MOLECULAR CLONING OF CYCLODEXTRIN GLUCANOTRANSFERASE
GENE FROM Bacillus sp. G1
ONG RUI MIN
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Master of Engineering (Bioprocess)
Faculty of Chemical and Natural Resources Engineering
Universiti Teknologi Malaysia
AUGUST 2005
iii
Dedicated to my beloved parents, siblings and brothers
and sisters in Christ for their love and encouragement
iv
ACKNOWLEDGEMENTS
My deepest thanks and praise to God for His love and blessings throughout
the research and preparation of this thesis.
I would like to express my sincere appreciation and gratitude to my
supervisor, Associate Professor Dr. Rosli bin Md. Illias for giving me an opportunity
to be involved in this research as well as for his invaluable advice, useful guidance,
encouragement and support through out the duration of the research. I would like to
extend my thanks to En. Nik Azmi for his helpful suggestions and advice.
I sincerely thank all the laboratory staff of the Department of Bioprocess
Engineering, UTM; Encik Yaakop, Encik Abdul Malek and Encik Muhamad for
their kind assistance and cooperation throughout the work. I am also extremely
grateful to my laboratory colleagues; Mohd. Khairizal, Ho Kek Sian, Nadzarah, Wan
Salwanis, Rozaimi, Azmil, Naqiah, Evelyn Wong, Kamalesh, Goh Kian Mau, Liew
Chong Wai, Lo Po Kim, Chee Chit Lai, Nadia, Amelia and Tiong Swee Ling for
their support, companionship and friendship.
I would also like to thank Universiti Teknologi Malaysia for providing
financial aid, facilities and opportunity for me to pursue my studies.
Last but not least, I would like to convey my gratitude and appreciation to my
beloved family for their love, support, help and understanding during my studies. I
would also be glad to extend my gratitude to Pastor Kwong Lie and family as well as
the brothers and sisters in Hope of God Johore Bahru for their love, encouragement,
support and prayers for me.
v
ABSTRACT
The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and sequenced. The 16s rRNA gene sequence of Bacillus sp. G1 was compared with 18 closet neighbours and it exhibited the closest match with that from Bacillus sp. NER (99%). Based on this finding, Bacillus sp. G1 are considered to be a member of the Bacillus rRNA group 5 and identified as Bacillus sp. Determination of the nucleotide sequence revealed the presence of an open reading frame of 2610 bp beginning with a TTG initiation codon which encodes a typical signal peptide of 29 amino acid residues followed by the mature enzyme of 674 amino acid residues. The mature CGTase corresponds to a calculated molecular weight of 75389 Da which is very close to the molecular weight of the wild type Bacillus sp G1 (75kDa) estimated through SDS-Page. A putative Shine-Dalgarno sequence AAGG was located 5 bp upstream of the TTG codon. The deduced amino acid sequence of the mature gene Bacillus sp G1 showed the highest homology of 98.3%, with 95% identity to alkalophilic Bacillus sp 1-1. The three catalytic residues Asp221, Glu249 and Asp320 in conserved regions II, III and IV respectively was found in CGTase from Bacillus sp. G1. 11 strictly conserved residues of the raw-starch binding motif were also found in Domain E. Some of the key residues and regions to product specificity are identified which are Tyr188, His47, Phe252, 38ETNPNY44 and 82HP---SGY85. The recombinant CGTase was expressed in the same pUC19 vector in E.coli and partially purified with ammonium sulphate precipitation. The optimum pH and temperature of the partially purified recombinant CGTase were 6.0 and 60oC. The pH stability was from pH 7.0 to pH 9.0 and the activity was retained up to 50oC after 30 minutes incubation at pH 6.0 in 0.1 M phosphate buffer without any substrate. The partially purified recombinant CGTase was able to prolong its thermal stability up to 60oC in the presence of 20 mM Ca2+. The CGTase was strongly inhibited by Zn2+, Cu2+, Co2+, Fe2+ and Fe3+.The CGTase produced - and -CD in a ratio of 0.11: 0.89 to total CD produced from 50 g/l tapioca starch after 24 hours incubation at 60oC, without adding any selective agents. The amount of -CD produced was 3.79 g/l. The partially purified CGTase from the recombinant E.coli retained properties quite similar to those of the wild type CGTase Bacillus sp. G1 in terms of molecular mass, reaction conditions, stability and the production of cyclodextrins.
