Chapter-2

CLONING OF CELLULASE GENES FROM

CELLULOLYTIC BACTERIA

2.1 Introduction

The cellulase enzymes have attracted considerable attention in recent years due to their

great biotechnological and industrial applications. Conversion of food, industrial and agricultural

wastes in to valuable sugars is the potential use of cellulase enzymes (Bothast and Saha, 1997).

Cellulases are being studied because of their application in the hydrolysis of cellulose, the most

abundant biopolymer and potential source of utilizable sugars. These serve as raw materials in

the microbial production of a wide variety of chemicals, food and fuel. Cellulose is hydrolysed

by a multicomponent cellulase system of microorganisms to glucose.

The major enzymes involved in cellulose hydrolysis are endo and exo- glucanases which

act synergistically to convert crystalline cellulose into cellobiose which is then converted to

glucose by the action of β-glucosidase (Mandels, 1982). The structural genes encoding these

enzymes have been isolated and identified from cellulolytic bacteria and expression of the

cellulase genes in a cellulase free host microorganism has also been achieved (Penttila et al.,

1986).

Enzymatic hydrolysis of cellulosic wastes may give a relatively pure product with the

consumption of less energy during the process (Fennington et al., 1982). Substantial efforts have

been made by enzyme suppliers and industrial users to improve existing cellulase enzymes.

Consequently, thermophiles are being aggressively pursued to provide new enzymes that are

highly thermostable depending on the environment of the native organism. Since biomass is

abundant and reasonably inexpensive, the key to successful commercialization of the process

utilizing biomass is the development of efficient and economical conversion methods such as

enzyme hydrolysis. Cellulolytic enzymes are synthesized by a number of fungi and bacteria are

the major natural agents responsible for cellulose degradation. The bioconversion of cellulosic

material into useful products has become one of the priority areas of research (Lederberg, 1992).

In recent years recombinant DNA technology has provided techniques for isolating,

characterizing and manipulating the genes of interest. It is expected that the use of recombinant

DNA technology applied to the cellulase system will facilitate a better understanding of the

catalytic and regulatory activity of the cellulase enzymes. This would also give insights into the

nature of the cooperative interactions between different enzymes that are involved in the

complete hydrolysis of cellulose, the development of expression systems for the utilization of

natural cellulose into usable products. Development of novel cellulolytic microorganisms by the

introduction of exogenous genes would enable other properties of the host organism to be

exploited more fully. Much of the recent success in isolating different endoglucanase genes can

be attributed to the use of the simple yet powerful Congo Red-carboxymethylcellulose overlay

technique for selecting recombinant clones that express endoglucanase activity (Teather and

Wood, 1982). By genetic manipulation of the genes and coordinate expression of enzymes it

shall be possible to transform a noncellulolytic organism to cellulose utilizing microorganisms.

The noncellulolytic organisms that are used for the expression of cellulase genes are

mainly S. cerevisiae, Z. mobilis and E. coli. They are all proved suitable for industrial

applications. The present effort is to express a functional cellulase system, in order to enable

non-cellulolytic bacteria to hydrolyse and utilize cellulosic biomass. There are good tools for the

genetic manipulation of them, but as cellulase systems are very complex and is not fully

understood, it is still not so easy to achieve this goal. Nevertheless a lot of work has been done

for cloning of cellulase genes and studying the cellulolytic strategy and it is still progressing.

This chapter explains the cloning of cellulase gene from five different cellulolytic bacteria

isolated from insects gut into E. coli using a high copy number stable plasmid, pET20b(+).

2.2 Materials and Methods

2.2.1 Bacterial strains and culture media

The isolated cellulolytic bacterial strains such as, P. mirabilis, P. aeruginosa,

K. pneumoniae, P. fluorescens and E. cloaceae were used for isolating cellulase gene. E.coli

harboring pET20b(+) (AmpR, T7 expression vector, size of plasmid 3.7 kb) was kindly provided

by Dr. S. Krishanaswamy, School of Biotechnology, Madurai Kamaraj University, Madurai.

E. coli cells harbouring (pET20b(+) plasmid grown on Luria agar with ampicillin (50μg/ml)

under static condition at 37oC and the plasmid was used for cloning of cellulase gene.

2.2.2 Genomic DNA isolation from cellulolytic bacteria

Genomic DNA of cellulolytic bacteria such as P. mirabilis, P. fluorescens,

P. aeruginosa, K. pneumoniae and E. cloacae was isolated as described by Sambrook and Russel

(2003). About 1.5ml of overnight culture of bacteria was centrifuged at 10,000 rpm for 5 min.

