and alkaline aqueous conditions.
Figure 4.44: pH stability (a) TnBglA and (b) TnBglB was determined by pre-incubating enzyme at different
pH values (3.0-11.0) at 70°C for 60 minutes, using McIlvaine buffer (closed square, pH 3.0-7.0), HEPES
(closed triangle, pH 8.0) and CAPS (closed circle, pH 9.0-11.0) buffers, activity of enzyme without pre-
incubation is taken as 100%. Y-error bars indicate standard deviation (+SD) among the triplicate
experimental reactions, which differ significantly at p≤0.05.
0
20
40
60
80
100
120
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
Rel
ativ
e ac
tivi
ty %
pH
(a) pH stability of TnBglA
0
20
40
60
80
100
120
3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5
Rel
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e ac
tivi
ty %
pH
(b) pH stability of TnBglB
Results
159
4.10.7. Shelf life of recombinant TnBglA and TnBglB
Storage stability of both enzymes was an important and crucial parameter for commercial
utilization, analyzed in crude media supernatant, after heat precipitation (70°C for 1 h) and
purified samples. TnBglA and TnBglB enzymes had an exceptional storage stability,
retaining 100% initial activity after the storage for two year (till now) at 4°C, in all the
three ways of storage (crude supernatant, heat treated and purified). Heat treated and
purified TnBglA and TnBglB had retained 100% initial activity at room temperature up to
175 days but after 35 days the crude supernatant enzymes showed marginal decline of 5-
7%. Both enzymes showed similar shelf life stability.
4.10.8. Effect of metallic ions and different chaotropic agents
Generally industrial enzymes cannot perform their catalytic work proficiently in the
presence of metallic cations, detergents and chemical inhibitors, because the toxic metallic
cations and chemical reagents interact and alter the enzymes activity in different ways. The
manipulation of metal ions (10 mM) on TnBglA and TnBglB activities were only nominal,
and exhibited optimal hydrolytic efficiency even by the addition of different tri-/divalent
metallic cations. Although Mn2+, Zn2+ , Co2+ and Ca2+ stimulate the activity of TnBglB up
to 115%, 120%, 110% and 125%, respectively. Only Hg2+ and SDS effectively inhibited
the TnBglA and TnBglB activities up to certain limit. However EDTA, Urea, Triton X-100,
Tween-80 and β-mercaptoethanol did not show any conspicuous influence on TnBglA and
TnBglB enzymes activities (Figure 4.45a and 4.45b).
Results
160
Figure 4.45: Effect of metal ions and different inhibitors on purified TnBglA and TnBglB activity. Residual
enzyme activity was expressed as a percentage with pNPG (4 mg mL-1) as substrate, under standard assay
conditions after the addition of metal ions (10 mM), EDTA (10 mM) and chemical reagents (1%) in reaction
mixture (a) TnBglA did not show any significant influence in the presence of inhibitors, while Hg2+ and SDS
detergent cause an obvious inhibitory effect. (b) TnBglB did not reveal any significant influence in the
presence of inhibitors, although Zn2+ and Co2+ cause simulative effect, only Hg2+ and SDS inhibited the
enzyme activity. Y-error bars specify the standard deviation (±SD) of parallel triplicate experimental
reactions. Probability of all the variables is < 5% significance level which entail that elected variables are
significant at 95% confidence interval.
0
20
40
60
80
100
120
Con
tro
l
Ca²⁺
Ni²⁺
Zn²⁺
Mn²⁺
Pb²⁺
Cd²⁺
Co²⁺
Fe²⁺
Mg²⁺
Cu²⁺
Hg²⁺
ED
TA
SD
S
Ure
a
Tw
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Tri
ton-
X-1
00
β-M
erca
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etha
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Rel
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ivit
y %
Metal ions (10 mM), EDTA (10 mM) and chemical reagents (1%)
(a) Effect of Metal ions, chelating agent and chemical reagents on TnBglA
0
20
40
60
80
100
120
140
160
Con
tro
l
Ca²⁺
Ni²⁺
Zn²⁺
Mn²⁺
Pb
²⁺
Cd²⁺
Co²⁺
Fe²⁺
Mg²⁺
Cu²⁺
Hg²⁺
ED
TA
SD
S
Ure
a
Tw
een
-80
Tri
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X-1
00
β-M
erca
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etha
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Rel
ativ
e ac
tivi
ty %
Metal ions (10 mM), EDTA (10 mM) and chemical reagents (1%)
(b) Effect of Metal ions, chelating agent and chemical reagents on TnBglB
Results
161
4.10.9. Effect of organic solvent
All organic solvents cause a noticeable influence on purified TnBglA and TnBglB enzymes
activity as the concentration increased. Acetonitrile and Ethyl acetate repressed the activity
of purified recombinant TnBglA enzyme up to certain limit. Purified TnBglA activity was
significantly inhibited by addition of 40% (v/v) acetone, ethyl acetate, acetonitrile,
dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF) up to 18%, 38%, 50%, 52%
and 50%, respectively (Figure 4.46a). Whereas, 50% (v/v) acetone, ethyl acetate,
acetonitrile, dimethyl sulfoxide (DMSO) and Dimethylformamide (DMF) influenced the
activity of TnBglB up to 50%, 56%, 54%, 50% and 48% respectively (Figure 4.46b).
4.10.10. Effect of Alcohol
Effect of straight chain aliphatic alcohols (methanol, ethanol, n-butanol and isopropanol)
on TnBglA and TnBglB activity were scrutinized by addition of the test short chain length
alcohols at the final concentration of 2-30% (v/v) in the reaction mixture. These alcohols
have proven an obvious inhibitory additives for the recombinant TnBglA, and drastically
reduced the activity up to 8-10% at the final concentration of 30% (v/v) (Figure 4.47a).
TnBglB showed more resistant behavior as compared to TnBglA, these alcohols did not
exhibit any significant influence on the activity of purified TnBglB at the final
concentration of 20-25% (v/v), whereas at high concentration of 50% (v/v) methanol,
ethanol, n-butanol and isopropanol repressed the TnBglB activity up to 24%, 26%, 22%
and 25%, respectively (Figure 4.47b).
Results
162
Figure 4.46: Effect of organic solvent (acetone, ethyl acetate and acetonitrile) on purified TnBglA and
TnBglB activity. Residual enzyme activity was determined using pNPG (4 mg mL-1) as substrate, under
standard assay conditions and expressed as a percentage (a) TnBglA significant influence in the presence of
50% organic solvents. (b) Organic solvents slightly less inhibited TnBglB activity. Y-error bars specify the
standard deviation (±SD) of parallel triplicate experimental reactions. Probability of all the variables is < 5%
significance level which entail that elected variables are significant at 95% confidence interval.
0
20
40
60
80
100
120
0 5 10 20 30 40 50
Rel
ati
ve
Act
ivit
y %
% Reagent (v/v)
(a) Effect of Organic Solvent on TnBglA activity
Acetone Ethyl acetate Acetonitrile DMSO DMF
0
20
40
60
80
100
120
0 5 10 20 30 40 50
Rel
ativ
e A
ctiv
ity
%
% Reagents (v/v)
(b) Effect of Organic Solvent on TnBglB activity
Acetone Ethyl acetate Acetonitrile DMSO DMF
Results
163
Figure 4.47: Effect of short chain alcohol (ethanol, methanol and isopropanol) on purified TnBglA and
TnBglB activity. Residual enzyme activity was expressed as a percentage with pNPG (4 mg mL-1) as
substrate, under standard assay conditions (a) TnBglA showed obvious constrain in the presence of short
chain alcohol. (b) TnBglB did not displayed any significant influence in the presence of 0-20% alcohols. Y-
error bars specify the standard deviation (±SD) of parallel triplicate experimental reactions. Probability of all
the variables is < 5% significance level which entail that elected variables are significant at 95% confidence
level.
0
20
40
60
80
100
120
0 2 5 10 15 20 25 30
Rel
ati
ve
Act
ivit
y %
% Alcohol (v/v)
(a) Effect of short chain alcohol on purified TnBglA
Ethanol Methanol Isopropanol n-Butanol
0
20
40
60
80
100
120
140
160
0 2 5 10 20 30 40 50
Rel
ativ
e A
ctiv
ity
%
% Alcohol (v/v)
(b) Effect of short chain alcohol on purified TnBglB
Ethanol Methanol Isopropanol n-Butanol
Results
164
4.10.11. Effect of glucose and xylose
Mostly, glucose cause an inhibitory effect on β-glucosidases, to reconnoiter the effect of
glucose and xylose on purified TnBglA and TnBglB activities, various concentration of
glucose and xylose (20-1500 mM) was used to find the inhibition constant (Ki) values of
glucose and xylose for both enzymes. TnBglA catalytic activity was enhanced 160% and
148% by the addition of 100 mM and 200 mM glucose (at final concentration in reaction
mixture), respectively and enzyme remained active even in the presence of 600 mM
glucose. However, TnBglA activity was gradually repressed with the increase
concentration of glucose, and Ki value for glucose was found to be 1200 mM (Figure
4.48a). Similar behavior was observed with the addition of xylose, TnBglA activity was
increased 170% and 154% by adding 100 mM and 200 mM xylose, respectively. TnBglA
catalytic efficiency was gradually repressed with the increase concentration of xylose,
whereas the Ki value was 1300 mM (Figure 4.48b). While, TnBglB enzyme did not show
any stimulatory effect in the presence of glucose and xylose, the relative activity declined
continuously with increased concentration of glucose and its relative activity was repressed
to 12% and 18% in the presence of 600 mM glucose and xylose, respectively. The Ki value
of TnBglB for glucose inhibition was estimated 150 mM, whereas Ki value for xylose was
200 mM (Figure 4.48a and 4.48b).
0
20
40
60
80
100
120
140
160
180
0 200 400 600 800 1000 1200 1400 1600
Rel
ativ
e ac
tivi
ty %
Glucose concentration (mM)
(a) Effect of glucose on TnBglA and TnBglB activity
TnBglA TnBglB
Results
165
Figure 4.48: Effect of glucose and xylose on purified TnBglA and TnBglB activity. (a) Influence of different
glucose concentration (20-1500 mM) on TnBglA and TnBglB activity. (b) Effect of different concentration
of xylose (20-1500 mM) on TnBglA and TnBglB catalytic activity using pNPG (4 mg mL-1) as substrate. Y-
error bars specify the standard deviation (±SD) of parallel triplicate experimental reactions. Probability of all
the variables is < 5% significance level which entail that elected variables are significant at 95% confidence
interval.
4.10.12. Substrate Specificity of TnBglA and TnBglB
Substrate specificity of recombinant TnBglA and TnBglB was examined with various para-
and ortho-nitrophenyl glycoside and heteroglycans substrates. Recombinant highly
thermo-efficient TnBglA displayed great affinity towards pNP-β-D-glucopyranoside
(pNPG) and pNP-β-D-fucopyranoside (pNPF), with highest ever reported specific
activities of 255 kU mg-1 and 253 kU mg-1, respectively. Hydrolytic activity was also
observed with pNP-β-D-xylopyranoside (pNPX, 11 kU mg-1), pNP-β-D-cellobioside
(pNPC, 16 kU mg-1), pNP-β-D-galactopyranoside (pNPGal, 102 kU mg-1) and cellobiose
(177.78 U mg-1). Similarly, TnBglB also showed maximum affinity towards pNPG with
high specific activity of 130 kU mg-1. TnBglB catalytic activity was also found with pNPX
(26 kU mg-1), pNPF (10 kU mg-1), pNPC (8 kU mg-1), pNPGal (7 kU mg-1) and cellobiose
with 150 U mg-1. However, TnBglA and TnBglB were not able to cleave o-nitrophenyl
glycoside substrates, sucrose, carboxymethyl cellulose (CMC), avicel, laminarin and xylan
(Table 4.8).
0
20
40
60
80
100
120
140
160
180
0 200 400 600 800 1000 1200 1400 1600
Rel
ati
ve
act
ivit
y %
Xylose concentration (mM)
(b) Effect of xylose on TnBglA and TnBglB activity
TnBglA TnBglB
Results
166
Table 4.8: Substrate specificity of the recombinant β-glucosidases (TnBglA and TnBglB)
Substrates
TnBglA TnBglB
Specific Activity
(kU mg-1)
Relative
activity
(%)
Specific Activity
(kU mg-1)
Relative
activity
(%)
pNP-β-D-glucopyranoside (pNPG) 255 + 0.12 100 130 + 0.08 100
pNP-α-D-glucopyranoside (pNPαG) 0.005+ 0.14 0.0196 0.004 + 0.03 0.0037
pNP-β-D-fucopyranoside (pNPF) 253 + 0.13 99.2 10 + 0.05 7.7
pNP-α-D-fucopyranoside (pNPF) 0.003+ 0.11 0.012 ND ND
pNP-β-D-galactopyranoside (pNPGal) 102 + 0.03 40 7 + 0.06 5.4
pNP-α-D-galactopyranoside (pNPαGal) ND ND ND ND
pNP-β-D-xylopyranoside (pNPX) 16 + 0.03 6.27 26 + 0.1 10.2
pNP-β-D-cellobioside (pNPC) 11 + 0.19 4.3 8 + 0.04 3.14
pNP-α-D-cellobioside (pNPαC) ND ND ND ND
pNP-β-D-glucuronide (pNPGn) ND ND ND ND
pNP-β-D-mannoside (pNPM) ND ND ND ND
pNP-α-L-arabinofuranoside (pNPαA) ND ND ND ND
p-NP-α-L-fucopyranoside (pNPαLF) ND ND ND ND
oNP-β-D-galactopyranoside (oNPGal) ND ND ND ND
oNP-β-D-fucopyranoside (oNPF) ND ND ND ND
oNP-β-D-glucopyranoside (oNPG) 0.09 + 0.02 0.035 0.6 + 0.03 0.46
Cellobiose 0.177 + 0.001 0.07 0.15 + 0.002 0.12
Sucrose ND ND ND ND
Carboxymethyl cellulose (CMC) ND ND ND ND
Avicel ND ND ND ND
Laminarin ND ND ND ND
Xylan from Birch wood ND ND ND ND
Xylan from Beech wood ND ND ND ND
To analyzed TnBglA and TnBglB catalytic activity, different substrates were tested in McIlvaine buffer
having pH 7.0 at 95°C and pH 5.0 at 85°C, respectively. All data represents the average of three experiments
done in triplicate and the + represents the standard deviation. ND means not detected, specific activity is not
detected by the analytical methods used in this study.
Results
167
4.10.13. Kinetic studies for the hydrolysis of various substrates
Kinetics parameters (Km, Vmax, kcat, and kcat Km-1) of purified TnBglA and TnBglB for the
hydrolysis of various substrates such as pNPG, pNPF, pNPGal, pNPX, pNPC and
cellobiose were analyzed using Lineweaver-Burk plot under standard assay conditions, to
investigate the recombinant enzymes efficiency towards these substrates (Figure 4.49a and
4.49b). Reactions rate were determined by using various concentration of substrates during
activity assay. All kinetic parameters for each substrate that were computed from their
respective Lineweaver-Burk plot and both recombinant TnBglA and TnBglB enzymes
exhibited distinctive affinity towards substrates, results are demonstrated in table 4.9a and
4.9b. Purified TnBglA and TnBglB displayed high Km value 7.76 mM and 7.53 mM against
cellobiose, respectively. Whereas, TnBglA revealed lowest Km value (1.07 mM) with
pNPC, while TnBglB showed lowest Km value (0.45) with pNPG. The highest Vmax 297
mmol mg-1 min-1 of TnBglA was obtained for pNPG with turnover number 1527778 sec-1,
and similarly TnBglB had maximum Vmax value 153 mmol mg-1 min-1 for pNPG with
turnover number 1214285.7 sec-1. Both TnBglA and TnBglB displayed high specificity
constant (kcat Km-1) for pNPG and lowest for cellobiose.
y = 0.5041x + 0.337R² = 0.9793
y = 0.1254x + 0.0849R² = 0.9786
y = 0.5152x + 0.4773R² = 0.8939
y = 0.1558x + 0.0655R² = 0.9821
y = 0.5781x + 0.3381R² = 0.971
y = 1.6479x + 0.0521R² = 0.9832
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
-1 -0.5 0 0.5 1 1.5 2 2.5 3
1/V
1/[S]
(a) Lineweaver–Burk plot for the hydrolysis of various substrates using TnBglA
pNPG pNPGal pNPC pNPX pNPF Cellobiose
Results
168
Figure 4.49: (a) Lineweaver–Burk plot for the hydrolysis of various substrates using TnBglA (b)
Lineweaver–Burk plot for the hydrolysis of various substrates using TnBglB.
Table 4.9 (a): Kinetic parameters of β-glucosidase from T. naphthophila (TnBglA)
Substrates Km (mM) Vmax (mmol mg-1 min-1) kcat (sec-1) kcat Km-1 (mM-1 s-1)
pNPG 1.5 + 0.24 297 + 0.12 1527778 1018518.67
pNPF 1.71 + 0.23 295.7 + 0.13 1521090 889526.31
pNPGal 1.47 + 0.24 117.8 + 0.03 107767.50 73311.2
pNPX 2.37 + 0.21 15.3 + 0.03 78703.70 33208.3
pNPC 1.07 + 0.35 20.95 + 0.19 605967 566324.3
Cellobiose 7.76 + 0.04 0.263 + 0.001 1352.88 174.34
Concentration of TnBglA [e] = 0.00324 µmol.