vi
ABSTRAK
Skop penyelidikan ini termasuk mengenalpasti 16S rRNA bacteria dari Bacillus sp. G1, memencilkan dan mengklon gen siklodekstrin glukanotransferase (CGTase, EC 2.4.1.19) dari Bacillus sp. G1, menganalisa jujukan nukleotida dan jujukan asid amino gen CGTase, mengekpres CGTase rekombinan di dalam E.colidan pencirian CGTase rekombinan separa tulen. Jujukan nukleotida 16S rRNA Bacillus sp. G1 telah dibandingkan dengan 18 jujukan nukleotide yang hampir sama dan Bacillus sp. NER menunjukkan peratus persamaan yang tertinggi (99%). Berdasarkan ini, Bacillus sp. G1 dikategorikan dalam kumpulan 5 Bacillus rRNAand dikenalpasti sebagai Bacillus sp. Gen CGTase dari Bacillus sp. G1 telah dipencilkan dan jujukan nukleotidanya telah ditentukan. Analisis jujukan nukleotida ini menunjukkan satu rangka bacaan terbuka sepanjang 2160 bp, dengan TTG sebagai kodon permulaan dan peptida isyarat yang mempunyai 46 asid amino diikuti oleh jujukan peptida CGTase sebanyak 674 asid amino. Satu jujukan nukleotida Shine-Dalgarno, AAGG telah dikenalpasti kira-kira 5 bp sebelum kodon TTG. Jujukan asid amino CGTase daripada Bacillus sp. G1 menunjukkan 98.3% homologi dan 95% identiti dengan CGTase daripada alkalofilik Bacillus sp. 1-1. Tiga asid amino katalitik iaitu Asp221, Glu249 dan Asp320 di kawasan II, III dan IV telah dikenalpasti wujud di dalam CGTase Bacillus sp. G1. Siklodekstrin glukanotransferase rekombinan telah diekspres di dalam E.coli dan penulenan separa telah dilakukan melalui pemendakan ammonium sulfat. Enzim separa tulen siklodekstrin glukanotransferase menunjukkan pH optimum 6.0 dan suhu optimum 60ºC. Enzim ini stabil dari pH 6.0 ke 10.0 dan mengekalkan aktivitinya apabila di eram sehingga suhu 50ºC selama 30 minit dalam penimbal fosfat pH 6.0 tanpa kanji. Kestabilan suhu telah meningkat dengan penambahan 20mM Ca2+ tetapi aktiviti direncatkan dengan penambahan Zn2+, Cu2+, Co2+, Fe2+ dan Fe3+. Siklodekstrin glukanotransferase ini menghasilkan -CD dan -CD dalam nisbah 0.15:0.85 dari jumlah CD yang dihasilkan apabila ditindakbalas dengan 70 g/l kanji ubi kayu selama 24 jam pada suhu 60ºC, tanpa panambahan sebarang agen selektif. Jumlah -CD yang terhasil adalah 16.47 g/l. Gen CGTase dari Bacillus sp. G1 alkalophilik telah diklonkan dengan berjaya dan telah diekpres ke luar medium kultur.