The pellet was taken after decanting the culture supernatant. The cells were resuspended and

lysed in 200 µl of lysis buffer by vigorous pipetting. Followed by 66µl of 5M NaCl was added to

remove most of the cell debris and proteins. The tubes were incubated at -20oC for 10min and

were centrifuged for 10min at 12000 rpm at 40C. The supernatant was carefully transferred to a

fresh sterile tube without disturbing the pellet (debris) using a micropipette and extracted once

with equal volume of phenol: chloroform (1:1 mixture). Subsequently the upper aqueous phase

was extracted once with chloroform: isoamyl alcohol (24:1) and once with ether. Final aqueous

phase containing the DNA was transferred to a sterile tube and the DNA was precipitated by

adding 4 volumes of ice cold 100% ethanol and incubating 1 hr in -200C incubator. DNA was

collected by centrifuging the content at 10,000 rpm, 10 min at 100C. The DNA pellet was

collected and washed once with 70% ethanol and air dried for 5min. Finally, the DNA pellet

obtained was dissolved in 100μl TE buffer and quantified by UV spectrophotometrically

(Sambrook and Russel, 2003).

2.2.3 Plasmid DNA isolation from E. coli contain pET 20b (+) by mini preparation method

Single colony harbouring E.coli-pET20b (+) plasmid was cultured overnight with

ampicillin (100μg/ml) at 37oC in 3 ml LB. It was subcultured at the rate of 10 ml of overnight

culture/ liter and grown till 1 OD at 600 nm. Cells from 1.5ml of the culture were harvested by

centrifugation at 10,000 rpm for 5min at 40C. The supernatant has been decanted and the cells

were resuspended in 100µl of TEG buffer (pH8.0).After the cells were resuspended in 200μl of

NaOH/ SDS buffer was added and mixed well by gentle inversion till the cells lysed completely.

Then 150μl of 3M sodium acetate (pH 4.6) was added and gently mixed by inversion. The tubes

were incubated in ice for 30 min and were centrifuged for 10min at 12,000 rpm at 4oC. The

supernatant was carefully transferred to a fresh sterile tube without disturbing the pelletted debris

precipitate using a micropipette. Then, 720μl of isopropanol was added to the supernatant and

mixed gently, kept at room temperature for 10 min and centrifuged at 12,000 rpm for 10 min at

10oC. The pellet (DNA) was collected and washed once with 70% ethanol and air dried for 5min.

Finally, the pellet obtained was dissolved in 100μl TE buffer and stored at 4oC. The DNA was

further purified by extraction with phenol: chloroform (1:1) once and once with chloroform:

isoamyl alcohol (24:1) and finally reprecipitaed with ethanol. The DNA sample was checked on

an agarose gel and quantified using UV absorbance spectrophotometry at 260 nm (Sambrook and

Russel, 2003).

2.2.4 Restriction digestion of genomic DNA of cellulolytic bacteria

2 μg of purified chromosomal DNA of cellulolytic bacteria was digested with BamHI

enzyme for 1 hr at 370C and the reaction was stopped by heating to 65oC for 10 min and purified

by extraction through phenol choloroform and reprecipitated by ethanol.

2.2.5 Restriction digestion of plasmid pET20b(+)

The purified 2 μg of plasmid pET20b(+) DNA was digested with 10 units of BamHI

enzyme for 1 hr at 370C and the reaction was stopped by heating to 65oC for 10 min and by

extraction through phenol choloroform and reprecipitated by ethanol (Sambrook and Russel,

2003).

2.2.6 Ligation of restricted chromosomal and plasmid DNA

The BamHI digested genomic as well as plasmid DNA were purified further and ethanol

precipitated, resuspended in TE buffer and used for ligation. Both genomic and plasmid DNAs

were mixed and ligated using T4 DNA ligase (Fermentase, Germany) at 16oC for 16 hr

(Sambrook and Russel, 2003).