The Vmax and Km values were determined by plotting the corresponding substrate concentration versus the
initial velocity for each reaction. All values are the average of two experiments done in triplicate.
y = 0.2933x + 0.6547R² = 0.8258
y = 0.2363x + 0.1007R² = 0.9268
y = 0.1052x + 0.0948R² = 0.9045
y = 0.4569x + 0.341R² = 0.9506
y = 0.2054x + 0.0696R² = 0.948
y = 0.3967x + 0.0535R² = 0.9644
0
0.2
0.4
0.6
0.8
1
1.2
1.4
-1 -0.5 0 0.5 1 1.5 2 2.5
1/V
1/[S]
(b) Lineweaver–Burk plot for the hydrolysis of various substrates using TnBglB
pNPG pNPGal pNPC pNPX pNPF Cellobiose
Results
169
Table 4.9 (b): Kinetic parameters of β-glucosidase from T. naphthophila (TnBglB)
Substrates Km (mM) Vmax (mmol mg-1 min-1) kcat (sec-1) kcat Km-1 (mM-1 s-1)
pNPG 0.45 + 0.24 153 + 0.08 1214285.7 2698413
pNPF 2.95 + 0.23 14.37 + 0.05 114047.6 38660
pNPGal 2.35 + 0.24 9.93 + 0.06 78809.5 33536
pNPX 1.34 + 0.21 29.3 + 0.1 232539.7 173537
pNPC 1.11 + 0.35 10.55 + 0.04 83730.2 75433
Cellobiose 7.35 + 0.04 0.185 + 0.002 1469.8 200
Concentration of TnBglB [e] = 0.0021 µmol
The Vmax and Km values were determined by plotting the corresponding substrate concentration versus the
initial velocity for each reaction. All values are the average of two experiments done in triplicate.
4.10.14. Kinetic studies for pNPG hydrolysis at different temperature
Both recombinant TnBglA and TnBglB enzyme were used to find the various Vmax values
at different temperatures (with the increment of 5°C) for pNPG hydrolysis (Table 4.10a
and 4.10b). Maximum Vmax value of TnBglA and TnBglB was observed at 95°C and 85°C,
respectively.
Table 4.10 (a): Kinetic parameters of TnBglA for the hydrolysis of pNPG substrate.
Temp. (K) Km (mM) Vmax (mmol mg-1min-1) kcat (sec-1) kcat Km-1 (mM-1 s-1)
368 (95°C) 1.5 + 0.24 297 + 0.12 1527778 1018518.67
363 (90°C) 1.55 + 0.21 285 + 0.14 1466049 945838.06
358 (85°C) 1.61 + 0.22 254+ 0.11 1306584 811542.86
353 (80°C) 1.02 + 0.24 192+ 0.09 987654 968288.24
Table 4.10 (b): Kinetic parameters of TnBglB for the hydrolysis of pNPG substrate
Temp. (K) Km (mM) Vmax (mmol mg-1min-1) kcat (sec-1) kcat Km-1 (mM-1 s-1)
358 (85°C) 0.45 + 0.24 153 + 0.08 1214285.7 2698413
353 (80°C) 0.24 + 0.21 130 + 0.09 1031746 4298942
348 (75°C) 0.173 + 0.21 108 + 0.04 857143 4954584
343 (70°C) 0.39 + 0.23 102+ 0.06 809524 2075703
Results
170
4.10.15. Thermodynamic of substrate (pNPG) hydrolysis
Thermodynamic study was conducted for the hydrolysis of pNPG substrate, various Vmax
values at different temperatures for pNPG hydrolysis, were applied to construct Arrhenius
plot by taking In Vmax along Y-axis and 1/T along X-axis (Figure 4.50a and 4.50b). The
relationship of temperature-dependent transition state equilibrium of substrate (pNPG)
hydrolysis was explained by Arrhenius approach. Gibb’s free energy (∆G*), Enthalpy
(∆H*) and entropy (∆S*) were determined for the hydrolysis of pNPG by using purified
TnBglA and TnBglB. Activation energy (Ea) for TnBglA and TnBglB was calculated from
the value of slope of Arrhenius plot, which values were 28.74 kJ mol-1 for TnBglA and
27.07 kJ mol-1 for TnBglB. For TnBglA, thermodynamic parameters like ∆H*, ∆G* and
∆S* were calculated by Eyring’s absolute rate equation for 80°C to 95°C, however at
optimal temperature (95°C) of TnBglA as 25.7 kJ mol-1, 47.24 kJ mol-1 and -58.58 J mol-1
K-1, respectively (Table 4.11a). At 85°C optimal temperature of TnBglB, ∆H*, ∆G* and
∆S* values were 24.09 kJ mol-1, 46.55 kJ mol-1 and -62.74 J mol-1 K-1, respectively (Table
4.11b).
y = -3456.8x + 22.033R² = 0.9757
12
12.1
12.2
12.3
12.4
12.5
12.6
12.7
0.0027 0.00272 0.00274 0.00276 0.00278 0.0028 0.00282 0.00284
ln V
max
1/Temperature (1/K)
(a) Arrhenius Plot for pNPG hydrolysis using TnBglA
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171
Figure 4.50: Arrhenius plot of recombinant enzymes from T. naphthophila. (a) Arrhenius plot to calculate
activation energy Ea of recombinant TnBglA for the hydrolysis of pNPG (substrate). (b) Arrhenius plot to
calculate activation energy Ea of recombinant TnBglB for the hydrolysis of pNPG (substrate). Ea= –slope ×
R, where R (gas constant) = 8.314 JK-1 mol-1. Error bars show standard deviation among three observations.
Table 4.11 (a): Thermodynamic parameters of TnBglA for pNP-G hydrolysis. Ea = 28.74 kJ mol-1
Temperature
(K)
Vmax
(mmol mg-1 min-1)
kcat
(sec-1)
ΔH*
(kJ mol-1)
ΔG*
(kJ mol-1)
ΔS*
(J mol-1K-1)
368 (95°C) 297 1527778 25.68 47.24 -58.58
363 (90°C) 285 1466049 25.72 46.66 -57.68
358 (85°C) 254 1306584 25.76 46.31 -57.41
353 (80°C) 192 987654 25.80 46.47 -58.55
Concentration of the enzyme [e] = 0.00324 µmol
y = -3256.1x + 20.998R² = 0.9401
11
11.2
11.4
11.6
11.8
12
12.2
0.00278 0.0028 0.00282 0.00284 0.00286 0.00288 0.0029 0.00292 0.00294
ln V
ma
x
1/Temperature (1/K)
(b) Arrhenius Plot for pNPG hydrolysis using TnBglB
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Table 4.11 (b): Thermodynamic parameters of TnBglB for pNP-G hydrolysis. Ea = 27.07 kJ mol-1
Temperature
(K)
Vmax
(mmol mg-1 min-1)
kcat
(sec-1)
ΔH*
(kJ mol-1)
ΔG*
(kJ mol-1)
ΔS*
(J mol-1K-1)
358 (85°C) 153 1214285.7 24.09 46.55 -62.74
353 (80°C) 130 1031746 24.13 46.31 -62.83
348 (75°C) 108 857143 24.17 46.18 -63.25
343 (70°C) 102 809523.8 24.22 45.65 -62.48
Concentration of the enzyme [e] = 0.0021 µmol
4.10.16. Thermodynamics stability of TnBglA and TnBglB
Thermodynamic parameters for irreversible thermal inactivation of purified recombinant
TnBglA and TnBglB enzyme were calculated. To evaluate the thermal denaturation, the
purified enzyme TnBglA was incubated at temperatures ranging from 97-99°C (in an
increment of 1°C), nearer to the optimal temperature of TnBglA activity and similarly
TnBglB purified enzyme was incubated at temperatures ranging from 94-96°C (in an
increment of 1°C). Pseudo-first-order plots were applied to assess the thermal inactivation
of TnBglA and TnBglB (Figure 4.51a and 4.51b).
Recombinant enzyme TnBglA was found highly stable and showed half-life (t1/2) of 5.2
minutes at 97°C and TnBglB enzyme showed high stability with a half-life (t1/2) of 4.44
minutes at 94°C. Thermodynamic parameters for denaturation of purified TnBglA enzyme
like enthalpy of denaturation (ΔH*D), free energy of denaturation (ΔG*D) and entropy of
denaturation (ΔS*D) at 97°C were found to be 662.04 kJ mol-1, 110.1 kJ mol-1 and 1.491 kJ
mol-1K-1, respectively (Table 4.12a). All these parameters were determined after
calculating the activation energy EaD for irreversible thermal inactivation of TnBglA by
Arrhenius plot (Figure 4.52a). While, thermodynamic parameters for denaturation of
TnBglB enzyme such as enthalpy of denaturation (ΔH*D), free energy of denaturation
(ΔG*D) and entropy of denaturation (ΔS*D) at 94°C were found to be 283.78 kJ mol-1,
108.69 kJ mol-1 and 0.477 kJ mol-1K-1, respectively (Table 4.12b). These parameters were
determined after calculating the activation energy EaD for irreversible thermal inactivation
of TnBglB by Arrhenius plot (Figure 4.52b).
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Figure 4.51: Pseudo-first-order plots for irreversible thermal denaturation of recombinant thermostable
enzymes. (a) Pseudo-first-order plots for irreversible thermal denaturation of TnBglA. The purified TnBglA
enzyme was pre-incubated at 94°C, 97°C, 98°C and 99°C in the absence of pNPG substrate, after appropriate
time interval TnBglA activity was determined in McIlvaine buffer (pH 7.0) at 95°C, before this incubating
enzyme on ice for 30 minutes. (b) Pseudo-first-order plots for irreversible thermal denaturation of TnBglB.
The purified TnBglB enzyme was pre-incubated at 92°C, 94°C, 95°C and 96°C in the absence of pNPG
substrate, after appropriate time interval TnBglB activity was determined in McIlvaine buffer (pH 5.0) at
85°C, before this incubating enzyme on ice for 30 minutes. Error bars show standard deviation among three
observations.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 5 10 15 20 25 30
In R
esid
ua
l a
ctiv
ity
%
Time (min)
(a) Pseudo-first-order plots of TnBglA
94°C 97°C 98°C 99°C
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 5 10 15 20 25 30
In R
esid
ual
act
ivit
y %
Time (min)
(b) Pseudo-first-order plots of TnBglB
92°C 94°C 95°C 96°C
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174
Figure 4.52: Arrhenius plot for irreversible thermal inactivation of purified recombinant enzymes. (a)
Arrhenius plot to calculate activation energy EaD for irreversible thermal inactivation of TnBglA. (b)
Arrhenius plot to calculate activation energy EaD for irreversible thermal inactivation of TnBglB. Error bars
show standard deviation among three observations.
y = -80000x + 214.08R² = 0.9552
-2.5
-2
-1.5
-1
-0.5
00.002676 0.002681 0.002686 0.002691 0.002696 0.002701
lnK
d
1/T
(a) Arrhenius plot for irreversible thermal inactivation of TnBglA
y = -34500x + 92.317R² = 0.9983
-2
-1.9
-1.8
-1.7
-1.6
-1.5
-1.4
-1.3
-1.2
-1.1
-10.002705 0.00271 0.002715 0.00272 0.002725 0.00273 0.002735
lnK
d
1/T
(b) Arrhenius plot for irreversible thermal inactivation of TnBglB
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175
Table 4.12 (a): Thermodynamic parameters for denaturation of recombinant β-1,4- glucosidase
(TnBglA) enzyme. EaD = 665.12 kJ mol-1
Temperature
(K)
kd
(min-1)
Half-life
(min)
ΔH*D
(kJ mol-1)
ΔG*D
(kJ mol-1)
ΔS*D
(kJ mol-1K-1)
370 (97°C) 0.133 5.21 662.04 110.10 1.491
371 (98°C) 0.399 1.74 662.03 107.02 1.496
372 (99°C) 0.656 1.06 662.02 105.78 1.495
Table 4.12 (b): Thermodynamic parameters for denaturation of recombinant β-1,4- glucosidase
(TnBglB) enzyme. EaD = 286.83 kJ mol-1
Temperature
(K)
kd
(min-1)
Half-life
(min)
ΔH*D
(kJ mol-1)
ΔG*D
(kJ mol-1)
ΔS*D
(kJ mol-1K-1)
367 (94°C) 0.156 4.44 283.78 108.69 0.477
368 (95°C) 0.215 3.22 283.83 108.02 0.478
369 (96°C) 0.319 2.24 283.76 107.20 0.479
4.11. Structure Analysis of TnBglA and TnBglB
Secondary and three-dimensional (3-D) structures of TnBglA and TnBglB were analyzed
using various online software. In the following section secondary and 3-D structures were
discussed separately for both proteins.
4.11.1.1. Secondary structure of TnBglA
All details related to the secondary structure of TnBglA was retrieved after submitting the
structure in PDB-format to the PDBsum server, the α-helices, β-sheets and random coils
were clearly evident in the secondary structure of TnBglA (Figure 4.53a). According to
the secondary structure of TnBglA, a chain had 446 amino acid residues, in which 21
helices, 2 sheets, 5 gamma turns, 27 beta turns, 11 strands, 2 beta hairpins, 6 beta bulges,
34 helix-helix interactions and 6 beta-alpha-beta units were present. Majority amino acid
residues were involved in the formation of β-turns and α-helices. The amino acid residues
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176
in helix were buried (hydrophobic) therefore, not exposed to the surrounding environment
(aqueous).
Figure 4.53 (a): Secondary structure of TnBglA. Predominance of strands and helices along with 2 beta-
hairpins.
4.11.1.2. Secondary structure of TnBglB
The detail secondary structure of TnBglB was retrieved after submitting the structure in
PDB-format to the PDBsum server, the α-helices, β-sheets and random coils were clearly
evident in the secondary structure of TnBglB, as shown in figure 4.53b. According to the
secondary structure of TnBglB, 721 amino acid residues were arranged in 29 helices, 6
sheets, 10 gamma turns, 58 beta turns, 26 strands, 6 beta hairpins, 7 beta bulges, 35 helix-
helix interactions and 5 beta-alpha-beta units. The amino acid residues in helix were buried
(hydrophobic) therefore, not exposed to the surrounding environment (aqueous).
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177
Figure 4.53 (b): Secondary structure of TnBglB. Predominance of strands and helices along with 6 beta-
hairpins.
4.11.2.1. Procheck Analysis for TnBglA
The statistics study of Ramachandran plot of TnBglA contained 446 amino acid residues
displayed in the figure 4.54a, which showed the presence of 92.5% amino acid residues in
most favorable regions, 6.4% residues present in additional allowed regions with only one
amino acid in disallowed region (Table 4.13a). All results related to the TnBglA structure
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178
is based on 118 structures analysis of resolution of 2.0 Aͦ (Angstroms) approximately and
R-factor no greater than 20%. A model having good quality, would be estimated to have
more than 90% amino acid residues in the most favoured regions.
Figure 4.54 (a): Ramachandran Plot for TnBglA on Procheck using PDBsum
Table 4.13 (a): Statistics of Ramachandran Plot. Results computed after Procheck analysis of
TnBglA protein structure using PDBsum.
Residues No. of
Residues
Frequency of amino acid residues in
Ramachandran plot (%)
In most favoured regions 359 92.5
In additional allowed regions 25 6.4
In generously allowed regions 3 0.8
In disallowed regions 1 0.3
No. of non-glycine and non-proline 388 100
No. of End amino acid 02 -
No. of glycine 35 -
No. of proline 21 -
Total No. of amino acid 446 -
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179
4.11.2.2. Procheck Analysis for TnBglB
Ramachandran plot statistics study of TnBglB that consisted of 721 amino acid residues
showed the presence of 92.3% amino acid residues in most favorable regions, in additional
allowed regions 6.6% residues and in disallowed region 0.3% amino acids residues present
(Table 4.13b). Triangles as shown in figure 4.54b displayed the presence of 60 glycine
residues, and total 37 proline residues were present in the structure. All results related to
the TnBglB structure is based on 118 structures analysis of resolution of 2.0 Aͦ (Angstroms)
approximately and R-factor no greater than 20%. A model having good quality, would be
estimated to have more than 90% amino acid residues in the most favoured regions.
Figure 4.54 (b): Ramachandran Plot for TnBglB on Procheck using PDBsum
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180
Table 4.13 (b): Statistics of Ramachandran Plot. Results computed after Procheck analysis of
TnBglB protein structure using PDBsum.