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TABLE OF CONTENTS
CHAPTER TITLE PAGE
TITLE i
DECLARATION ii
DEDICATION iii
ACKNOWLEDGEMENTS iv
ABSTRACT v
ABSTRAK vi
TABLE OF CONTENTS vii
LIST OF TABLES xiii
LIST OF FIGURES xiv
LIST OF SYMBOLS / ABBREVIATIONS xvii
LIST OF APPENDICES xx
1 INTRODUCTION 1
1.1 Introduction 1
1.2 Objective of The Research 3
1.3 Scope of The Research 3
2 LITERATURE REVIEW 4
2.1 Microorganism 4
2.1.1 Alkalophilic Microorganism 4
viii
2.1.2 Alkalophilic Enzyme 5
2.1.3 The Genera of Bacillus 6
2.1.4 Classification and Phylogeny 7
2.1.5 The -amylase family 8
2.2 Cyclodextrin Glucanotransferase 10
2.2.1 Properties and Sources 11
2.2.2 Reactions Catalyzed by Cyclodextrin 12
Glucanotransferase
2.2.3 Cloning of CGTase Gene 14
2.2.4 Sequence Comparison of Various 16
CGTase and -amylase
2.2.5 Structural Characteristics of CGTase 17
2.2.6 Catalytic Mechanism of CGTase 21
2.2.7 Cyclization Activity of CGTase 24
2.3 Cyclodextrin 26
2.3.1 History of Cyclodextrins 26
2.3.2 Properties of Cyclodextrins 28
2.3.3 Applications of Cyclodextrin 30
2.2.3.1 Pharmaceuticals 31
2.2.3.2 Food 31
2.2.3.3 Environmental 32
2.3.3.4 Analytical Chemistry 32
2.3.3.5 Pesticide 32
2.3.3.6 Cosmetics and Toiletries 33
3 MATERIALS AND METHODS 34
3.1 Bacterial Strains 34
3.2 Chemicals 34
3.3 Preparation of bacterial glycerol stocks 35
3.4 Medium and Agar Plates Preparation 35
3.4.1 Horikoshi Broth and Agar 35
3.4.2 Luria-Bertani Broth and Agar 35
ix
3.5 General DNA Techiques 36
3.5.1 Agarose gel preparation 36
3.5.2 Genomic DNA Extraction 36
3.5.3 Isolation of Plasmid DNA 37
3.5.4 Restriction Digest of DNA 38
3.5.5 Ligation of DNA 38
3.5.6 DNA Quantification 38
3.5.7 Elution into Troughs 39
3.5.8 Design and Synthesis of 39
Oligodeoxyribonucleotide for DNA Sequencing
3.6 Bacterial Identification by 16S rRNA 40
3.6.1 PCR Amplification of 16S rRNA Gene 40
3.6.2 16S rRNA Sequencing 41
3.6.3 16S rRNA Sequence analysis 42
3.7 Synthesis of the CGTase Probe 43
3.7.1 Polymerase Chain Reaction (PCR) 43
3.7.2 Cloning of the PCR fragment 44
3.7.3 Nucleotide Sequence Determination and 44
Analysis
3.7.4 Southern Blotting 45
3.7.5 Pre-Hybridization and Hybridization 46
3.7.6 Detection of Nucleic Acid 47
3.8 Genomic Library Construction 47
3.8.1 Restriction digest of DNA 47
3.8.2 Dephosphorylation of DNA 48
3.8.3 Preparation of Competence Cells 48
3.8.4 Ligation of DNA 49
3.8.5 Transformation into E.coli Cells 50
3.8.6 Screening of Transformants 50
3.8.7 Nucleotide Sequence Determination and 51
Analysis
3.8.8 Secondary Structure Prediction 51
x
3.9 Partial Purification of Recombinant CGTase 53
from Bacillus sp. G1
3.9.1 Preparation of Bacterial Inoculum 53
3.9.2 Preparation of Crude Enzyme 53
3.9.3 Partial Purification of Recombinant CGTase 54
3.9.4 CGTase Enzyme Assay 54
3.9.5 Protein Determination 55
3.10 Characterization of Partially Purified CGTase 56
3.10.1 Molecular Weight Determination of 56
CGTase by SDS-PAGE
3.10.2 Effect of pH on Partially Purified 57
CGTase Activity
3.10.3 Effect of Temperature on Partially 57
Purified CGTase Activity
3.10.4 Effect of pH on the Stability of Partially 58
Purified CGTase Activity
3.10.5 Effect of Temperature on the Stability 58
of Partially Purified CGTase Activity
3.10.6 Effect of Calcium Ion on the Thermal 59
Stability of Partially Purified CGTase
3.10.7 Effect of Metal Ions and Reagants 59
on CGTase Activity
3.11 Analysis by HPLC 59
3.11.1 The analysis conditions of HPLC 59
3.11.2 Analysis of Cyclodextrins Production 60
by HPLC
4 RESULTS AND DISCUSSION 61
4.1 Bacterial 16S rRNA Identification 61
4.1.1 PCR Amplification of 16S rRNA from 61
Bacillus sp. G1
4.1.2 Sequencing and Analysis of the 16S rRNA 62
PCR Amplified Fragment
xi
4.2 Isolation of CGTase Gene from Bacillus sp. G1 67
4.2.1 Amplification of Bacillus sp.G1 fragment 67
4.2.2 Cloning of the PCR fragment 68
4.2.3 Nucleotide Sequence Determination and 69