2.2.7 Preparation of competent cells

Single colony of E. coli strain DH5α was inoculated into 3ml of Luria broth and

incubated for overnight in a rotatory shaker platform at a speed of 160 to 200 rpm. 0.5ml of the

overnight culture was subcultured in 50ml of fresh Luria broth. The culture was grown to an

O.D. of 0.6 at 600nm. The culture was chilled in ice for 30min and centrifuged at 6000rpm for 5

min at 4oC. The cell pellet was gently resuspended in equal volume of 0.1M ice-cold calcium

chloride (CaCl2) solution and was incubated in ice for 20-30 min. Then the cells were

centrifuged at 6000 rpm for 5 min at 4oC and the supernatant was discarded and the cell pellet

was taken and resuspended in 1/10th volume of original culture in 0.1M CaCl2 (ice-cold) and was

incubated in ice for 30 min (Sambrook and Russel, 2003).

2.2.8 Transformation of E. coli competent cells with ligated mix

100μl of competent cell suspension was taken in a sterile and chilled eppendorf tube. To

the competent cell suspension 10 μl of ligated mix was added and mixed well. The tube was

incubated in ice for 30 min. The cells were given a heat shocked at 42oC for 2min and were again

incubated on ice for 3min. 0.9ml of freshly prepared LB was added and incubated at 37oC with

gentle shaking. Then an aliquot of the expressed culture was plated on to a selective medium

containing ampicillin (100 μg/ml), IPTG (0.1mM/ml), 0.6% CMC and incubated at 37oC

incubator for 16 hr and the transformants were scored (Sambrook and Russel, 2003). The

recombinant colonies were subcultured on ampicillin plates for further screening.

2.2.9 Screening and selection of transformants by congo-red method

Congo red overlay technique (Teather and Wood, 1982) was used for screening and

selection of recombinant E. coli clones that have cellulase gene coding sequence. The positive

clones were selected by measuring their cellulase activity by cellulose hydrolysis assay. The

assay was performed in 25ml flask with 1x1 cm filter paper suspended in 5ml of 50mM sodium

acetate buffer (pH-4.8), to which the crude cellulase enzyme preparation (culture supernatant)

solution was added. The mixture was incubated at 450C for overnight in a shaking water bath

adjusted to 150 rpm. After incubation reducing sugar content in the supernatant was analysed by

dinitrosalicylic acid method (Ghose, 1987).

2.2.10 Restriction analysis of cellulase gene clones

Plasmids DNA from positive clones were isolated by alkaline lysis method (Sambrook

and Russel, 2003). The isolated plasmid was purified and restricted with BamHI enzyme. The

BamHI digested cellulase gene clones were analysed on 0.7% agarose gel along with BamHI

digested plasmid pET20b(+) and a molecular weight marker DNA. The size of the cellulase gene

insert was studied.

2.2.11 DNA sequencing

DNA sequencing was done through commercial DNA sequencing services (MWG,

Bangalore) as follows; the cycle sequencing reaction was performed using BigDye terminator

V3.1 cycle sequencing Kit containing AmpliTac DNA polymerase (from Applied Biosystems).

The sequencing reaction - mix was prepared by adding 1μl of BigDye v3.1, 2μl of 5x sequencing

buffer and 1μl of 50% DMSO. To 4µl of sequencing reaction mix was added 4 Pico moles of

primer (2μl) and sufficient amount of plasmid. The constituted reaction was denatured at 95°C

for 5 min. Cycling began with denaturing at 95°C for 30 sec, annealing at 52°C for 30 seconds

and extension for 4 min at 60°C and cycle repeated for a total 30 cycles in a MWG thermocycler.

The reaction was then purified on sepheadex plate (Edge Biosystems) by centrifugation to

remove unbound labelled and unlabelled nucleotides and salts. The purified reaction was loaded

on to 96 capillary ABI 3700 DNA analyzer and electrophoresis was carried out for 4 hr and the

sequence was read through automated DNA sequencing system.

2.3 Results

2.3.1 Cloning of cellulase gene in pET 20b(+)

The genomic DNA of E. cloacae, P. mirabilis, P. fluorescens, P. aeruginosa and

K.. pneumoniae were isolated by standard method (Sambrook and Russel, 2003). Plasmid pET

20b (+) was isolated by alkaline denaturation method. Both genomic DNA and pET20b (+)

plasmids were restricted with BamHI enzyme. The restricted fragments were ligated with

T4DNA ligase and the ligated DNA was confirmed by gel electrophoresis. The ligated DNA was

transformed into E. coli strain DH5α competent cells by CaCl2 method competent cell

transformation. The transformation efficiency was found as 103 transformants/ μg plasmid DNA.