Residues No. of
Residues
Frequency of amino acid residues in
Ramachandran plot (%)
In most favoured regions 575 92.3
In additional allowed regions 41 6.6
In generously allowed regions 5 0.8
In disallowed regions 2 0.3
No. of non-glycine and non-proline 623 100
No. of End amino acid 01 -
No. of glycine 60 -
No. of proline 37 -
Total No. of amino acid 721 -
4.11.3.1. Three Dimensional Structure of TnBglA protein
ExPASy-proteomics (GENO-3-D) explained that TnBglA has (α/β)8 triose phosphate
isomerase (TIM) barrel scaffold, which is one of the key feature of GH family 1 enzymes
especially β-glucosidases, are connected through a chain and gives stability to the protein
structure. The 3-D structure of β-glucosidase (TnBglA) represents an interesting pattern of
conserved structural elements in the way that the outer part of the structural fold contains
alpha helices while inner part contains almost all parallel beta sheets forming the catalytic
cleft. In 3-D structure, glutamic acids (Glu166, Glu351 and Glu405) residues were present in
a pocket shaped structure with hydrophobic amino acid residues at the entrance point
leading to the active site pocket (Figure 4.55a). According to the 3-D structure, TnBglA is
a single domain protein, and has 42% helix (H), 10% β-sheet (E) and 47% loop (C). The
quality of 3-D structure was determined by using RaptorX server, and computed the p-
value, uGDT, SeqID and score after submitting amino acid sequence of TnBglA. Model
structure got 519 score, relative quality of a structure was estimated by p-value, TnBglA
computed small 2.83e-18 value, which showed that structure had higher quality. The value
of uSeqID was higher 430(96) which explained that predicted structure folds were correct.
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181
uGDT (unnormalized Global Distance Test) measured the absolute model quality, TnBglA
model retrieved a high 492(110) value, showed that model was fairly good. TnBglA model
displayed solvent accessibility (ACC) values of 46% buried (B), 28% medium (M), and
24% exposed (E).
Figure 4.55 (a): Three-dimensional (3-D) model structure of TnBglA with glutamic acid (Glu) residues
(Glu351 and Glu405) as catalytic nucleophile as well as catalytic proton donor (Glu166). The arrows in the center
shows the catalytic cavity for substrate binding covered by parallel β sheets. The structure was predicted
using an automatic protein modeling sever, GENO-3-D, and results analyzed by PyMOL (molecular system
for visualization and graphics).
4.11.3.1.1. Catalytic Triad of TnBglA
Catalytic triad of TnBglA comprise on three well-conserved catalytic residues which are
Glu166, Glu351 and Glu405, the distance between proton donor catalytic residue Glu166 and
nucleophile active catalytic residue Glu351 is 4.7 Aͦ and the distance between proton donor
Glu166 and catalytic nucleophile Glu405 is 10.1 Aͦ, all the values calculated by using PyMOL
molecular graphics system. Although the distance between Glu351 and Glu405 is 8.6 Aͦ as
shown in figure 4.55b.
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182
Figure 4.55 (b): Catalytic triad of TnBglA, distance between Glu166 act as proton donor and nucleophile
active residues Glu351 and Glu405
4.11.3.2. Three Dimensional Structure of TnBglB protein
ExPASy-proteomics (GENO-3-D) explained that TnBglB has three-domain structure,
composed of 721 amino acid residues that are arranged in three domains. These domains
are connected by two linkers (307-320 and 537-599 residues), domain-I (1-306 residues)
has an (α/β)8 barrel structure similar to a TIM barrel, domain-II (321-536 residues) has
five-stranded α/β sandwich (α/β)5, both domains are important because have a direct and
crucial role for the active-site organization. Domain-III (600-721 residues) has a
fibronectin type III (FnIII) fold whose functions is still not clear (unknown) (Figure 4.55c).
All domains of 3-D predicted structure of TnBglB (β-glucosidase) are well organized in
which active site present at the interface of domain-I and II forming the catalytic pocket.
Domain-I has an essential catalytic residue aspartic acid (Asp242) which act as nucleophile
while domain-II contains a (α/β)5 sandwich fold and has an acid/base residue glutamic
acids (Glu458) (Figure 4.55c). The 3-D structure of TnBglB composed of 25% helix (H),
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183
19% β-sheet (E) and 55% loop (C). The quality of 3-D structure was determined by using
RaptorX server, and computed parameters such as p-value, uGDT, SeqID and score after
submitting amino acid sequence of TnBglB. Model structure got 729 score, relative quality
of a structure was estimated by p-value, TnBglB computed small 7.40e-22 value, which
showed that structure had high quality. The value of uSeqID was higher 634(88), which
elucidated that predicted structure folds were correct. uGDT (unnormalized Global
Distance Test) estimated the absolute model quality, TnBglB model retrieved a high
629(87) value, showed that model was fairly good. TnBglB model displayed solvent
accessibility (ACC) values of 41% buried (B), 28% medium (M), and 30% exposed (E).
Figure 4.55 (c): 3-D model structure of TnBglB colored by domain, along with catalytic residues. Domain-
I, red color (1-306 residues); Domain-II, cyan color (321-536 residues); Domain-III, orange color (600-721).
The linker (307-320 residues) between domain-I and domain-II is shown in green; the linker (537-599
residues) between domain-II and domain-III is represented in yellow. Catalytic active site present at the
interface of domain-I and domain-II, in a pocket like structure in which aspartic acid residues (Asp242) act as
nucleophile and glutamic acid residue (Glu458) act as catalytic proton donor. The structure was predicted
using an automatic protein modeling sever, GENO-3-D, and conserved domains or coding sequence within
a protein studied by using NCBI, results analyzed by PyMOL (molecular system graphic system).
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184
4.11.3.2.1. Catalytic residues of TnBglB
TnBglB consist of well-conserved two catalytic residues Asp242 and Glu458, distance
between the proton donor catalytic residue Glu458 and nucleophile active catalytic residue
Asp242 is 5.8 Aͦ (Figure 4.55d). The distance between catalytic amino acid residues
measured by PyMOL molecular system for visualization and graphics.
Figure 4.55 (d): Catalytic residues of TnBglB, distance between Glu458 act as proton donor and nucleophile
active residues Asp242.
4.11.4.1. Docking Studies of TnBglA
The docking studies of enzyme-substrate interaction by PatchDock generated result give
more than 100 possible solutions of docking. All these solutions were evaluated for their
accuracy by Procheck and Ramachandran plots on PDBSum which revealed their stability
(Laskowski, 2009). Additionally, the analysis of enzyme-substrate complexes were also
performed by studying the ligplot (prediction tool for H-bonds) which showed the possible
non-covalent interaction detail of enzyme with the substrates. One of the best complexes
amongst the first 100, for each substrate-TnBglA docking results, having at least more than
80% residues in favored and allowed regions of Ramachandran plots and also having two
conserved glutamic acid (Glu) residues, one in N-terminal site and the other in middle or
C-terminal site was selected for each of the enzyme-substrate complex. The best docking
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185
complex of TnBglA with cellobiose substrate was used to analyze the enzyme substrate
interaction (Figure 4.56a). In this reaction complex Tyr295, Asn165, Glu166, Glu351 and Glu405
active-site residues are involved in making hydrogen bonds with ligand, suggested these
amino acid residues may play a crucial role in the substrate hydrolysis process. The
surrounding amino acids Trp324, Phe414, Trp398, Trp122, Try406, His180, Glu408 and Met322
provide a hydrophobic environment to improve reactivity in the interaction. However,
pNPG substrate molecules in the complex system were coordinately linked with the Met322,
Tyr295, Glu351 and Glu405 active-site residues (Figure 4.56b). This interacting system
surrounded by Glu166, Trp398, Trp324, Trp122, Trp406, His180 and Glu408 involved in
hydrophobicity to enhance reactivity and thermophily. All finest complexes for each
substrate revealed that at least two glutamic acidic (Glu) amino acids present in the active
cleft or catalytic site (Figure 4.56c). One important fact that is evident from all these
docking models is that in each docking models there were at least two or at highest three
glutamic acid residues (Glu166, Glu351 and Glu405) were conserved; and these residues were
observed in close contact to the substrate to perform the hydrolytic function.
(a)
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186
(b)
Figure 4.56: Ligplot output for H-bond interactions of the amino acid residues of TnBglA (a) involved in
binding with cellobiose (b) with the p-phenyl sugars as substrates.
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187
Figure 4.56 (c): Surface view of 3-D structure of TnBglA, the circle (in red colour) represents the catalytic
cleft for substrate binding.
4.11.4.2. Docking Studies of TnBglB
The docking studies of TnBglB enzyme-substrate interaction by PatchDock generated
result give more than 100 possible solutions of docking. All these solutions were evaluated
for their accuracy by Procheck and Ramachandran plots on PDBSum which revealed their
stability (Laskowski, 2009). Additionally, the analysis of enzyme-substrate complexes
were also performed by studying the ligplot (prediction tool for H-bonds) which showed
the possible non-covalent interaction detail of enzyme with the substrates. One of the best
complexes amongst the first 100, for each substrate-TnBglB docking results, having at least
more than 80% residues in favored regions of Ramachandran plots, and having one
conserved nucleophile aspartic acid (Asp242) residue in domain-I and one conserved
catalytic proton donor glutamic acid (Glu458) residue in domain-II, was selected for each
of the enzyme-substrate complex. Both substrates locked in a tight network of hydrogen
bonds in which active-site residues Asp58, Arg64, Glu458 and Asp242 involved. The side
chain of Ser370 and Try243 may form weak hydrogen bond with substrates (Figure 4.57a and
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188
4.57b). All best complexes for each substrate demonstrated that two catalytic residues
present in the active cleft or catalytic pocket (Figure 4.57c). Moreover, evident from all
these docking models, it is predicted that in each docking models there were at least two
catalytic residues Asp242 and Glu458 were conserved; and these residues were observed in
close contact to the substrate to perform the hydrolytic function.
(a)
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189
(b)
Figure 4.57: Ligplot output for H-bond interactions of the amino acid residues of TnBglB (a) involved in
binding with cellobiose (b) with the p-phenyl sugars as substrates.
Figure 4.57 (c): Surface view of 3-D structure of TnBglB, the circle (in red colour) shows the catalytic cleft
for substrate binding.
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190
4.12. Enhanced Production of Recombinant TnBglA and TnBglB
After purification and characterization, the recombinant TnBglA and TnBglB enzymes
production was further enhanced in a mesophilic E. coli BL21 CodonPlus DE3-(RIPL)
host through various cultivation and induction strategies. To enhance the production of
engineered host cells and over-expression of recombinant enzymes, different inducing
media were used which induced individually with various concentration of IPTG and
lactose. Culture conditions and other parameters were also optimized to achieve maximum
yield of heterologous proteins.
4.12.1. Production and Expression of recombinant enzymes
Ten different inducing media including LB, LB+, ZB, 4×ZB, ZBM, ZYB, 3×ZYB,
ZYBM9, 3×ZYBM9, and M9 with some modifications in their composition were used,
which induced individually either with IPTG (0.1-1.0 mM) or lactose (10-300 mM), and
studied the effect of each medium on both TnBglA and TnBglB expression and activity.
Therefore, 1% of all respective seed cultures of both cloned host strains harboring pET-
21a–TnbglA and pET-21a–TnbglB plasmids were used to inoculate their respective
sterilized media containing both antibiotics. Cultivation conditions and parameters
including pre-induction optical density, agitation, concentration of both inducer,
temperature and time of induction, were again optimized in all modified and complex
inducing media to achieve the maximum yield of heterologous proteins. All experimental
results (from various media) are the means of triplicate experiments. Enzymes percentage
(%) were detected by densitometric scanning of Coomassie stained gel (Syngene, Gene
Tools, UK).
4.12.2. Screening of optimal inducing media for TnBglA and TnBglB
The host cells harboring pET-21a–TnbglA and pET-21a–TnbglB plasmid, were grown in
ten different media (LB, LB+, ZB, ZBM, ZYB, 3×ZYB, 4×ZB, M9, ZYBM9, and
3×ZYBM9) supplemented with ampicillin (100 µg mL-1) and chloramphenicol (50 µg mL-
1) induced individually with IPTG and lactose to screen the best medium for both TnBglA
and TnBglB proteins production and expression in terms of activity. Maximum
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191
extracellular TnBglA production and activity was found in 4×ZB modified medium
induced with IPTG (0.5 mM) after 72 h induction at 22°C, seemed to be due to greater
recombinant cell density and dry cell weight. However, lowest culture density and activity
was observed in M9 medium (Figure 4.58a). Similarly, optimal production and TnBglB
extracellular enzyme activity found in 4×ZB modified medium induced with 0.5 mM IPTG
(Figure 4.58b), seemed to be due to greater recombinant cell density and dry cell weight.
Figure 4.58: Effect of media on recombinant enzymes activity induced with 0.5 mM IPTG (a): Effect of
inducing media on recombinant TnBglA activity induced with 0.5 mM IPTG after 72 h incubation at 22°C,
optimal activity was observed in 4×ZB medium and least in M9 medium. (b): Effect of inducing media on
recombinant TnBglB activity after induction with 0.5 mM IPTG at 22°C for 72 h, optimal activity was
observed in 4×ZB medium and least in M9 medium. Y-error bars specify the standard deviation (±SD) of
parallel triplicate experimental reactions. Probability of all the variables is < 5% significant level which entail
that elected variables are significant at 95% confidence interval.
0
20
40
60
80
100
120
LB
LB
+
ZB
4Z
B
ZY
B
3Z
YB
ZY
BM
9
3Z
YB
M9
ZB
M
M9
Rel
ativ
e a
ctiv
ity
%
Media
(a) Optimization of Media Induced with 0.5 mM IPTG for TnBglA
24 h 48 h 72 h
0
20
40
60
80
100
120
LB
LB
+
ZB
4ZB
ZY
B
3Z
YB
ZY
BM
9
3ZY
BM
9
ZB
M
M9
Rel
ativ
e ac
tivi
ty %
Media
(b) Optimization of Media Induced with 0.5 mM IPTG for TnBglB
24 h 48 h 72 h
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192
Induced with lactose (150 mM), peak TnBglA activity and cultural density were also
observed in 4×ZB after 72 h as compared with other media (Figure 4.59a). Similarly
maximum TnBglB activity was obtained with 150 mM lactose in 4×ZB medium after 72 h
incubation at optimal conditions as compared with other inducing media (Figure 4.59b).
Figure 4.59: Effect of media on recombinant enzymes activity induced with 150 mM lactose (a): Effect of
inducing media on recombinant TnBglA activity induced with 150 mM lactose after 72 h incubation at 22°C,
optimal activity was observed in 4×ZB medium and least in M9 medium. (b): Effect of inducing media on
TnBglB activity after induction with 150 mM lactose at 22°C for 72 h, optimal activity was observed in 4×ZB
medium and least in M9 medium. Y-error bars specify the standard deviation (±SD) of parallel triplicate
experimental reactions. Probability of all the variables is < 5% significant level which entail that elected
variables are significant at 95% confidence level.
0
20
40
60
80
100
120L
B
LB
+
ZB
4ZB
ZY
B
3Z
YB
ZY
BM
9
3ZY
BM
9
ZB
M
M9
Rel
ati
ve
act
ivit
y %
Media
(a) Optimization of Media Induced with 150 mM Lactose for TnBglA
24 h 48 h 72 h
0
20
40
60
80
100
120
LB
LB
+
ZB
4Z
B
ZY
B
3ZY
B
ZY
BM
9
3ZY
BM
9
ZB
M
M9
Rel
ativ
e ac
tivi
ty %
Media
(b) Optimization of Media Induced with 150 mM Lactose for TnBglB
24 h 48 h 72 h
Results
193
4.12.2.1. Expression of TnBglA in various Media induced with IPTG
The effect of various cultivation media induced with 0.5 mM IPTG, on the expression of
TnBglA was examined by comparing the heterologous protein profiles of recombinant
E.coli BL21 CodonPlus (DE3) cultivated in LB, ZB, ZBM, ZYB, 3×ZYB, 4×ZB, M9,
ZYBM9, and 3×ZYBM9 inducing media (Figure 4.60a and 4.60b).
Figure 4.60: SDS-PAGE analysis of recombinant TnBglA in E. coli BL 21 grown in various 0.5 mM IPTG
inducing media (a) Lane M, Protein marker; Lane C, Crude control supernatant harboring pET-21a (+)
without induction; Lane 1, Crude supernatant proteins of cells cultivated in LB; Lane 2, Crude heat treated
LB; Lane 3, supernatant cultivated in ZB; Lane 4, heat treated supernatant of ZB; Lane 5, supernatant protein
of ZBM; Lane 6, heat treated supernatant of ZBM; Lane 7, Supernatant of 4×ZB; Lane 8, heat treated
supernatant of 4×ZB. (b) Lane C, Crude control supernatant; Lane 1, supernatant protein of ZYB; Lane 2,
heat treated supernatant of ZYB; Lane 3, supernatant of 3×ZYB; Lane 4, heat treated supernatant of 3×ZYB;
Lane 5, supernatant of ZYBM9; Lane 6, heat treated supernatant of ZYBM9; Lane 7, supernatant of
3×ZYBM9; Lane 8, heat treated supernatant of 3×ZYBM9.
kDa
100
TnBglA 51.5 kDa
75
50
35
25
(a)
TnBglA 51.5 kDa
(b)
Results
194
4.12.2.2. Expression of TnBglB in various Media induced with IPTG
Various cultivation inducing media LB, ZB, ZBM, ZYB, 3×ZYB, 4×ZB, M9, ZYBM9,
and 3×ZYBM9 were used to analyzed the effect of growth media induced with 0.5 mM
IPTG on the expression of TnBglB by comparing the heterologous protein profiles of
recombinant E.coli BL21 CodonPlus (DE3) cultivated in all these inducing media (Figure
4.61a and 4.61b).