Analysis
4.2.4 Southern Blotting 73
4.3 Genomic Library Constuction 74
4.3.1 Complete Digestion of Genomic DNA 74
4.3.2 Complete digestion of pUC 19 and 74
dephosphorylation
4.3.3 Screening of Transformants 75
4.3.4 Nucleotide Sequence of CGTase gene 77
4.3.5 Codon Usage 82
4.3.6 Amino Acid Composition 87
4.3.7 Amino Acid Sequence Comparison 89
4.3.8 Secondary Structure 95
4.3.9 The CGTase Protein 95
4.3.9.1 Domains 95
4.3.9.2 Active Site Residues 99
4.3.9.3 Raw Starch Binding Domain 100
4.3.9.4 Cyclization 101
4.3.9.5 Product Specificity 103
4.3.9.6 Thermostability 113
4.3.9.7 pH Optimum 113
4.4 Expression and Partial Characterization of recombinant 114
Bacillus sp. G1 CGTase
4.4.1 Partial Purification of recombinant Bacillus sp.
G1 CGTase 116
4.4.2 Characterization of the Partially Pure 116
Recombinant CGTase
4.4.2.1 Molecular Weight Determination 116
4.4.2.2 Effect of Temperature on Enzyme 121
Activity and Stability
xii
4.4.2.3 Effect of pH on Enzyme Activity 121
and Stability
4.4.2.4 Effect of Metal Ions 124
4.4.3 Analysis by HPLC 126
4.4.3.1 Effects of Different Type of Starches 126
on Cyclodextrin Production
4.4.3.2 Effects of Tapioca Starch 127
Concentration on CDs Production
5 CONCLUSIONS 131
5.1 Conclusions 131
5.2 Further Works 133
LIST OF REFERENCES 135
APPENDICES A-C 154
xiii
LIST OF TABLES
TABLE TITLE PAGE
2.1 Ribosomal RNAs in Prokaryotes 7
2.2 The members in the -amylase family (Jana ek, 1997) 9
2.3 CGTases that were cloned 15
2.4 Properties of -, - and -cyclodextrins 28
3.1 The primers for sequencing of the 16S rRNA sequence 42
3.2 The primer used for sequencing of the 800kb PCR product 45
3.3 The primers for sequencing of the 2kb PCR product 52
4.1 Statistical report of Nucleotide Sequence Identities and 65
Similarities
4.2 Codon usage in E.coli (Sharp and Li, 1986) 84
4.3 Codon usage of Bacillus sp. G1 86
4.4 Comparison for amino acid usage 88
4.5 Statistical report of Amino Acid Sequence Identities 93
and Similarities
4.6 Comparison of the amino acid residue around the active 105
site in 20 CGTases grouped into six types of CGTase,
-CGTase, -CGTase-, - CGTase, / -CGTase,
/ -CGTase (no -CD) and hyperthermophilic CGTase
4.7 The amino acid sequence data 106
4.8 Distribution of CGTase activity in different fractions for 115
E.coli harboring the Bacillus sp. G1
4.9 Summary of the purification of the CGTase from Bacillus sp. G1 116
4.10 Effects of metal ions & reagents on recombinant CGTase 125
activity
4.11 Effects of tapioca starch concentration on CDs production 130
xiv
LIST OF FIGURES
FIGURE TITLE PAGE
2.1 Schematic representation of CGTase catalysed reactions 13
2.2 Views of calcium binding sites of CGTase from 19
alkalophilic Bacillus sp. 1011
2.3 Ribbon drawing of the structure of CGTase Bacillus 20
circulans strain 251.
2.4 The CGTase reaction mechanism 22
2.5 Schematic representation of cyclization reaction 23
catalyzed by CGTase
2.6 Schematic representation of the hydrogen bonds between 25
the CGTase Bacillus circulans 251 and a maltononaose
inhibitor bound at the active site
2.7 Structure of -, - and -cyclodextrins (CDs) 28
2.8 Schematic representation of the formation of 30
inclusion complex
3.1 The blotting apparatus 46
4.1 Amplified 1.5 kb PCR band in agarose gel 62
4.2 16S rRNA gene of Bacillus sp G1 consisting of 1540 bp 63
4.3 Sequence Match results showing Taxonomic Hierarchy 63
4.4 Sequence Match results showing phylogeny of Bacteria 64
4.5 Unrooted phylogenetic tree based on 16S rRNA 66
sequences showing the relationship of Bacillus sp. G1
with 18 closest sequence match
4.6 PCR band in agarose gel 68
4.7 Recombinant plasmids after digestion in agarose gel 69
xv
4.8 Nucleotide sequence of 831 bp and corresponding 277 70
deduced amino acid of plasmid PP2
4.9 Alignment of amino acid sequence of the four highly 71
conserved regions for members of -amylase family
of glycosylases.