2.3.2 Screening and selection of transformants

The transformants were selected on selective agar plates containing ampicillin (100

µg/ml) and CMC (0.6% w/v). The transformants forming clear zone around the colony during

Congored overlay method were further selected (Figs. 15 to 19). Efficient cellulase expressing

clones were further selected by cellulose hydrolysis method. The total cellulase enzyme activity

of selected clone is tabulated (Table 7 and Fig. 20). The number of cellulolytic clones in each

cloning experiments varied with bacteria. The number of cellulolytic clones obtained from

E. cloacae, P. mirabilis, P. fluorescens, P. aeruginosa and K. pneumoniae were 123, 101, 93, 67

and 53 respectively.

2.3.3 Restriction analysis

The plasmid DNA of transformants showing higher cellulolytic activity were isolated and

restricted with BamHI enzyme. The sizes of the insert DNA in each cellulolytic clone in the

genomic DNA library were determined by restriction analysis. The size of the insert were as

follows: pET-cel-Ec cellulase gene insert from E. cloacae (2.25kb) (Fig. 21), pET-cel-Pa

cellulase gene insert from P. aeruginosa (3 kb) (Fig. 22), pET-cel-Pf cellulase gene insert from

P. fluorescens (2.7 kb) (Fig. 23), pET-cel-Kp cellulase gene insert from K. pneumoniae (1.3 kb)

(Fig. 24) and pET-cel-Pm cellulase gene insert from P. mirabilis (1.3kb) (Fig. 25). The gene

cloning strategy used for cloning cellulase gene is explained in figure (26). The positive

cellulolytic clones selected from the cellulolytic bacteria were named based on their bacterial

origin. Accordingly recombinant pET20b(+) plasmid with the cellulase gene inserts from

E. cloacae was named as pET-cel-Ec, P. mirabilis was named as pET-cel-Pm, P. fluorescens was

named as pET-cel-Pf, P. aeruginosa was named as pET-cel-Pa and K. pneumoniae was named

as pET-cel-Kp.

2.3.4 Sequencing of cellulase gene

The cellulase gene clones such as pET-cel-Ec, pET-cel-Pa, pET-cel-Pm, pET-cel-Pf and

pET-cel-Kp were sequenced by automated sequencing and the sequences were analysed through

BLAST software. The cellulase gene sequences from the above five cellulolytic bacteria were

deposited in NCBI database. The accession numbers of the cellulase gene sequences are as

follows pET-cel-Ec is GQ368735, pET-cel-pf is HM235919, pET-cel-Kp is HM235918, pET-

cel-Pa is GQ872426 and pET-cel-Pm is HM235922. The cellulase gene cloned from

Enterobacter cloacae has a coding sequence of 1083 nucleotide, gene from P. mirabilis has a

coding sequence of 1196 nucleotide, gene from P. fluorescens has a coding sequence of 1104

nucleotide, gene from P. aeruginosa has a coding sequence of 2883 nucleotide and gene from K.

pneumoniae has a coding sequence of 1196 nucleotide. The nucleotide and amino acid sequences

of insert DNAs of the above five clones are given (Figs. 27 to 46).

Fig. 15 Screening of recombinant clones from Enterobacter cloacae on cellulose supplemented

agar

Fig. 16 Screening of recombinant clones from Proteus mirabilis on cellulose supplemented agar

Fig. 17 Screening of recombinant clones from Pseudomonas fluorescens on cellulose

supplemented agar

Fig. 18 Screening of recombinant clones from Pseudomonas aeruginosa on cellulose

supplemented agar

Fig. 19 Screening of recombinant clones from Klebsiella pneumoniae on cellulose supplemented

agar

Table 7 Total cellulase activity of cellulase gene clones

TransformantsFilter paper

activity (FPU/ml)Endoglucanase

activity (CMC/ml)Cellobiase (CB/ml)

pET-cel- Ec 0.171±0.09 8.68±0.02 2.7±0.05

pET-cel-Pf 0.146±0.01 5.77±-0.03 2±0.012

pET-cel-Pa 0.171±0.02 3.49±0.01 1.4±0.02

pET-cel-Kp 0.091±0.03 3.3±0.04 1.34±0.01

pET-cel-Pm 0.159±0.05 2.53±0.06 1.01±0.03

E. coli (pET20b(+)) 0 0 0

Fig. 20 Exoglucanase, endoglucanase and cellobiase activity of cellulase gene clones

0

1

2

3

4

5

6

7

8

9

10

pET-

cel-

Ec

pET-

cel-

Pf

pET-

cel-

Pa

pET-

cel-

Kp

pET-

cel-

Pm

E. c

oli

(pE

T20b

(+)