Figure 4.61: SDS-PAGE analysis of TnBglB in E. coli grown in various IPTG inducing media (a) Lane M,
Protein marker; Lane 1, Control supernatant harboring pET-21a (+) without induction; Lane 2, Crude
supernatant cells proteins cultivated in ZYB; Lane 3, Crude heat treated ZYB; Lane 4, supernatant cultivated
in ZB; Lane 5, heat treated supernatant of ZB; Lane 6, supernatant protein of ZBM; Lane 7, heat treated
supernatant of ZBM; Lane 8, Supernatant of 3×ZYBM9; Lane 8, heat treated supernatant of 3×ZYBM9. (b)
Lane M, Protein marker; Lane 1, Crude control supernatant; Lane 2, supernatant of 4×ZB; Lane 3, heat
treated supernatant of 4×ZB; Lane 4, supernatant of LB; Lane 5, heat treated supernatant of LB; Lane 6,
supernatant of 3×ZYB; Lane 7, heat treated supernatant of 3×ZYB; Lane 8, supernatant of ZYBM9; Lane 9,
heat treated supernatant of ZYBM9.
TnBglB
81 kDa
(a)
75
100 kDa
50
35
25
(b)
50
75
100 kDa
35
25
TnBglB
81 kDa
Results
195
4.12.2.3. Expression of TnBglA in various Media induced with Lactose
The effect of various cultivation media induced with 150 mM lactose, on the expression of
TnBglA was studied by comparing the heterologous protein profiles of E. coli BL21
CodonPlus (DE3) cultivated in LB, ZB, ZBM, ZYB, 3×ZYB, 4×ZB, M9, ZYBM9, and
3×ZYBM9 inducing media (Figure 4.62).
Figure 4.62: SDS-PAGE analysis of TnBglA (51.5 kDa) in E. coli grown in various lactose inducing media.
Lane M, Protein marker; Lane 1, Control supernatant harboring pET-21a (+) with lactose induction; Lane 2,
Control supernatant harboring pET-21a (+) without induction; Lane 3, Crude supernatant cells proteins
cultivated in ZBM; Lane 4, supernatant cultivated in 3×ZYB; Lane 5, supernatant of ZYB; Lane 6,
supernatant of ZB; Lane 7, supernatant of LB; Lane 8, heat treated supernatant of LB; Lane 9, supernatant of
ZYBM9; Lane 10, heat treated supernatant of ZYBM9; Lane 11, supernatant of 4×ZB; Lane 12, heat treated
supernatant of 4×ZB; Lane 13, supernatant of 3×ZYBM9; Lane 14, heat treated supernatant of 3×ZYBM9.
4.12.2.4. Expression of TnBglB in various Media induced with Lactose
The influence of various cultivation media LB, ZB, ZBM, ZYB, 3×ZYB, 4×ZB, M9,
ZYBM9, and 3×ZYBM9 induced with 150 mM lactose, on the expression of TnBglB was
observed by comparing the heterologous protein profiles of E.coli BL21 CodonPlus (DE3)
cultivated in various inducing media (Figure 4.63).
Results
196
Figure 4.63: SDS-PAGE analysis of TnBglB (81 kDa) in E. coli grown in various lactose inducing media.
Lane M, Protein marker; Lane C, Control supernatant harboring pET-21a (+) without induction; Lane 1,
Crude supernatant cells proteins cultivated in ZBM; Lane 2, heat treated supernatant in ZBM; Lane 3,
supernatant of 3×ZYB; Lane 4, heat treated supernatant of 3×ZYB; Lane 5, supernatant of 3×ZYBM9; Lane
6, heat treated supernatant of 3×ZYBM9; Lane 7, heat treated supernatant of ZB; Lane 8, supernatant of ZB;
Lane 9, supernatant cultivated in 4×ZB; Lane 10, supernatant of 4×ZB; Lane 11, supernatant of LB; Lane
12, heat treated supernatant of LB; Lane 13, supernatant of ZYB; Lane 14, heat treated supernatant of ZYB;
Lane 15, supernatant of ZYBM9; Lane 16, heat treated supernatant of ZYBM9.
4.12.3. Pre-induction optimal cell density
Optimum culture density of both recombinant E. coli BL21 CodonPlus cultures at the time
of induction were determined by using all complex and defined media supplied with both
antibiotics. Recombinant genes was induced at different optical density of culture
(OD600nm) 0.5 to 0.9, and the optimal results were observed when culture was induced at
0.6 optical density in all inducing media, this result was same as described in LB medium.
4.12.4. Effect of Heat Shock
Direct induction of recombinant bacterial host cells at optimal density resulted in
comparatively lower enzymes expression in term of activity as compared to when bacterial
culture was given a heat shock for 60 minutes in a shaking water bath at 42°C with 200 rev
min-1 and after heat shock the culture was induced either with specific concentration of
IPTG or lactose. TnBglA and TnBglB activity were also less when heat shock (42°C) was
kDa
225150100 75 50
35 25
Results
197
given after induction. Similar results were obtained for all inducing media including LB
medium.
4.12.5. Optimal induction temperature, agitation speed
Immediately after heat shock and induction, the bacterial culture was incubated at various
temperatures ranging from 16-40°C in a shaking incubator with different agitation speed
(50-250 rev min-1). Samples were withdrawn periodically from each incubated flasks for
the analysis of both recombinant enzymes activity. The optimal temperature for incubation
was 22°C with an agitation speed of 200 rev min-1, in all studied media. The results were
same as in case of LB medium.
4.12.6. Optimal incubation time
After induction, the incubation time period at which both recombinant culture showed
maximum growth, expression and activity were optimized in all inducing media.
Recombinant enzymes activities and cell growth were monitored up to 96 hours after
regular intervals. TnBglA and TnBglB activity increased up to 72 hours post induction
beyond which further increment in enzymes activities were not detected in any of the media
under study. Therefore, maximum activity was observed after 72 hours of induction at 22°C
in all inducing media (all results were similar with the LB medium finding).
4.12.7. Optimal IPTG concentration
To over-express both heterologous proteins (TnBglA and TnBglB), induced the cloned
culture cells with different concentration of IPTG (0.1-1.0 mM), which revealed that
optimal level of recombinant enzymes activity, expression and bacterial cell density was
observed with 0.5 mM concentration, beyond 0.5 mM, there was no increase in
recombinant genes expression in all used inducing media during fermentation. Moreover,
further increment in IPTG concentration (0.9 mM) retarded the growth of recombinant E.
coli BL21 cells as well as lowered the TnBglA and TnBglB activities (Figure 4.64a and
4.64b). Using IPTG, high cell density (OD600nm) of TnBglA and TnBglB were 16.64 with
9.88 g DCW L-1 and 16.91 with 10.11 g DCW L-1 attained in 4×ZB, respectively which
considerably higher than that observe in LB medium (Table 4.14a and 4.15a).
Results
198
Figure 4.64: Effect of IPTG on recombinant enzymes expression in 4×ZB medium (a) Optimal TnBglA
activity and total cell protein was observed at 0.5 mM IPTG after 72 h incubation at 22°C temperature. (b)
TnBglB showed optimal activity and total cell protein at 0.5 mM IPTG after 72 h incubation at 22°C
temperature, both are defined as 100% activity to calculate the relative activity. Y-error bars show the
standard deviation (±SD) of parallel three experimental reactions, which significant at probability 5%.
4.12.8. Optimal lactose concentration (an alternative inducer)
Recombinant culture production and enzymes expression were studied using lactose a
natural inducer of T7 lac promoter, to scrutinize the effect of lactose various concentrations
(50-300 mM) were used to induce the both cloned TnBglA and TnBglB cultures.
Recombinant TnBglA and TnBglB activities were enhanced gradually with the increment
of lactose concentration up to 150 mM, whereas more increase in concentration appeared
0
1
2
3
4
0
20
40
60
80
100
120
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Pro
tein
Co
nc.
(m
g m
L-1
)
Rel
ati
ve
act
ivit
y %
IPTG Concentration (mM)
(a) Optimization of IPTG conc. for TnBglA
IPTG Conc. Protein Conc.
0
1
2
3
4
0
20
40
60
80
100
120
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Pro
tein
Con
c. (
mg
mL
-1)
Rel
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e ac
tivi
ty %
IPTG Concentration (mM)
(b) Optimization of IPTG conc. for TnBglB
IPTG Conc. Protein Conc.
Results
199
to be a slightly negative effect or no more increase in activity and expression of enzymes,
approximately in all inducing media (Figure 4.65a and 4.65b). Induction with 150 mM
lactose enhanced the extracellular expression and activity of TnBglA as compared to IPTG
(0.5 mM) induction. While, induction with 150 mM lactose exhibited a same level of
expression in term of activity of TnBglB as in the case of 0.5 mM IPTG.
Figure 4.65: Effect of lactose on the expression of recombinant TnBglA and TnBglB enzymes in 4×ZB
medium (a) Optimal TnBglA activity (expression) and total cell protein was observed at 150 mM lactose
conc. after 72 h incubation at 22°C temperature. (b) TnBglB showed optimal activity and total cell protein at
150 mM lactose conc. after 72 h incubation at 22°C temperature, both are defined as 100% activity to
calculate the relative activity. Y-error bars specify the standard deviation (±SD) of parallel triplicate
experimental reactions. Probability of all the variables is < 5% significance level which entail that elected
variables are significant at 95% confidence interval.
00.511.522.533.544.5
0
20
40
60
80
100
120
50 100 150 200 250 300
Pro
tein
Con
c. (
mg
mL
-1)
Rel
ativ
e a
ctiv
ity
%
Lactose Concentration (mM)
(a) Optimization of Lactose conc. for TnBglA
Lactose Conc. Protein Conc.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0
20
40
60
80
100
120
50 100 150 200 250 300
Pro
tein
Con
c. (
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mL
-1)
Rel
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tivi
ty %
Lactose Concentration (mM)
(b) Optimization of Lactose conc. for TnBglB
Lactose Conc. Protein Conc.
Results
200
4.12.9. Comparison of IPTG and Lactose as inducer
Lactose has been found as effective as IPTG for inducing heterologous TnBglA and
TnBglB proteins. Lactose induction, as compared to IPTG, supported exponential growth
of E. coli BL21 CodonPlus (DE3)-RIPL harboring pET-21a–TnbglA and pET-21a–TnbglB
(expression plasmids) to relatively higher cell densities in all inducing media under study
except M9 medium. Maximum optical density (OD600nm) and DCW attained with lactose
inducing medium 18.96 (TnBglA) and 18.66 (TnBglB) which was considerably higher as
compared with 0.5 mM IPTG induction. The culture growth never attained such high-level
optical density as with lactose, even if induced with high level of IPTG concentration,
greater than 0.9 mM IPTG retarded the bacterial growth. Highest cell density (OD600nm) of
E. coli BL21 harboring pET-21a–TnbglA obtained after IPTG induction (0.5 mM), was
16.64 with 9.88 g DCW L-1 in 4×ZB medium, which was noticeably greater than all other
inducing media (Table 4.14a). While, maximum cell density (OD600nm) of recombinant host
cell obtained after lactose induction (150 mM), which was 18.96 with 11.30 g DCW L-1 in
4×ZB medium, significantly greater than all other inducing media (Table 4.14b). TnBglA
enzyme activity was 3.8 fold enhanced in 4×ZB medium when recombinant culture was
induced with 150 mM lactose, and 3.74 fold enhanced in 4×ZB medium with 0.5 mM IPTG
induction as compared to the LB medium.
Optimal cell density (OD600nm) of E. coli BL21 (DE3) harboring pET-21a–TnbglB obtained
after IPTG induction (0.5 mM), was 16.91 with 10.11 g DCW L-1 in 4×ZB medium that
was significantly greater than all other inducing media (Table 4.15a). However, induction
with lactose (150 mM) enhanced the cell density and dry cell weight were 18.66 and 11.08
g DCW L-1 in 4×ZB medium, respectively, which was considerably greater than all other
inducing media used for this recombinant strain (Table 4.15b). In case of TnBglB, lactose
induction (150 mM) exhibited a same level of expression in term of extracellular activity
as in the case of 0.5 mM IPTG; and TnBglB activity was only 0.096 and 0.085 fold
enhanced in 4×ZB medium with lactose and IPTG inducers, respectively as compared to
the LB medium.
Results
201
Table 4.14 (a): Recombinant TnBglA production, induced with 0.5 mM IPTG when cultivated in
different neutral pH media, at 22°C after 72 h induction in a shaker with 200 rev min-1.
Media
0.5 mM IPTG Inducer
Max.
OD600nm
DCW*
(g L-1)
a TCP*
(mg L-1)
Relative activity
(%)
b TnBglA % of
total protein
LB 08.11 04.81 2130 21 22
LB+ 08.19 04.91 2125 19 22
ZB 08.92 05.34 1937 44 22
4×ZB 16.64 09.88 2680 100 30
ZYB 10.16 06.04 2161 61 26
3×ZYB 13.09 07.71 2346 68 26
ZYBM9 08.40 04.98 1917 41 22
3×ZYBM9 09.08 05.38 1987 47 23
ZBM 06.77 04.01 1821 29 21
M9 03.11 01.81 0663 05 11
Table 4.14 (b): Recombinant TnBglA production, induced with 150 mM lactose when cultivated in
different neutral pH media, at 22°C after 72 h induction in a shaker with 200 rev min-1.
Media
150 mM Lactose Inducer
Max.
OD600nm
DCW*
(g L-1)
a TCP*
(mg L-1)
Relative activity
(%)
b TnBglA % of
total protein
LB 09.86 05.86 2116 20 22
LB+ 09.79 05.78 2011 22 22
ZB 10.76 06.39 2086 51 24
4×ZB 18.96 11.30 2865 100 34
ZYB 13.21 07.84 2344 61 32
3×ZYB 14.83 08.81 2463 79 32
ZYBM9 10.45 06.19 2115 47 28
3×ZYBM9 11.16 06.61 2168 63 33
ZBM 10.47 06.20 2294 44 22
M9 03.22 01.88 0756 07 17
*Dry Cell Weight (DCW); *Total Cell Protein (TCP). a Total call protein was estimated by Bradford method.
b Expression levels were computed by densitometric scanning of total cell protein resolved on 12% SDS-PAGE.
Results
202
Table 4.15 (a): Recombinant TnBglB production, induced with 0.5 mM IPTG when cultivated in
different neutral pH media, at 22°C after 72 h induction in a shaker with 200 rev min-1.
Media
0.5 mM IPTG Inducer
Max.
OD600nm
DCW*
(g L-1)
a TCP*
(mg L-1)
Relative activity
(%)
b TnBglB % of
total protein
LB 08.41 05.04 2184 91 28
LB+ 08.29 04.97 2178 82 28
ZB 09.82 05.81 1817 51 18
4×ZB 16.91 10.11 2708 100 30
ZYB 08.16 04.87 2161 69 20
3×ZYB 09.09 05.41 2253 73 21
ZYBM9 11.40 06.84 2417 94 28
3×ZYBM9 07.51 04.48 1754 42 16
ZBM 06.47 03.88 1633 31 16
M9 02.71 01.52 0616 08 09
Table 4.15 (b): Recombinant TnBglB production, induced with 150 mM lactose when cultivated in
different neutral pH media, at 22°C after 72 h induction in a shaker with 200 rev min-1.
Media
150 mM Lactose Inducer
Max.
OD600nm
DCW*
(g L-1)
a TCP*
(mg L-1)
Relative activity
(%)
b TnBglB % of
total protein
LB 09.82 05.78 2183 92 28
LB+ 09.59 05.69 2103 85 28
ZB 10.66 06.29 1935 49 16
4×ZB 18.66 11.08 2817 100 30
ZYB 10.09 06.02 2444 69 20
3×ZYB 11.23 06.66 2574 73 20
ZYBM9 12.37 07.34 2621 86 22
3×ZYBM9 08.26 04.81 2214 43 16
ZBM 07.38 04.34 2188 34 15
M9 03.41 01.96 0684 08 08
*Dry Cell Weight (DCW); *Total Cell Protein (TCP). a Total call protein was estimated by Bradford method.
b Expression levels were computed by densitometric scanning of total cell protein resolved on 12% SDS-PAGE.
Results
203
4.12.10. Purification of Recombinant TnBglA and TnBglB
For the purification of soluble extracellular unrefined TnBglA and TnBglB proteins were
cultivated individually under optimal conditions in fresh sterilized 4×ZB medium (5 L
flasks) supplemented with both antibiotic (as describe earlier). Optimal heterologous
expression and efficient folding of proteins were observed when both neutral pH culture
media were induced at 0.6 OD600nm with lactose (150 mM) after heat shock treatment for 1
h at 42°C followed by incubated for 72 h at 22°C in a rotary shaker (200 rev min-1).