4.10 Alignment of plasmid PP2 with other known CGTases 71
and -amylase.
4.11 Relationship among the known CGTase gene. 72
Dendrogram created with Clustal X (1.83)
4.12 Complete digestion of DNA and the result of Southern Blot 73
4.13 Digested Genomic DNA 74
4.14(a) Blue/white selection on LB-IPTG-XGal supplemented 76
with ampicillin
4.14(b) Screening for zone clearing colonies on 76
LB-IPTG-XGal-starch plate
4.15(a) Top row - Uncut plasmids 77
4.15(b) Bottom row - XbaI digested plasmid 77
4.16 Nucleotide sequence of the cyclodextrin 79
glucanotransferase (CGTase) gene of Bacillus sp.G1
4.17 Identifiable domains of signal peptide of secretory 81
proteins – the amino (n-) region, the hydrophobic (h-) region
and the carboxyl-terminal (c-) region.
4.18 Comparison of the deduced amino acid sequences of 92
typical -, -, - CGTases and -amylase to the
-CGTase from Bacillus sp. G1
4.19 Dendrogram constructed using Clustal X (1.83) 94
4.20 Secondary structure predictions 96
4.21 Domain organization of CGTase and -amylase 98
4.22 Putative conserved domain detected by BLASTX 98
4.23 Amino acid sequence alignment of the region from 107
amino acid residue 154 to 159
4.24 Amino acid sequence alignment of the region from 108
amino acid residue 87 to 93 at subsite
xvi
4.25 Amino acid sequence alignment of the region 109
around residue 47 at subsite -3
4.26 Amino acid sequence alignment of the region 110
around residue 89 at subsite -3
4.27 Amino acid sequence alignment of the region 111
around residue 259 at subsite +2
4.28 SDS-PAGE 117
4.29 A cleared zone formed on phenolphthalein plate 1 118
indicates the presence of recombinant -CD
4.30 Optimum temperature of the recombinant CGTase. 120
The optimum temperature of the partially purified
CGTase was determined to be 60ºC
4.31 Thermal stability of the recombinant CGTase. 120
4.32 Optimum pH of the recombinant CGTase. The 122
optimum pH of the partially purified CGTase was
determined as pH 6.0
4.33 pH stability of the recombinant CGTase. The partially 122
purified enzyme was stable from pH 7.0 – 10.0 at 60ºC
in 30 minutes incubation.
4.34 Production of CD by recombinant Bacillus sp. G1 127
CGTase on various starch sources, namely tapioca,
soluble, sago, potato and corn.
4.35 Time course of CDs production by recombinant 128
Bacillus sp. G1 CGTase with 10 g/l tapioca starch as substrate
4.36 Time course of CDs production by recombinant 128
Bacillus sp. G1 CGTase with 50 g/l tapioca starch as substrate
4.37 Time course of CDs production by recombinant 129
Bacillus sp. G1 CGTase with 10 g/l tapioca starch as substrate
xvii
LIST OF SYMBOLS/ABBREVIATIONS
g - Microgram
m - Micrometer
Å - Angstrom
°C - Degree Celcius
% - Percentage
A - Absorbance
Ala - alanine
Amp - Ampicillin
Arg - arginine
Asn - asparagine
Asp - aspartic acid
blast - Basic local alignment search tool
BSA - bovine serum albumin
bp - base pairs
Cys - cysteine
CD - Cyclodextrin
CGTase - Cyclodextrin glucanotransferase
DNA - deoxyribonucleic acid
DNase - deoxyribonuclease
DMSO - dimethyl sulphoxide
E.coli - Escherichia coli
EC - Enzyme Commission
EDTA - ethylene diamine tetraacetic acid
g - Gram
Gln - glutamine
xviii
Glu - glutamic acid
Gly - glycine
HCl - hydrochloric acid
His - histidine
HPLC - high performance liquid chromatography
Ile - isoleusine
IPTG - isopropyl- -D-thiogalactopyranoside
kb - kilo base
kDa - kilo Dalton
L - Liter
LB - Luria Bertani
Leu - leusine
Lys - lysine
M - Molar
MBS - maltose binding site
Met - methionine
mg - Miligram
min - minute(s)
ml - Milliliter
MW - molecular weight
NaCl - sodium chloride
NaOH - sodium hydroxide
NCBI - National Center for Biotechnology Information
ng - Nanogram
ORF - Open reading frame
OD - optical density
pmol - picolmole
PAGE - polyacrylamide gel electrophoresis
PCR - polymerase chain reaction
Phe - phenylalanine
Pro - proline
RNase - ribonuclease
rpm - revolution per minute
rRNA - ribosomal RNA
xix
RNase - ribonuclease
SDS - sodium dodecyl sulphate
Ser - serine
TEMED - N,N,N’,N’-tetramethylene-ethylenediamine
Thr - threonine
Tm - melting point
Tris - 2-hydroxymethyl-2-methyl-1,3-propanediol
Trp - tryptophan
U - unit enzyme
UV - ultraviolet
Val - valine