Unrefined cloned TnBglA and TnBglB were purified by employing two subsequent
purification steps of IEC and HIC after heat-treatment (as described earlier). Purity and
activities of enzymes were monitored at each stage of purification by SDS-PAGE analysis,
7.37 fold with a recovery of 28.5% yield and 7.42 fold purification with a recovery of 41%
yield were achieved, respectively. The active fractions of TnBglA and TnBglB exhibited a
single prominent band with 51.50 kDa and 81 kDa on SDS-PAGE, respectively which
revealed that the target heterologous proteins were successfully purified (Figure 4.66a and
4.66b).
Figure 4.66 (a): SDS-PAGE analysis of recombinant purified TnBglA after lactose (150 mM) induction in
4×ZB medium. Lane M, Novagen Protein marker (cat # 69079-3); Lane 1: Purified TnBglA fraction (2 µg)
(b): SDS-PAGE analysis of recombinant purified TnBglB after lactose (150 mM) induction in 4×ZB medium.
Lane M, Novagen Protein marker (cat # 69079-3); Lane 1: Purified TnBglB fraction (2 µg); Lane 2: Purified
TnBglB fraction (2 µg).
75 kDa
100 kDa 81 kDa
(b) (a)
51.5 kDa 50 kDa
35 kDa
150 kDa
225 kDa
25 kDa
CHAPTER-V
DISCUSSION
Discussion
204
5. Discussion
Industrial biotechnology is ubiquitous and has caused a huge impact on diverse industries
especially for green energy generation along with sustainable production of highly valuable
genetically engineered products as compared to previously envisioned (Elleuche et al.,
2014). Recently, biotechnological products have gained significant interest all over the
world, which are proficient of enhancing the efficiency of bioenergy production, livestock,
agriculture, pharmacy, poultry, paper and pulp, beverage, food, detergent and textile
industries. The prevailing conditions in industrial processes are frequently far from the
properties of standard biocatalysts. Therefore, the demand of novel highly stable and
efficient biocatalysts is considerably increased globally, which are capable to reach the
goal (aim) by supplementing or replacing the traditional chemical approaches (Woodley,
2013).
Naturally, the members of genus Thermotoga have been reported to be a valuable home of
highly active and thermotolerant glycoside hydrolases (GHs) especially cellulolytic
enzymes (Mehmood et al., 2014). The highly heat stable cellulases from genus Thermotoga
are the most prominent candidates, which can perform their catalytic functions effectively
under extremely harsh conditions of biosaccharification process and numerous other
industries (Xie et al., 2015; Long et al., 2016). The complete genome (1809823 bp)
sequence of T. naphthophila RKU-10T (GenBank Accession No. CP001839.1) is available
on the NCBI online database. Two putative cellulolytic β-1,4-glucosidases genes TnbglA
(ADA66698.1) and TnbglB (ADA66752.1) from a hyperthermophilic anaerobic bacterium
T. naphthophila were selected for cloning and overexpressed in mesophilic host E. coli
BL21 CodonPlus (DE3)-RIPL using pET-21a(+) expression vector followed by respective
heterologous enzymes purified to homogeneity.
Initially, the amplicons of TnbglA (1.341 kb) and TnbglB (2.169 kb) genes were ligated
into pTZ57R/T vector for direct cloning and sequencing, TA cloning is one of the simplest
and highly proficient strategy to clone genes (Zafar et al., 2014; Haq et al., 2015b). During
primer designing, NdeI restriction site was introduced in forward primer at 5' end, for the
directional cloning and to make TnbglA and TnbglB genes compatible with open reading
Discussion
205
frame (ORF) of pET-expression vector, similar strategy was observed in many earlier
studies (Yang et al., 2015; Chan et al., 2016; Long et al., 2016). The recombinant pTZ-
TnbglA and pTZ-TnbglA plasmids were propagated in E. coli DH5α to maintain a high
copy number, subsequently positive transformants selected by blue white screening, and
positive clones of both genes (TnbglA and TnbglB) were subjected to sequencing.
GHs have been classified on the basis of nucleotide and amino acid sequence similarities,
in which β-glucosidases belong to either GH family 1 or GH family 3 (Liu et al., 2012). T.
naphthophila possesses two different β-glucosidases (TnbglA and TnbglB) genes. Many
thermophiles contain genes for two or more exo-acting β-specific glycosyl hydrolases,
including T. neapolitana (Turner et al., 2007), T. maritima (Xue et al., 2009), T. petrophila
(Haq et al., 2012b), Thermoanaerobacterium thermosaccharolyticum (Pei et al., 2012) and
T. petrophila (Xie et al., 2015). TnBglA and TnBglB displayed high amino acid sequence
homology range of 99-55% with the β-glucosidases of other microbes (thermophiles to
mesophiles). The deduced sequence homology of both β-glucosidases from T.
naphthophila revealed that TnBglA should be classified as the member of GH family 1
because it shared similar consensus sequence blocks (conserved region) with other β-
glucosidases of GH family 1 (Figure 4.11a); while, TnBglB should be classified as the
member of GH family 3 since it shared similar consensus sequence blocks (conserved
region) with other GH family 3 β-glucosidases (Figure 4.11b).
Sequence multi-alignments studies revealed that TnBglA has some conserved regions and
shared highest identity with other GH family 1 thermophilic β-glucosidases especially with
the members of genus Thermotoga. Mostly, conserved regions of TnBglA located near N
and C terminal, there were some conserved regions in which first region YQIEG from 19
to 23, second consensus sequence YRFS from position 76 to 79, third YHWDLP from 120
to 125, fourth NEPW from 165 to 168, fifth TENGAA from 350 to 355, and sixth
DNFEWA consensus sequence from 402 to 407 amino acid residues. In conserved regions,
there were conserved sequences of glycine (G), histidine (H), glutamic acid (E), asparagine
(N), serine (S), tryptophan (W) and phenylalanine (F); and for GH family 1 β-glucosidases,
two strictly conserved catalytic residues of glutamic acid (E), one in N-terminal and the
other in middle or C-terminal region, participate in the hydrolysis of glycosidic bond
Discussion
206
(Jenkins et al., 1995), by a general acid/base catalytic retaining mechanism. TnBglA
protein has conserved catalytic residues, one glutamic acid (E166) act as proton donor closer
to the N terminus and other two glutamic acid (E351 and E405) act as nucleophiles (Figure
4.11a) (Gräbnitz et al., 1991).
The catalytic active sites for GH family 1 β-glucosidases have been identified and
confirmed by site-directed mutagenesis (Wang et al., 1995; Moracci et al., 1996;
Vallmitjana et al., 2001). TnBglA belonged to GH family 1, which shares a retaining
catalytic mechanism by involving three active glutamic acid (E166, E351 and E405) residues.
However, Serine (S) also assists in the hydrolytic activity of β-glucosidase while Gly (G)
provides the turns which are essential and indispensable for the structural stability. The
sequence alignment revealed that the asparagine (N162) residue from TnBglA, is highly
conserved in clan A of GHs except GH family 26, which suggests that it provide a polar
interaction with glucose (Gloster and Davies, 2010). The study of conserved domains of
TnBglA using NCBI database explained that TnBglA is a monomeric protein (Marchler-
Bauer et al., 2015). Previously, a cloned monomeric GH family 1 β-glucosidase from
Thermotoga petrophila has been reported to contain three similar catalytic conserved
glutamic acid (Glu) residues (Haq et al., 2012b). Mostly, GH family 1 thermophilic
monomeric recombinant β-glucosidases have two comparable conserved glutamic acid
(Glu), which suggested as a nucleophile and catalytic acid/base residue (Fang et al., 2014;
Rajoka et al., 2015; Yang et al., 2015; Chan et al., 2016).
Multi-alignments studies of TnBglB with some other GH family 3 members, demonstrated
that it has some highly conserved regions and exhibited great sequence similarity with β-
glucosidases of other Thermotoga species (Goyal et al., 2001; Turner et al., 2007; Xie et
al., 2015). The conserved motifs of TnBglB was observed ADGPAG (57-62), TAFPV (79-
83), GRNFEYYSEDP (129-139), GVQS (152-155), NNQETNR (168-174), LREIYL
(186-191), YNKLNG (210-215), GFVMSDW (237-243), RISGEG (454-459),
NPSGKLPTTFP (537-547), IYVGYRYYDTF (576-586) and RVGASSRDI (697-705)
residues. In conserved regions, there were conserved sequences of glycine (G), lysine (K),
aspartic acid (D), glutamic acid (E), arginine (R), tryptophan (W), threonine (T), leucine
(L), serine (S), tyrosine (Y) and histidine (H) (Figure 4.11b). The sequence alignment of
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207
TnBglB with other β-glucosidases of GH family 3, showed that one catalytic residue
aspartic acid (D242) present in highly conserved region of domain-I, act as a fully conserved
catalytic nucleophile; and previously this residue has been predicted as active nucleophile
over 15 different GH family 3 β-glucosidases after sequence alignment (Dan et al., 2000;
Turner et al., 2007). Such conservation is consistent with a key catalytic nucleophilic role
in forming the glycosyl-enzyme intermediate by covalent linkage, stabilizing and
modulating the ionization state of proton donor catalytic residue and making a hydrogen
bond to sugar 2-hydroxy group at transition state (Parry et al., 2001). Another general
acid/base catalytic residue glutamic acid (E458) present in highly conserved region of
domain-II, which act as proton donor and display hydrolytic function to break glycosidic
bond. However, the importance of catalytic nucleophile (D242) and essential acid/base
residue (E458) have been demonstrated and confirmed by site-directed mutagenesis (or
selected active-site variants) at the catalytic pocket (Hong et al., 2006; Pozzo et al., 2010;
Zhao et al., 2015).
The signature amino acid sequence analysis revealed that TnBglB consists of a three-
modular domain protein, from the N-terminal to C-terminal, having glycosyl hydrolase
family 3 N-terminal catalytic domain (Glyco_hydro_3), a glycosyl hydrolase family 3 C-
terminal catalytic domain (Glyco_hydro_3_C), and a fibronectin type III (FnIII) module
found at C-terminal end, its actual function is unknown although FnIII domain is closely
associated with N- and C-terminal glycosyl hydrolase domains (Marchler-Bauer et al.,
2015). The conserved sequence KHFV (163 to 166) of TnBglB is a putative carbohydrate-
binding site (Thongpoo et al., 2013). GH family 3 β-glucosidases from Thermotoga
neapolitana (Pozzo et al., 2010), T. petrophila (Xie et al., 2015) and Myceliophthora
thermophila (Zhao et al., 2015) also contained three domains with similar pattern. All the
data presented here reveal that TnBglB follows a retention catalytic mechanism, and a
prominent member of GH family 3 composed of three domains with two highly active
residues, D242 and E458 in catalytic center.
Both genes TnbglA and TnbglA were sub-cloned in an efficient expression vector pET-
21a(+) under the control of a T7 lac promoter and the strong upstream translation signals
for target proteins, followed by optimally overexpressed in a most popular, well-
Discussion
208
established and excellent expression platform E. coli BL21 Codon Plus (DE3)-RIPL host
strain. For overexpression of thermostable β-glucosidases genes various expression vectors
and mesophilic hosts were efficaciously used, whereas the pET expression vector and E.
coli BL21 CodonPlus (RIPL) host is the most preferable expression coordination as
compared to others. Due to high growth rate, well characterized molecular genetics and
incredible expression level, many researcher have been selected pET vector and E. coli
BL21 CodonPlus strain to overexpressed β-glucosidases isolated from Dictyoglomus
thermophilum (Zou et al., 2012), Thermotoga thermarum (Long et al., 2016) and
Caldicellulosiruptor owensensis (Peng et al., 2016).
Positive transformants of E. coli BL21 harboring the pET-21a–TnbglA and pET-21a–
TnbglB plasmids were cultivated initially in LB medium and induced with IPTG, subjected
to SDS-PAGE for the analysis of heterologous expression of proteins along with crude
control supernatant of E. coli BL21 harboring pET-21a (+) without insert, which also
induced with IPTG. The crude media supernatant of TnBglA and TnBglB enzymes
displayed a prominent band of 51.5 kDa and 81.1 kDa on SDS-PAGE, respectively (Figure
4.23a and 4.23b). Both cloned enzymes from T. naphthophila did not have putative signal
peptide sequence; expression and activity were observed in all three cellular fractions but
extracellular soluble fraction exhibited maximum expression as compared to cell-free
lysate and cell-bound fractions (Figure 4.27a and 4.27b). Similar behavior of cloned
thermophilic β-glucosidases have been reported from Thermoascus aurantiacus (Parry et
al., 2001).
To attain a high level expression of recombinant enzymes and cultures density, a better
cultivation approach has developed by modifying and optimizing a few essential
parameters such as pre-induction optical cell density, growth temperature, better agitation,
heat shock treatment (42°C for 1 h) to the bacterial cultures before and after induction, and
optimal inducer concentration which played a critical role to improve the bacterial growth
and revamp the targeted proteins with proper folding. Best pre-induction optical density
(OD600nm) of TnBglA and TnBglB cultures for getting maximum expression were studied.
High level of expression and total cell proteins were observed when both cultures induced
at 0.6 (OD600nm) (Figure 4.29a and 4.29b), which indicated that host cell density effects the
Discussion
209
output of heterologous proteins, similar behavior had been reported in many earlier studies
(Schroder et al., 2014; Yang et al., 2015; Chan et al., 2016).
Effect of heat shock treatment on the expression of both enzymes were reconnoitered,
TnBglA and TnBglB expression and activities were greatly improved when cultures were
given heat shock at 42°C for 1 h just before induction (Figure 4.30a and 4.30b). Heat shock
treatment to the bacterial culture is accountable for the production of molecular chaperons
(heat shock protein), which are concerned primarily with protein folding and support the
appropriate expression, and constrain the formation of inclusion bodies (Oganesyan et al.,
2007; Hameed et al., 2014). After heat shock treatment, both cultures were induced with
different concentration of IPTG (0.1-0.9 mM), and optimal expression of TnBglA and
TnBglB were observed with 0.5 mM IPTG (Figure 4.31 and 4.32). Cell density and
enzymes expression were reduced with the further increment of IPTG molar concentration
because high amount of IPTG causes a toxic effect on host cells growth. Use of reduced
concentration of IPTG is an effective approach to get the high level of soluble protein
expression and host cell growth, which also make the fermentation process more
economical. Similar strategy has been applied by many researchers to achieve better
expression of β-glucosidases such as Kim et al. (2012), Schroder et al. (2014), Xie et al.
(2015), and Chan et al. (2016).
Inducing temperature is a critical parameter, as can be observed its effect on the
heterologous proteins expression and host cell growth. Therefore, effect of induction
temperature (16-42°C) on the expression of TnBglA and TnBglB were investigated by
incubating culture at various temperature after induction with IPTG (0.5 mM). Best
expression and production outputs of recombinant enzymes were obtained when culture
incubated at 22°C in a shaking incubator (Figure 4.33a and 4.33b). Low temperature
inducement is an effective method for proper protein folding and to achieve maximum
soluble expression. This strategy is also effectual to repress the production of inclusion
bodies. Several group of researchers have been reported that optimal expression of β-
glucosidases were observed at low temperature inducement as compared to 37°C and
reduced the inclusion bodies (Xue et al., 2009; An et al., 2010; Xie et al., 2015).
Discussion
210
Time of incubation, pH of medium and agitation speed are another important factors which
affect the production and expression of desired protein along with growth of engineered E.
coli CodonPlus. Therefore, time course profile up to 96 hours was studied in various pH
(5.5-8.0) of LB medium along with different agitation speed (50-250 rev min-1) after
induction. Optimal expression of both heterologous proteins were observed in pH 7.0
medium after 72 h inducement at 22°C with 200 rev min-1. Previously, a study
demonstrated that best expression of β-glucosidase was achieved when E. coli BL21 host
cells grown at 18°C for 20 hours in a shaking incubator with 200 rev min-1 after IPTG
induction (Wang et al., 2012). Hence, low temperature along with appropriate
environmental conditions and inducement time are necessary factors for the proper
recombinant protein folding into native conformation.
For purification of TnBglA and TnBglB, host cells grown individually at optimal
cultivation conditions followed by stockpiled culture supernatants. Partial purification of
both enzymes were achieved by heat treatment at 70°C for 1 h. At such high temperature
approximately all heat-liable proteins of mesophilic host were denatured and thermo-
tolerant cloned protein was partially purified. SDS-PAGE analysis revealed that heat-
precipitation step was alone sufficient to remove several host contaminating proteins
(Figure 4.38a and 4.38b). Heat denaturation is an inexpensive, highly effective and an
expedient prime enrichment step in refining of thermostable recombinant enzymes
expressed in mesophilic host. Formerly, several thermophilic bacterial β-glucosidases
partially purified using this strategy (Li et al., 2013; Xie et al., 2015; Gumerov et al., 2015;
Long et al., 2016).
TnBglA and TnBglB were further purified to homogeneity using anion-exchange (AEC)
and hydrophobic interaction (HIC) chromatography, activity of both enzymes were
monitored at each step of purification (Table 4.7a and 4.7b), similar scheme of purification
has been adopted by Haq et al. (2012b). TnBglA and TnBglB were purified with a final
purification of 7.28 and 7.38-fold, a yield of 40.8 and 39.5% with the highest specific
activity of 255×103 U mg-1 and 130×103 U mg-1, respectively. Purification folds are largely
consistent with the results obtained by Shin and Oh, (2014). The apparent molecular mass
and purity of TnBglA and TnBglB enzymes were analyzed by SDS-PAGE, which showed
Discussion
211
a single prominent band of 51.50 kDa and 81.1 kDa, respectively (Figure 4.38a and 4.38b).
Molecular mass of purified TnBglA is in agreement with those of many GH family 1 β-
glucosidases characterized from other thermophilic bacteria (Pei et al., 2012; Zou et al.,
2012; Yang et al., 2015). Purified TnBglB has a molecular mass of 81.1 kDa which is in
accordance to many other GH family 3 β-glucosidases characterized from other
thermophilic bacterial sources (Goyal et al., 2001; Turner et al., 2007; Xie et al., 2015).
Extracellular purified TnBglA displayed a single noticeable band on native-PAGE under
non-reduced conditions and after boiling (Figure 4.39a), which suggests that TnBglA is a
monomeric protein. This behavior is similar to β-glucosidases as from Thermotoga
neapolitana (Park et al., 2005) and T. thermarum (Long et al., 2016). While, under non-
denatured conditions and after boiling, TnBglB enzyme remained as a trimer with high
molecular mass monomeric conformation and showed a single band on native-PAGE
(Figure 4.39b). Similarly, Parry et al. (2001) had been reported a trimeric GH family 3 β-
glucosidase from Thermoascus auranticus with a 120 kDa monomer under non-reducing
conditions.
Individually, purified TnBglA and TnBglB were completely characterized; variation of
temperature and pH obviously influenced the activity of enzymes. To reconnoiter pH
dependence of both enzyme activity, employed an overlapping buffer systems to cover a
broad range of pH from 3.0 to 11.0 at 70°C, and also investigate the optimal buffer system
for the catalytic efficacy. Generally, different buffer system greatly influenced the enzymes
activities, hence McIlvaine buffer for pH 3.0-7.0, HEPES for pH 8.0 and for pH 9.0-11.0
CAPS were found the best buffer for both enzymes. Peak hydrolytic activity of TnBglA
and TnBglB were determined at pH 7.0 and 5.0, respectively using McIlvaine buffer, and
a noteworthy difference was observed in activity between McIlvaine and Tris-Cl buffer at
optimal pH of both enzymes. Maximum activity in McIlvaine buffer as compared to other
buffer systems, thought to be due to charge distribution or changes in conformation.
Similarly, reduction in catalytic activity of β-glucosidases in Tris-Cl and optimal in
McIlvaine buffer had been reported from Exiguobacterium sp. DAU5 (Chang et al., 2011)
and Sphingopyxis alaskensis (Shin and Oh, 2014).
Discussion
212
Purified TnBglA retained more than 90% of optimal activity at 6.0 to 9.0 broad range of
pH, and enzyme efficiency was completely repressed at high concentration of hydrogen
ions and more alkaline aqueous environment (Figure 4.40a). Whereas, purified TnBglB
exhibited 90% catalytic activity at pH 4.0 and reduced in alkaline pH range (Figure 4.40b).
Optimal pH of both enzymes are not unexpected, as most bacterial β-glucosidases showed
maximum activity at neutral or slightly acidic ranges (Fang et al., 2014). Any change in
ionic strength from optimal pH caused an obvious constrain on catalytic efficiency because
enzymes are extremely sensitive to change in pH that can alter structural conformation by
dissolving hydrogen and ionic bonds, which subsequently denature the catalytic site and
eventually disrupt the enzyme-substrate complex (Stoker, 2008; Ha and Bhagavan, 2011).
Comparison of the optimum pH of TnBglA with other thermophilic bacterial GH family 1
β-glucosidases revealed that it was quite similar to Dictyoglomus thermophilum (Zou et
al., 2012) and T. maritium (Mehmood et al., 2014); higher than the previously reported β-
glucosidases from Thermoanaerobacterium aotearoense (Yang et al., 2015), and
Acidilobus saccharovorans (Gumerov et al., 2015) that displayed peak activity at pH 6.0.
Although, some variant β-glucosidases from T. thermarum (Zhao et al., 2013) and
Anoxybacillus sp. (Chan et al., 2016) optimally active at pH 4.8 and 8.5, respectively.
Optimal pH of TnBglB is fairly similar to other bacterial GH3 β-glucosidases from T.
petrophila (Xie et al., 2015) and T. thermarum (Long et al., 2016). However, it was higher
than that of previously reported GH3 β-glucosidases from Thermofilum pendens (Li et al.,
2013) that displayed maximum activity at pH 3.5, and lower than from Terrabacter
ginosenosidimutans (An et al., 2010) that exhibited peak activity at pH 7.0.
Purified TnBglA and TnBglB displayed peak activity at 95 and 85°C, respectively. TnBglA
retained more than 90% catalytic activity at 100°C (Figure 4.41a), while TnBglB showed
more than 85% activity even at 90°C, and at 100°C TnBglB activity declined up to 66%
(Figure 4.41b). At optimal temperature, catalytic reaction rate become faster due to high
kinetic energy between the enzyme-substrate complexes. Whereas, enzyme denature
rapidly above the optimum temperature, due to the disruption of approximately all
hydrogen bonds and weak-interactions, which are accountable for the stability and proper
folding of enzyme catalytic site (Laidler and Peterman, 2009; Daniel et al., 2010).
Discussion
213
Optimal temperature of TnBglA (95°C) is analogous to the thermophilic recombinant GH1
β-glucosidases from T. maritima (Mehmood et al., 2014) and Acidilobus saccharovorans
(Gumerov et al., 2015); and higher than those reported from Thermoanaerobacterium
aotearoense (Yang et al., 2015) and Anoxybacillus sp. (Chan et al., 2016) that optimally
work at 60°C and 70°C, respectively. However, a β-glucosidase isolated from a
hyperthermophilic Pyrococcus furiosus archaeon had an exceptional 105°C optimal
temperature (Voorhorst et al., 1995). TnBglB optimal temperature (85°C) is similar to other
GH3 β-glucosidases cloned from Dictyoglomus turgidum (Kim et al., 2011) and T.
thermarum (Long et al., 2016). While, higher than β-glucosidases from Myceliophthora
thermophila (Zhao et al., 2015) and lower than from T. petrophila (Xie et al., 2015).
Optimal incubation time for the substrate hydrolysis is a crucial parameter. Therefore, time
course profile for the hydrolysis of substrate using purified TnBglA and TnBglB were
studied from 01 to 15 minutes. Both enzymes showed peak hydrolytic activity after 10
minutes incubation of enzyme-substrate mixture however further increase in incubation
time did not cause any perceptible effect (Figure 4.42). Optimal incubation time of TnBglA
and TnBglB is in accord with previously reported cloned GH1 β-glucosidases (Hong et al.,
2009; Fang et al., 2014; Schroder et al., 2014), and GH3 β-glucosidases (Goyal et al., 2001;
Zhao et al., 2015; Long et al., 2016).
Thermostability is the most prominent feature of β-glucosidases that cloned from
hyperthermophiles especially from the members of genus Thermotoga, which make them
an imperative candidates of several biotechnological and industrial applications. Thermal
stability profile revealed that TnBglA and TnBglB were thoroughly stable at 85°C for 720
minutes (12 h) and 480 minutes (8 h), respectively at neutral pH range from 6.5 to 7.5
(Figure 4.43a and 4.43b), while lowest thermostability of both enzymes were found at pH
4.0, which suggesting that the best ionization state and net charge for stabilization of
enzyme structure maintains at narrow range of pH (6.5 to 7.5), which is different from the
optimum net charge of TnBglB for catalysis. Net charge acquired at neutral pH is sufficient
to stabilize the secondary and tertiary structure of recombinant TnBglA and TnBglB even
at higher temperature (Gitlin et al., 2006; Colussi et al., 2015). Conversely, above 80°C
and at low pH 4.0, alternation and structural destabilization occur at both secondary and
Discussion
214
tertiary level, and net charge can influence their equilibrium properties such as free energy
of enzyme interactions with ligands.
Purified TnBglA showed astonishing thermostability as compare to TnBglB, at 90°C,
TnBglA retained 82% residual activity after being incubated for 300 minutes (5 h) at pH
6.5 to 7.5, whereas TnBglB lost 46% of its original hydrolytic efficiency after 240 minutes
(4 h) incubation. Thus, hyperthermostable TnBglA and TnBglB could be used efficaciously
in various fields such as in food processing and saccharification of biomass, etc. The
variant thermotolerant behavior of both enzymes with different pH level, is in harmony
with previously reported cloned β-glucosidases from T. petrophila (TpBgl1 from GH1 and
TpBgl3 from GH3) and Pyrococcus furiosus (PfBgl1 from GH3) that showed highest
thermostability at pH 6.0 and lowest at pH 4.0 (Cota et al., 2015).
Thermal stability of TnBglA was higher than the stability of other GH1 β-glucosidases
from Fervidobacterium islandicum (Jabbour et al., 2012) and T. thermarum (Zhao et al.,
2013) that displayed 100% thermal efficiency at 70°C for 3 h and 85°C for 2 h, respectively
at neutral pH. While, an exceptional β-glucosidase from T. maritima was fairly stable up
to 140°C (Mehmood et al., 2014). Thermal tolerance of TnBglB is greater than the several
GH3 β-glucosidases from Dictyoglomus turgidum (Kim et al., 2011) and T. thermarum
(Long et al., 2016) that remained active at pH 5.0 up to 85°C and 75°C for 5.57 h and 2 h,
respectively. But an exceptional acidophilic and thermo-active GH3 β-glucosidase from
Thermofilum pendens maintained nearly 100% of its peak activity at pH 4.0 after 2 h
incubation at 90°C (Li et al., 2013).
Both purified TnBglA and TnBglB displayed an astounding pH stability, which indicated
their potential as industrial candidates. Bu et al. (2013) suggested that enzymes catalytic
activities are directly linked with the balance between protonation and conformational
changes. In this present study, TnBglA remained stable over a broad range of pH from 6.0
to 9.0, with a drastic reduction to ≈3% at pH 3.0 (Figure 4.44a). While, TnBglB remained
stable at pH 6.5 to 7.5 and retained more than 70% of its optimal activity at pH 5.0 to 8.5
(Figure 4.44b). Hence, the experimental changes in the hydrolytic behavior of TnBglA and
TnBglB with variation in pH were due to modified protonation of enzyme’s amino acid
Discussion
215
residues and net charge that are directly affecting the interaction with ligands. In enzyme
catalysis, ionic groups are involved, for instance, acid-base amino acid residues in the
active site of β-glucosidases (Colussi et al., 2015). Therefore, the protonation state of the
carboxylate nucleophile and carboxylic acid amino acid residue are essential for enzymatic
reaction and a variation in pH could impair the hydrolytic mechanism (Gitlin et al., 2006;
Sørensen et al., 2013).
The result of TnBglA stability is in agreement to the numerous formerly reported GH1 β-
glucosidases from Lactococcus sp. FSJ4 (Fang et al., 2014), and Anoxybacillus sp. (Chan
et al., 2016) which showed stability within a broad range of pH. While, TnBglB showed
more pH stability at neutral range than the acidic and alkaline conditions, this phenomenon
is in accordance to other cloned GH3 β-glucosidases from T. petrophila (Xie et al., 2015)
and T. thermarum (Long et al., 2016). However, in contrast to TnBglB, an acidophilic β-
glucosidase from Thermofilum pendens retained 80% of its maximum activity at an acidic
range of pH 3.5 to 4.0 (Li et al., 2013).
Recombinant TnBglA and TnBglB enzymes exhibited remarkable thermal and pH stability,
can retained their catalytic activity for a longer time and reduce amount will need in the
reaction processes due to reusability. Steam explosion is commonly applied during the
biosaccharification process to make plant biomass more accessible and suitable for
dynamic cellulolytic hydrolysis, a highly thermostable β-glucosidase (either TnBglA and
TnBglB) could be used simultaneously along with other thermo-efficient cellulases
(endoglucanases and cellobiohydrolases) and directly devoid of a pre-cooling step thereby
can reduce the processing time and cost, save energy, provide an opportunity to increase
solubility of products and reactants, decrease the risk of contamination, improve qualities
and yields (Pei et al., 2012). In practical applications, thermal activation is desired since
longer active life means the less enzyme consumption, due to the incredible stability of
TnBglA and TnBglB at elevated temperature and pH, both are the potential candidates for
wide industrial bioconversion processes, including waste treatment, textiles, paper and
pulp for cellulose-degrading purpose (Kuhad et al., 2011).
Discussion
216
The great storage stability of crude supernatant, heat treated (at 70°C for 1 h) and purified
TnBglA and TnBglB were observed after storage at different conditions like room
temperature and 4°C, as shelf-life of an enzyme is an imperative parameter for the
commercial utilization. Providentially, enzymes exhibited an excellent storage stability and
retained 100% of original activity after storage at room temperature for about 175 days
while crude supernatant showed marginal decline in activity of 5-7%, and no substantial
loss of activities were observed at 4°C (up till now) in all the three ways of storage (crude
supernatant, heat treated and purified). Schroder et al. (2014) reported that a heat-active β-
glucosidase from hydrothermal spring was absolutely stable at 4°C. In contrast to the
present study, a cellulolytic enzyme from Trichoderma viridae had lost its original activity
up to 42% after 30 days storage at room temperature (Iqbal et al., 2011).
Usually chaotropic agents, trace metals and detergents modify the original activity of
biocatalyst to a certain limit or may terminate the catalytic efficiency completely.
Therefore, inhibitory effect of some chemical agents and metallic cations on both enzymes
were assessed. Divalent metallic cations (Ca2+, Cd2+, Co2+, Fe3+, Mg2+, Mn2+, Ni2+, Zn2+,
Pb2+ and Cu2+), chelating agent (EDTA), detergents (Triton X-100 and Tween-80) and urea
did not exhibit any sensitive influence on TnBglA and TnBglB catalytic efficiency (Figure
4.45a and 4.45b), which revealed that these additives were not necessary for the enzymes.
The results also explains that catalytic region of both proteins do not have a potential metal
ion binding site. Therefore, TnBglA and TnBglB are determined as metal-independent
proteins. However, their activities were intensely inhibited by Hg2+ and SDS which proved
to be the strong inhibitors; and suggested that the active site might have thiol groups which
involved in binding or catalysis and necessary for the proper and accurate tertiary structure
of enzyme (Liu et al., 2012).
Distinctly, TnBglA displayed the most favorable property that is the independence of metal
ions and various chemical additives. Largely congruent with the other reported bacterial
GH1 β-glucosidases as from T. thermarum (Zhao et al., 2013) and Anoxybacillus sp. (Chan
et al., 2016) recognized to be various additives tolerant. In contrast, hydrolytic activity of
multiple GH1 β-glucosidases modified to some extent or completely repressed by the
addition of these agents as have been explained in various studies (Chang et al., 2011; Lu
Discussion
217
et al., 2013; Yang et al., 2015). Conspicuously, TnBglB affinity was slightly improved by
the addition of Ca2+, Zn2+, Mn2+ and Co2+ cation by 125%, 120%, 115% and 110%,
respectively. These cations play a significant function in structural stability and improve
the binding affinity of substrate by stabilizing the active site conformation. Hence, it is
likely that β-glucosidase may has a metal ion binding loop, which contains the competency
to retain protein structure by binding with ions. An increased activity with metal ions and
repressed by Hg2+, were recently described for GH3 thermophilic β-glucosidases from
Thermofilum pendens (Li et al., 2013) and T. petrophila (Xie et al., 2015). TnBglB activity
was significantly reduced by SDS, but no obvious influence was detected with the addition
of EDTA (Long et al., 2016).
In addition, disulfide reducing agent β-mercaptoethanol enhanced the activity of TnBglA
and TnBglB up to 110% and 146%, respectively (Figure 4.45a and 4.45b). It is a well-
known inhibitor of thiol group, suggesting that sulfhydryl group (SH-group) may not be
engaged in catalytic center of both enzymes structure, while rather may be necessary for
maintaining three-dimensional structure and also play role in sustaining the enzyme in
reducing state. There is, although rare, precedent for the activation by β-mercaptoethanol
has been observed in β-glucosidases from T. neapolitana (Park et al., 2005) and
hydrothermal spring metagenome (Schroler et al., 2014); while Jabbour et al. (2012)
reported that enzyme activity was repressed by β-mercaptoethanol.
Mostly, organic solvents like acetone, ethyl acetate, acetonitrile, dimethyl sulfoxide
(DMSO) and dimethylformamide (DMF), and straight chain aliphatic alcohols (methanol,
ethanol, n-butanol and iso-propanol) caused strong inhibitory influence. TnBglA tolerated
competently a variety of organic solvents at low concentration, and no obvious effect has
been perceived with 15% (v/v) organic solvents though at high concentration the activity
decreased drastically (Figure 4.46a). This demeanor is largely congruent with the results
obtained by other GH1 β-glucosidases, where all these organic solvents revealed an
inhibitory effect on enzymes (Jabbour et al., 2012; Schroder et al., 2014). Moreover, Fang
et al. (2010) has also been reported that β-glucosidases lost its original activity to 30% with
5% (v/v) DMSO. In this study, purified TnBglB showed more resistance attitude towards
organic solvents as compared to TnBglA, and acts as a competitive inhibitors. No
Discussion
218
significant influence was found with 2-25% (v/v) organic solvents and caused little to
moderate reduction in TnBglB activity with the increase concentration (Figure 4.46b). This
agree with the data for GH3 β-glucosidase from T. maritima (Goyal et al., 2001); while
some other β-glucosidases also displayed similar reduction behavior with the increment of
DSMO (Li et al., 2013; Xie et al., 2015).