V - Volts
v/v - Volume per volume
w/v - Weight per volume
X-Gal - 5-bromo-4-chloro-3-indolyl- -D-galactopyranoside
xx
LIST OF APPENDICES
APPENDIX TITLE PAGE
A Materials and Methods 154
B Experiment Data 165
C Publications 176
CHAPTER 1
INTRODUCTION
1.1 Introduction
Cyclodextrin glucanotransferase (EC 2.4.1.19) is an industrially important
enzyme that produces -, - and -cyclodextrins (CDs) from starch through an
intramoleculer transglycosylation reaction. The -, - and -CD have closed ring
structures with six, seven and eight glucose units joined by - 1,4-glucosidic bonds
respectively. The exteriors of CDs are hydrophilic while the interiors are
hydrophobic, enabling it to easily form inclusion complexes with either organic or
inorganic molecules. The encapsulated guest undergoes advantageous physical and
chemical changes, such as improved stability and water solubility in a variety of fine
organic and inorganic compounds, sharply reducing volatility, chemical, thermal and
light reactivity of guest molecules, stabilization and protection of sensitive hosts such
as flavors, odor and aroma. As a result, CDs and its derivatives have wide
application in today’s industries such as food, cosmetics, chemicals, environmental,
agricultural, cosmetics, pharmaceuticals and toiletries.
The CGTase enzyme is generally found in bacteria and a wide variety of bacteria
have been determined as CGTase producers, namely aerobic mesophilic bacteria,
aerobic thermophilic, anaerobic thermophilic and aerobic alkalophilic bacteria.
Various genera of bacteria that are known as CGTase producer includes Bacillus,
Klebsiella, Pseudomonas, Brevibacterium, Thermoanaerobacterium,
Corynebacterium, Micrococcus, and Clostridium etc (Gawande et al., 1999).
2
Most CGTases produce a mixture of -, - and -CD in different ratios,
depending on the origin of the CGTase as well as the reaction conditions. CGTase is
classified into three different types, -CGTase, -CGTase and -CGTase according
to the major CD produced. As a result, industrial production process of CDs and the
subsequent separation process are rather elaborate and costly as expensive
purification procedures are needed. Furthermore, there is the consideration of
solvent toxicity, flammability and the need for a solvent recovery process which is an
added disadvantage. Besides, the complete removal of solvent from the CDs is
expensive, limiting the use of CDs in the pharmaceutical and food industries (Biwer
et al., 2002).
Therefore, the availability of CGTase enzymes that are capable of producing
an increased ratio of one particular type of CD and also with reduced product
inhibition would help to avoid using expensive and environmentally harmful
procedures involving organic solvents. On top of that, it is also desirable to develop
a novel CGTase that is better to produce CD in high proportion from starch for
industrial and biochemical studies. In order to achieve this, genetic engineering or
molecular biotechnology technique will facilitate the process of obtaining better
enzymes.
The attractive feature of CGTase from Bacillus sp. G1 is that it
predominantly produced -CD (89%) from tapioca starch and this high yield can be
increased to 100% yield of -CD with the addition of 4% (v/v) Triton X-100 (Ho et
al., 2005). Therefore, CGTase from Bacillus sp. G1 can be considered as a good
model enzyme for further studies of -CD production. It is also a potential candidate
for commercialization and industrial production of -CD due to its stability, high
specificity in -CD production, versatility and ease of handling.
3
1.2 Objectives of the Research
The main objective of this research is to clone a cyclodextrin
glucanotransferase gene (CGTase) from Bacillus sp. G1 and to express it.
1.3 Scope of Research
There are five scopes in this research:
(a) Bacterial 16S rRNA Identification
(b) Isolation and cloning of CGTase gene from Bacillus sp. G1
(c) Analysis of nucleotide sequence of CGTase gene and its deduced amino
acid sequence
(d) Expression of CGTase in E.coli
(e) Characterization of the partially purified recombinant CGTase
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