However, TnBglA was unaffected at low concentration of methanol, ethanol, n-butanol and
iso-propanol, but drastically reduced up to 8-10% in the presence of 30% (v/v) alcohols
(Figure 4.47a). This trend of reduction in activity by alcohols were in agreement with the
result of Schroder et al. (2014). Although rare, there is precedence in the enhancement of
GH1 β-glucosidase activity as has been reported by Zhao et al. (2013). TnBglB was not
highly influenced by straight chain aliphatic alcohols, although low concentration
considerably activated the enzyme followed by suppressed up to certain limit with 50%
(v/v) (Figure 4.47b). This result demonstrated that TnBglB can be used effectively in
organic biotransformation, and the stimulatory effect of alcohols is largely attributed to the
occurrence of transglycosylation or to allosteric interactions. Similarly, positive influence
by alcohol on recombinant GH3 β-glucosidases have been reported from Thermofilum
pendens (Li et al., 2013) and T. petrophila (Xie et al., 2015).
Cellobiose is a potent competitive inhibitor of endoglucanases and cellobiohydrolases, β-
glucosidase is commonly a rate-limiting factor during hydrolysis and highly sensitive to
glucose inhibition. Hence, a β-glucosidase with strong resistance to the end product
(glucose) inhibition is an attractive candidate in this field. By studying the effect of glucose
(20-1500 mM), it was revealed that TnBglA not only resistant to glucose inhibition, on the
contrary its activity is perceptibly enhanced by glucose (20-600 mM), and displayed the
high apparent affinity constant or inhibition constant (Ki) value of 1200 mM (Figure 4.48a).
Mostly, bacterial β-glucosidases did not exhibit resistance to glucose (end product),
however rare displayed some tolerance to glucose. Ki value of TnBglA for glucose
inhibition is higher as compared to many glucose-tolerant GH1 β-glucosidases (Cota et al.,
2015; Gumerov et al., 2015; Yang et al., 2015).
Discussion
219
Various concentration of glucose (20-600 mM) was used to investigate the effect of glucose
on TnBglB activity and determined the Ki value for glucose inhibition. Recombinant GH3
TnBglB exhibited less glucose tolerant behavior as compared to GH1 TnBglA, and the
activity of TnBglB decreased rapidly at high concentration of glucose. It has been observed
frequently that β-glucosidases belonging to GH family 3 showed less resistant to glucose
than family 1. However, TnBglB has a Ki value of 150 mM (Figure 4.48a), which is higher
than the previously reported recombinant bacterial GH3 β-glucosidase isolated from T.
petrophila with Ki value of 30.1 mM glucose (Cota et al., 2015). Ki value of TnBglB is
greater than many formerly published GH3 fungal β-glucosidases. For instance, β-
glucosidases from Myceliophthora thermophila with Ki of 0.282 mM (Karnaouri et al.,
2013), and Neosartorya fischeri with Ki of 13.4 mM (Yang et al., 2014).
Effect of xylose on activity of TnBglA and TnBglB was scrutinized. TnBglA revealed not
only great tolerance against xylose inhibition although its relative activity was highly
enhanced by 170% with 100 mM xylose. The stimulatory effect of xylose on TnBglA
activity was observed over a broad range of 50-700 mM xylose. Whereas, TnBglB
exhibited less tolerant behavior to xylose and its relative activity was gradually decreased
up to 18% in the presence of 600 mM xylose. TnBglA and TnBglB have a Ki value of 1300
mM and 200 mM xylose, respectively (Figure 4.48b). Despite the fact that minor β-
glucosidases from other bacteria displayed sufferance to xylose. Ki of TnBglA and TnBglB
for xylose inhibition are as high as compared to other xylose-activated β-glucosidase from
Scytalidium thermophilum that had a Ki of 43.24 mM (Zanoelo et al., 2004).
Substrate specificity classifies β-glucosidases into three main groups, the first group is
known as aryl- β-glucosidases which have high affinity to aryl-β-glucosides. Second group
contains true cellobiases which preferably hydrolyze only oligosaccharides to release
glucose, while third group consists of broad specific β-glucosidases that hydrolyze many
types of substrates, and these are the most frequently observed form of β-glucosidases (Pei
et al., 2012). In this present study, affinity of TnBglA and TnBglB was determined over a
large number of heteroglycans, xylan, various para- and ortho-nitrophenyl glycosides
substrates with both α and β confirmation. Both enzymes displayed maximum specificity
towards pNP-β-D-glycosides and cellobiose substrates, while no noticeable activity
Discussion
220
observed with pNP-α-D-glycosides and oNP-glycosides, these results suggested that both
enzymes specific for β-linkage and belong to first group aryl-β-glucosidases. The
hydrolyzing activity for substrates having β-1-4 linkages such as pNPG, pNPF and pNPGal
were much higher than for the substrates involving β-1-2 linkages such as oNPG, oNPF
and oNPGal. The presence of nitro group at the ortho position reduced the catalytic
efficiency, which indicated the steric hindrance with a hydroxyl group at C-6 (Parry et al.,
2001). Both enzymes exhibited no catalytic affinity with sucrose, xylan, carboxymethyl
cellulose (CMC), avicel and laminarin substrates (Table 4.8).
Several studies have been reported that β-glucosidases possess high affinity to aryl-β-
glycosides (pNPG, pNPGal, and pNPX) or β-linkage and the low affinity towards α-
linkage, which is a common property of GH1 (Pei et al., 2012; Fang et al., 2014). However,
a β-glucosidase from Sphinogopyxis alakensis had maximum activity with β-1,2 linked
substrate oNP-β-glucopyranoside (Shin and Oh, 2014). TnBglB has high affinity towards
aryl-β-glycosides of glucose and xylose. Xylosidase activity of TnBglB could contribute to
the degradation of xylan in pretreated plant biomass, and a β-glucosidase having β-
xylosidase activity can enhance the hydrolysis capacity. This phenomenon is consistent
with GH3 β-glucosidases from T. maritium (Goyal et al., 2001), while from Terrabacter
ginsenosidimutans revealed maximum activity towards oNPG instead of pNPG (An et al.,
2010).
Kinetics parameters of both enzymes, Km, Vmax, kcat, and kcat Km-1 for the hydrolysis of
substrates like pNPG, pNPF, pNPGal, pNPX, pNPC and cellobiose were calculated using
Lineweaver–Burk plot (Figure 4.49a and 4.49b), all values are provided in table 4.9a and
4.9b. Kinetic values of both enzymes clarified that pNPG is a more specific substrate than
cellobiose and other substrates. TnBglA has lower Km value (1.5 mM) against pNPG than
GH1 β-glucosidases from Caldicellulosiruptor bescii with Km of 3.71 mM (Bai et al.,
2013), demonstrating higher affinity for pNPG. Conversely, TnBglA exhibited Km of 7.76
mM for cellobiose, where this finding is lower than from Thermoanaerobacterium
aotearoense (Km value of 25.45 mM) (Yang et al., 2015). Catalytic efficiency (kcat Km-1)
ratio is often supposed as to measure enzyme efficiency, large value of kcat and high affinity
Discussion
221
for substrate make kcat Km-1 value large. TnBglA values of Vmax, kcat and kcat Km
-1 for pNPG
and cellobiose are highest from many previous studies (Bai et al., 2013; Yang et al., 2015).
TnBglB revealed the lowest Km value (0.45 mM) for pNPG representing greater affinity for
substrate as compared to the Km values of other bacterial GH3 thermophilic β-glucosidases
included Thermus sp. IB-21 (Kang et al., 2005) Terrabacter ginsenosidimutans (An et al.,
2010), and T. thermarum (Long et al., 2016) that exhibited Km values of 11.8 mM, 4.2 mM
and 2.41 mM, respectively. TnBglB presented the high Km value of 7.35 mM for cellobiose,
which indicated less affinity to the substrate. This finding is in contrast to GH3 β-
glucosidases with low Km values for cellobiose obtained from Phanerochaete
chrysosporium (Km of 5.05 mM) (Kawai et al., 2003), and Penicillium funiculosum NCL1
(Km of 1.25 mM) (Ramani et al., 2015) had been reported earlier. TnBglB has the highest
kcat and kcat Km-1 values for pNPG as compared to the values for GH3 β-glucosidases from
T. neapolitana (Pozzo et al., 2010), Dictyoglomus turgidum (Kim et al., 2011), and T.
petrophila (Cota et al., 2015).
Genomic analysis of various thermotolerant β-glucosidases revealed that amino acid
composition is the fundamental factor which associated with the thermostability of protein,
when compared to their heat labile or mesophilic counterparts. Usually heat loving β-
glucosidase have more hydrophobic and helix forming residues, and less thermolabile
residues (Suhre and Claverie, 2003; Gupta et al., 2014). In general, the stability of β-
glucosidase increases with the insertion of helix-forming residues into α-helices, and
stability decreases with the addition of helix-breaking residues (Singh and Hayashi, 1995).
There are 193 helix-forming residues present out of 446 amino acids in TnBglA, and 233
helix-forming residues present out of 721 residues in TnBglB, suggesting this could be one
of the factor influencing the thermal stability.
Thermostability of β-glucosidase is enhanced in the presence of high percentage of charged
glutamic acid (E) and proline (P) residues (Matthews et al., 1987) whereas, lower
percentage of glutamine (Q) along with neutral residues methionine (M) and cysteine (C)
account for the formation of salt-bridges/ion pairs and H-bonds which play important role
in structure stabilization. Earlier, Voorhorst and colleagues (1995) described that an
Discussion
222
increase in acidic amino acids residues particularly glutamic acid (E) and a decrease in
number of neutral residues notably cysteine (C), are thought to the account for heat stability
of enzyme. Godde et al. (2005) also demonstrated that increases proportion of acidic
residues enhance the optimal temperature of enzyme. Thermotolerant TnBglA and TnBglB
have high percentage of charged residues, 63 and 116 negatively charged residues (aspartic
acid and glutamic acid) while 51 and 94 positively charged residues (arginine and lysine),
respectively.
TnBglA protein contains only 1 cysteine (C), 29 aspartic acid (D) and 34 glutamic acid (E)
residues, however TnBglB protein has only 4 cysteine (C), 41 aspartic acid (D) and 75
glutamic acid (E) residues, which is fairly similar to other thermophilic β-glucosidases
(Jabbour et al., 2012). Both proteins have lower frequency of hydrophilic and thermolabile
residues glutamine (Q) due to its propensity to undergo deamination, methionine (M) and
cysteine (C) due to their tendency to undergo oxidation at high temperature (Gupta et al.,
2014). Overall high hydrophobicity is also influenced the stability (Voorhorst et al., 1995).
TnBglA and TnBglB have 66 and 69 aromatic residues, about 15% and 10% of the
sequences, respectively. On average, the sequence of non-thermostable β-glucosidases
randomly picked from NCBI database, are composed of 6% aromatic acids. This finding
is in accord to the analysis of thermostable β-glucosidase from Fervidobacterium
islandicum (Jabbour et al., 2012).
Further non-polar residues such as alanine (A) regard as one of the best helix forming
residue, presence of isoleucine (I) and leucine (L) and valine (V) provide the
conformational stability and also contribute to enhance the thermostability of globular
proteins. Thermostable TnBglA and TnBglB have higher values of aliphatic index 82.60
and 84.17, respectively which is due to high percentage of non-polar residues (aliphatic
side chains), is a statistical approach used to determine enzyme thermostability by Ikai
(1980). Aliphatic index is defined as the relative volume occupied by aliphatic side chains
(alanine, leucine, valine, and isoleucine) which is conspicuously higher for thermophilic as
compared to mesophilic proteins.
Discussion
223
Stability can also be estimated by instability index, which plays an essential role in
determining half-life of a protein. A stable protein has an instability index less than 40,
while a protein is predicted to be unstable if its value is above 40 (Gupta et al., 2014).
TnBglA and TnBglB have 31.23 and 30.83 instability index, respectively. Mostly
thermostable β-glucosidases have instability index below 40 which makes them heat-stable
according to the study of Guruprasad et al. (1990). Grand average hydropathicity
(GRAVY) measures the hydropathicity of a specific protein, lower the GRAVY value,
better will be the protein interaction with water. TnBglA and TnBglB have -0.373 and -
0.427 values of GRAVY because all thermophilic proteins have low GRAVY value (Gupta
et al., 2014).
Aliphatic index, instability index and GRAVY values of TnBglA and TnBglB are in
accordance to the several heat tolerant GH1 and GH3 β-glucosidases and comparable to
the thermolabile β-glucosidases. For instance, GH1 thermophilic β-glucosidase T.
neapolitana had 80.99 aliphatic index, 33.45 instability index and -0.423 GRAVY values
(Park et al., 2005). Whereas, a mesophilic GH1 β-glucosidase from Bifidobacterium breve
displayed 77.63 aliphatic index, 26.75 instability index and -0.330 GRAVY (Nunoura et
al., 1996). Thermophilic GH3 β-glucosidase from Clostridium thermocellum had 89.59
aliphatic index, 37.14 instability index and -0.310 GRAVY values (Graebnitz et al., 1989),
which are comparable with TnBglB. Whereas, thermolabile GH3 β-glucosidase from
Terrabacter ginsenosidimutans showed 88.92 aliphatic index, 42.37 instability index and
-0.198 GRAVY (An et al., 2010).
Hyperthermophilic proteins (β-glucosidases) have more intramolecular interactions (H-
bonding, hydrophobic interactions, electrostatic interactions and disulfide bonding), strong
network of salt bridges, higher packing efficiency, greater rigidity, increased helical
content, shortened/less loops, conformation strain release and reduced entropy of unfolding
than mesophilic proteins which increase their stability (Li et al., 2005; Gupta et al., 2014).
In this study, the inherent thermostability of TnBglA and TnBglB is further verified and
corroborated by their kinetic and thermodynamic study.
Discussion
224
Thermodynamic study of TnBglA and TnBglB was performed to measure the
thermostability of substrate hydrolysis system. Transition state stabilization was described
using an alternative approach as a description for the rate enhancement with assay
temperature (Benkovic and Hammes-Schiffer, 2003). Thermodynamic parameters: Gibbs
free energy (ΔG*), enthalpy (ΔH*), entropy (ΔS*) and activation energy (Ea) for pNPG
hydrolysis by TnBglA and TnBglB were calculated using Arrhenius approach in a
temperature range from 80°C to 95°C and 70°C to 85°C, respectively (Table 4.11a and
4.11b). The result displayed that both enzymes have a highly thermotolerant system
because these parameters are nearly constant at various temperatures. Negative entropy and
low enthalpy values explain the formation of ordered and effective enzyme substrate
transition state complex (Akolkar and Desai, 2010). These parameters at high temperature
are comparable to the thermodynamic studies carried out for thermostable β-glucosidases
from Fusarium solani (Bhatti et al., 2013) and Thermotoga maritima (Mehmood et al.,
2014).
Thermal stability of protein (β-glucosidases) is relevant for biological function and
molecular evolution. Although complex, the thermal denaturation process of TnBglA and
TnBglB can be simplified as the classical two-step process in which the first step involves
unfolding of the protein's native structure. The unfolded polypeptide may refold reversibly
to the native conformation or in a second step undergo irreversible denaturation to
permanent inactivation due to protein aggregation, misfolding and covalent changes such
as the deamidation of asparagine (N) or glutamine (Q) residues and oxidation of cysteine
(C) or methionine (M) residues (Volkin and Klibanov, 1989). This denaturation is
accompanied by the disruption of non-covalent linkages, including hydrophobic
interactions, with concomitant increase in the enthalpy of activation (ΔH*D) and increase
in the disorder or entropy (ΔS*D) of activation (Marín et al., 2003).
Enzyme thermostability encompasses thermodynamic and kinetic stabilities (Sanchez-
Ruiz, 2010; Bommarius and Paye, 2013). Thermodynamic stability is governed by the
enzyme free-energy of stabilization, reflecting the difference between the free energies of
the folded and the unfolded states of the protein, and by its melting temperature (Tm,
temperature at which 50% of the protein is unfolded). Kinetic or long-term stability
Discussion
225
depends on the energy barrier to irreversible inactivation and is generally expressed as the
enzyme's half-life (t1/2) at a defined temperature. An increase in the enzyme resistance to
unfolding (higher Tm) also increases its resistance to inactivation (higher t1/2) as increase in
the stability of the native state leads to slower accumulation of the unfolded state and
secondly, the unfolded state is usually the ground state leading to irreversible denaturation
or inactivation.
In order to determine the thermodynamic parameters for thermal stability, energy of
activation (EaD) for thermal denaturation of TnBglA and TnBglB were determined by
applying the Arrhenius plot. The plot of residual activity versus incubation time in a
temperature range of 97°C to 99°C for TnBglA and 94°C to 96°C for TnBglB are linear
(R2>95%) indicating first order kinetics of inactivation (Figure 4.51a and 4.51b). With
increasing temperature, t1/2 value decreased and the first order deactivation rate constant
(Kd) increased indicating that enzyme is less thermostable at high temperature. EaD values
for TnBglA and TnBglB are found to be very high, 665.12 kJ mol-1 and 286.83 kJ mol-1,
respectively. These values indicating both enzymes are very stable and compact, which are
highly resistant to heat denaturation. The higher value of EaD means that more energy is
required to denature the enzyme (Tayefi-Nasrabadi and Asadpour, 2008) and that the
enzyme might have undergone considerable conformational changes during denaturation
(Marín et al., 2003).
The value of free energy of thermal denaturation (ΔG*D) for TnBglA and TnBglB decreased
with the increase in temperature as shown in table 4.12a and 4.12b, respectively. When
entropy of inactivation (ΔS*D) of TnBglA and TnBglB was calculated at each temperature,
it showed positive values, which indicates that there are no significant processes of
aggregation, since had this happened, the values would have been negative (Marín et al.,
2003). TnBglA and TnBglB established high stability and displayed a half-life (t1/2) of 5.21
and 4.44 minutes at temperature 97°C and 94°C, respectively. Thermodynamic analysis of
TnBglA and TnBglB are in line with some recent studies of recombinant enzymes (endo-
1,4-β-xylanase and endo-1,4-β-glucanase) from T. petrophila (Haq et al., 2012a; Haq et
al., 2015a). However, limited literature is available on the thermodynamic study of
recombinant β-glucosidases.
Discussion
226
The predicted secondary structure of TnBglA has 42% helices (H), 10% β-sheet (E) and
47% loop (C). Three-dimension structure modeling of monomeric TnBglA was elucidated
by GENO-3-D and validated by Procheck. Ramachandran plot suggested that 92.5%
residues are in the most favorable regions (Figure 4.54a). The predicted 3-D structure
showed that it consists of 12 α-helices (major) which is a common motif in hydrolases
secondary structure and has 12 β-sheets, in which 8 β-sheets are present in the core and
making barrel shape structure while 4 β-sheets are at the surface of the protein (Figure
4.55a). The predicted structure of TnBglA represents an interesting pattern of conserved
structural elements in the way that outer part of the structural fold contains α-helices
whereas inner part contains approximately all parallel β-sheets form catalytic cleft or
pocket.
The α-helices and β barrel are connected with each other through a chain that provides
structural stability and active site is placed at top face of the barrel. One of the structural
characteristic is that the loops at C-terminal end of the sheets frequently involve in active
site of protein (Wierenga, 2001). TnBglA has conserved structural loops and classical (α/β)8
triose phosphate isomerase (TIM) barrel structure/scaffold, commonly found in proteins,
span in cell membranes and binds hydrophobic ligands in the barrel center. The TIM barrel
scaffold is one of the key characteristics and highly conserved fold of GH1 protein and
probably the most commonly present folding motif in proteins from mesophiles to
thermophiles (Branden, 1991; Wierenga, 2001). Structural modeling studies of TnBglA
demonstrated that it is quite similar to GH1 β-glucosidases such as from Fervidobacterium
islandicum (Jabbour et al., 2012), and Thermoanaerobacterium aotearoense (Yang et al.,
2015).
In the case of GH1 β-glucosidases, the two essential catalytic glutamic acids (E) are about
200 residues away from each other in the sequence. While, in TnBglA, catalytic acid/base
residue E166 is located in the conserved TLNEP motif at C-terminal end of β-strand 4, and
nucleophile catalysts of TnBglA are E351 and E405, located in the conserved ITENG and
DNFEW motifs at C-terminal end of β-strand 7 (Henrissat et al., 1995; Jenkins et al.,
1995). Active-site glutamic acids (Glu) are present in a pocket shaped structure with
hydrophobic residues at the entrance point (Gasteiger et al., 2003). The distance between
Discussion
227
the active glutamic acid E166 and E351 is 4.7 Aͦ and distance between E166 and E405 is 10.1 Aͦ
(Figure 4.55b). TnBglA is typical of a retaining β-glucosidase and permits for the formation
of a glycosyl-enzyme intermediate (McCarter and Withers, 1994).
The predicted secondary structure of TnBglB is composed of 25% helices (H), 19% β-sheet
(E) and 55% loop (C). Three-dimension profile structure modeling of TnBglB was
elucidated by GENO-3-D and validated by Procheck. Ramachandran plot statistics of
TnBglB suggested that 92.3% residues are in the most favorable regions (Figure 4.54b).
The predicted 3-D structure of TnBglB showed that enzyme consists of three domains
including an (α/β)8 TIM barrel domain-I, 5-stranded α/β sandwich domain-II, and
fibronectin type III (FnIII) domain is sequentially inserted between first two domains
(Figure 4.55c). At C-terminal, 120 residues are involved to form a FnIII domain and
believed to provide heat stability to GH3 β-glucosidases by managing the relative
movement of domain I and II, though all three domains are connected to each other by two
linkers regions that provides structural stability and play key role in folding of protein into
its active form (Ramírez-Escudero et al., 2016).
The structural fold and catalytic cleft for substrate of TnBglB is highly conserved with
other GH3 β-glucosidases in the evolutionary history. All part of TnbglB structure are well
organized and allows all residues involved in hydrolytic activity to be identified. The
active-site residues are found at the interface of domain-I and II, and formed a catalytic
pocket or crater which is very large and good enough to accommodate large substrates.
Domain-I (α/β)8 TIM barrel fold is highly conserved and probably the most frequently
present folding motif in GH3 proteins from mesophiles to thermophiles. Surprisingly, all
β-strands in domain-I are not parallel as would be predicted in a TIM barrel, the direction
of some β-strands is reversed (anti parallel) with respect to TIM barrel topology in general
(Pozzo et al., 2010). FnIII domain is located on TnBglB side remotest from the active
region, its actual function has rarely been demonstrated unambiguously. Domain-III have
been found previously in a number of bacterial extracellular GHs displaying different
specificities including chitinases, pullulanases and cellobiohydrolases (Irwin et al., 2000;
Kataeva et al., 2002).
Discussion
228
In 3-D structure of TnBglB, domain-I contains a catalytic nucleophile aspartic acid (D242),
which is located in the conserved GFVMSDW region at C-terminal. However, a catalytic
acid/base glutamic acid residue (E458) is located in a conserved SGEG region of domain-
II, both active-site residues aspartic acid (D242) and glutamic acids (E458) are present in an
organized pocket shaped structure with hydrophobic amino acid residues at the entrance
point leading to the catalytic pocket (Pozzo et al., 2010). The distance between nucleophile
D242 and acid/base residue E458 is 5.8 Aͦ (Figure 4.55d). TnBglB like other GH3 β-
glucosidases has a retaining catalytic mechanism, the main catalytic function of enzyme is
to hydrolyze β-linkages between glucose and other moieties of diverse substrates structure
(Turner et al., 2006; Turner et al., 2007). From the structural modeling studies, it is
concluded that TnBglB has conserved structural loops and identical folds, highly analogous
to other GH3 β-glucosidases as from T. neapolitana (Pozzo et al., 2010), T. petrophila (Xie
et al., 2015).
Docking studies of TnBglA by PatchDock, more than 100 possible solution of docking
were predicted, which were evaluated through Procheck and further analysed the enzyme-
substrate complexes by ligplot. All of the best docking models showed catalytic mechanism
of TnBglA in which at least two or three conserved glutamic acid residues (Glu166, Glu351
and Glu405) were found in close contact to the substrate for hydrolysis. Docking data of
enzyme-substrate also in harmony with the earlier findings of GH1 β-glucosidases (Gloster
et al., 2007; Haq et al., 2012b). Furthermore, with cellobiose W324 (Tryptophan), F414
(Phenylalanine), W398 (Tryptophan), W122 (Tryptophan), W406 (Tryptophan), H180
(Histidine), E408 (Glutamic acid) and M322 (Methionine) amino acid residues present on the
periphery of the reaction complex (Figure 4.56a). Whereas, in case of pNPG, E166
(Glutamic acid), W398 (Tryptophan), W324 (Tryptophan), W122 (Tryptophan), W406
(Tryptophan), H180 (Histidine) and E408 (Glutamic acid) residues present on the periphery
of reaction complex (Figure 4.56b). These residues provide a hydrophobic surrounding to
improve reactivity and thermostability, as suggested previously for GH1 thermostable β-
glucosidases (Bai et al., 2013; Rajoka et al., 2015; Yang et al., 2015).
Docking studies of TnBglB by PatchDock, more than 100 possible solution of docking
were generated, which were evaluated through Procheck and additionally analysed the
Discussion
229
enzyme-substrate complexes by ligplot. All of the best docking models showed that
cellobiose and pNPG substrates can be docked into the catalytic pocket of TnBglB very
well, in which D58 (Aspartic acid), R64 (Arginine), E458 (Glutamic acid) and D242 (Aspartic
acid) active-site residues are involved in catalytic mechanism of TnBglB and tightly locked
the substrates in a network of hydrogen bonds (Figure 4.57a and 4.57b). However, two
highly conserved essential catalytic residues are found in close contact to the both
substrates, in which D242 (Aspartic acid) act as nucleophile and E458 Glutamic acid as an
acid/base in the hydrolytic reactions. Furthermore, side chain of S370 (Serine), L25
(Leucine), L28 (Leucine) and W243 (Tryptophan) residues present on the periphery of
reaction complex and may form weak hydrogen bond with cellobiose. Likewise, the
surrounding residues M207 (Methionine), S370 (Serine), L25 (Leucine), L116 (Leucine) and
W243 (Tryptophan) may form weak hydrogen bond with pNPG. These residues provide a
hydrophobic platform to enhance thermal stability and reactivity. This finding is largely
congruent with the docking results of GH3 β-glucosidases from T. neapolitana (Pozzo et
al., 2010) and ruminal microbe (Ramírez-Escudero et al., 2016).
Enhancing the production, expression and activity of heterologous proteins in microbial
host expression system is the most prominent aspect of modernized and revolutionized
biochemistry (Rosano and Ceccarelli, 2014). Therefore, production and expression of
TnBglA and TnBglB were enhanced by using modifying media and lactose inducer instead
of IPTG. All cultivation parameters were optimized again with all inducing media as
described above such as pre-induction optical cell density, growth temperature, medium
composition, better agitation, heat shock treatment (at 42°C) to the recombinant culture
and inducer concentration.
Hyperthermophilic β-glucosidases from T. naphthophila (TnBglA and TnBglB) are highly
effective and valuable proteins, hence to augment the volumetric yield of desired products
and bacterial cultures, ten various nutrient complex and modified inducing media (LB,
LB+, ZB, ZBM, ZYB, 3×ZYB, 4×ZB, M9, ZYBM9, and 3×ZYBM9) were used. Induced
them individually with different concentration of IPTG and lactose at appropriate induction
point. Mostly, all these media were designed for T7 expression system using pET vectors
in E. coli BL21 (Studier, 2005). Individually, comparative analysis of IPTG and lactose
Discussion
230
inducers in above mentioned media revealed that the maximum culture production and
activity of both enzymes were obtained in 4×ZB medium with 0.5 mM IPTG and 150 mM
lactose after 72 h induction at 22°C with 200 rev min-1 (Figure 4.58 and 4.59).
The extracellular activity of TnBglA is enhanced in 4×ZB medium by 3.74 fold with 0.5
mM IPTG and 3.8 fold with 150 mM lactose as compared to the LB medium induced with
IPTG in a shake flask. Whereas, extracellular TnBglB activity is enhanced by 0.096 and
0.085 fold in 4×ZB medium using lactose and IPTG inducers, respectively. Approximately
same level of TnBglB expression was observed with both inducers. Similarly, the
production and expression in term of activity of several recombinant proteins have been
enhanced by applying optimal cultivation parameters and lactose induction strategy (Cheng
et al., 2011; Hameed et al., 2014; Su et al., 2015). The inducing modified medium, 4×ZB
is a tryptone rich nutritional medium, which help host cells growth and improve the
heterologous protein production. Similarly, Basar et al. (2010) has been reported that
thermophilic cloned enzyme activity and E. coli culture production was enhanced by using
complex cultivation media.
However, IPTG is significantly effective and a stable inducer, commonly used in
laboratory to induce T7 lac promoter for the heterologous proteins expression in host but
must be removed from the targeted proteins due to its potential toxicity. Lactose inducer
of lac operon is quite different from IPTG, it causes less metabolic burden on host (E. coli
BL21) machinery, acts as carbon and energy source to promote growth and increase the
culture density, and may improve the output of desired protein. Lactose, unable to enter
the host cells, requires an enzyme primase which converts into allolactose by β-
galactosidase and finally able to start T7 lac promoter. Lactose inducing procedure is
complex than IPTG, therefore if lactose is selected as inducer, then inducing parameters
(inducer concentration and incubation time, etc.) must be optimized. It has been proved
that IPTG is highly toxic for bacterial growth whereas lactose is an alternative natural,
efficient, non-toxic and inexpensive inducer which has numerous incredible advantages
and successfully use for the industrial production of engineering products (Kilikian et al.,
2000; Yan et al., 2004).
Discussion
231
In the present study, after lactose induction, the growth of cloned TnBglA expressing
bacteria was high than that achieved with IPTG, however maximum culture density of
18.96 with 11.30 g DCW L-1 was acquired in 4×ZB medium induced with 150 mM lactose
(Table 4.14b). In all inducing media, growth of recombinant TnBglB producing bacteria
was high with lactose induction as compared to IPTG, and maximum culture density of
18.66 with 11.08 g DCW L-1 was achieved in 4×ZB medium with 150 mM lactose
induction (Table 4.14b). This behavior is according to the previous study of enhanced
production of cloned enzyme from Pyrococcus furiosus (Ikram et al., 2009) and Bacillus
halodurans (Naz et al., 2010).
The yield of TnBglA and TnBglB greatly improved when cultures were given a heat shock
at 42°C (1 h) just before IPTG/lactose induction followed by incubation at 22°C for 72 h
in a shaking incubator (200 rev min-1), in all inducing media the similar results were
observed as in case of LB medium induced with IPTG (described earlier in this chapter).
Induction strategy benefits the balance of bacterial growth, as well as cloned protein
synthesis, folding and transportation. Optimal TnBglA and TnBglB expression observed
with 0.5 mM IPTG in all media including optimal 4×ZB; and 150 mM lactose determined
to be ideal concentration for induction in all media including 4×ZB. Great amount of
allolactose is needed at the time of induction, thus more amount of lactose in growth
medium is mandatory. Similar strategy of lactose induction has been applied to enhance
the expression of proteins in engineered E. coli BL21 (Tran et al., 2010; Bashir et al., 2015;
Su et al., 2015).
Highly thermostable, xylose and glucose-tolerant cloned TnBglA and TnBglB expectedly
increases the competency of monosugar (glucose) production by synergistic interaction
with other thermostable cellulases (EGs and CBHs) during saccharification process of
biomass (data not shown) for consequent production of bioethanol. Saccharification of
pretreated biomass (bagasse, wheat bran and rice straw) is highly improved, when either
TnBglA or TnBglB used with other enzymes in cocktail, indicating both are strong
candidates for the bioconversion process. Use of these enzymes, with a low degree of
inhibition might improve the yield and reduce the cost of process, and also have several
biotechnological and industrial applications.
CHAPTER-VI
CONCLUSION
Conclusion
232
6. Conclusion
As biocatalysts and analytical tools, thermozymes have to date gained a great deal of
interest as novel enzymes with unique properties, and are expected to fill the gap between
biological and chemical industrial processes. In this present research work, focus was on
the development of such industrially relevant thermostable and persuasive enzymes.
Therefore, two novel cellulolytic genes (TnbglA and TnbglB) from a hyperthermophilic
bacterium T. naphthophila RKU-10T, were cloned and overexpressed in E. coli BL21
CodonPlus (DE3)-RIPL, a mesophilic expression host. Sequencing and alignment studies
demonstrated that β-glucosidase A (TnBglA) and β-glucosidase B (TnBglB) are belonged
to GH family 1 and 3, respectively. Several cultivation and induction strategies were
adopted to achieve the optimal production and expression of recombinant TnBglA and
TnBglB. Culturing in optimal modified medium (4×ZB) and induction with lactose (150
mM) significantly enhanced the transformant growth (optical density) and heterologous
expression of both proteins. Hence lactose in contrast to IPTG, has proved as an efficient,
cheap and non-toxic alternative inducer. It has also recognized that optimal fermentation
conditions such as pre-induction optimal density, heat shock treatment, inducer
concentration, agitation, pH of medium, inducement temperature and time, would be
valuable aspect to increase the production, expression and proper folding of recombinant
proteins. This research provides two useful novel β-glucosidases (TnBglA and TnBglB)
which displayed favorable properties as high thermostability, glucose and xylose tolerance,
independence of metallic cations, and high catalytic activity. All these prominent features
make both enzymes potential candidates to be used in SSF processes for production of
bioethanol as well as suitable for paper, textile, pharmaceutical, cosmetic, animal feed,
food and juices, and these enzymes have many other industrial and biotechnological
applications.
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