Cellulase gene clones

Tot

al c

ellu

lase

en

zym

e ac

tivi

ty

Filter paper activity (FPU/ml)

Endoglucanase activity (CMC/ml)

Cellobiase (CB/ml)

Fig. 21 Restriction analysis of cellulase gene cloned from Enterobacter cloacae

Lanes:

1. BamH1 digested pET-cel-Ec clone

2. Molecular weight marker (1Kb ladder)

3. BamH1 digested pET20b(+) plasmid vector

Fig. 22 Restriction analysis of cellulase gene cloned from Pseudomonas aeruginosa

Lanes:

1. BamH1 digested pET-cel-Pa clone

2. Molecular weight marker(1Kb ladder)

3. BamH1 digested pET20b(+) plasmid vector

Fig. 23 Restriction analysis of cellulase gene cloned from Pseudomonas fluorescens

Lanes:

1. BamH1 digested pET-cel-Pf clone

2. Molecular weight marker(1Kb ladder)

3. BamH1 digested pET20b(+) plasmid vector

Fig. 24 Restriction analysis of cellulase gene cloned from Klebsiella pneumonia

Lanes: 1. Molecular weight marker(1Kb ladder)

2. BamH1 digested pET20b(+) plasmid vector

3. BamH1 digested pET-cel-Kp clone

Fig. 25 Restriction analysis of cellulase gene cloned from Proteus mirabilis

Lanes:

1. BamH1 digested

pET-cel-Pm clone

2. Molecular weight

marker(1Kb ladder)

3. BamH1 digested

pET20b(+) plasmid

vector

Fig. 26 General strategy

used for cloning of

cellulase gene from

cellulolytic bacteria

Fig. 27 Nucleotide sequence of insert DNA in pET cel Ec (GQ368735) cloned from

Enterobacter cloacae strain JV

GGATCCTTATTCTGCACGGGCGATTACAGGGCAGCGAGCGTGAAACCTCGATTGGTCTGACGAAAGA

TAAGCAGGGCGACAGCAAAGTGCGCATCGACGGCACTGACGGGCACAAAGTCGCTGAGCTGGCTTTA

TTGATGCCGATGCAACTGATTACGCCTGAGGGATTTACATTACTCAACGGCGGCCCCAAATACAGAAG

AGCATTCCTTGATTGGGGATGTTTTCACAACGAAGCGGGTTTCTTTAACGCCTGGAGCAATCTCAAGCG

ACTGCTCAAACAGCGCAATGCCGCGCTGCGACAGGTCACCCGTTACGCTCAGGTGCGCCCGTGGGATA

TGGAACTGGTCCCCCTTGCGGAGCAAATTAGCCGCTGGCGCGCCGAATACAGCGCAGGTATTGCTGAA

GATATGGCGGATACCTGCAAACAATTTCTACCCGAGTTTTCTCTCACCTTCTCCTTCCAGCGTGGCTGG

GAGAAAGAGACGGATTATGCCGAAGTGCTGGAGAGAAGCTTCGAACGCGATCGCATGCTGACCTATA

CCGCGCACGGCCCACACAAAGCGGACTTCCGCATTCTCACCCGTGAAAGCGGGCGGCGCTGTCTGTAT

CTGATAGATGATTTTGCCTCGGAACTCGACGACGCACGGCGCGGACTGCTTGCCAGCCGCTTAAAAGC

CACGCAGTCGCAGGTTTTCGTCAGCGCCATTAGCGCTGAACACGTTCTGGACATGTCGGACAAAAATT

CGAAGATGTTCACCGTGGAAAAGGGTAAAATAACGGATTAACCCAAGAATAAATGAGCGTTCGTGCC

GACGGCGCACCGGTCGAAGATACCTTGTCGCGCGGACAGCTCAAACTCCTGATGTGCGCGCTGCGTTT

GGCGCAGGGGGAGTAGAAACGTTGATGGTCGCACTGGTCCTGGCGGCAGCGAATGCGCGTGCGGCCT

GTAGCTGGCCCGCGTGGGAGCAGTTTAAACAGGACTACATCAGCGATGGCGGGCGCGTGATTGATCCC

AGTGACGCGCGGAAAATCAGCACTTCGGAAGGGCAAAGCTATGCGCTGTTCTTTGCCCTGGCCGCCAA

CGATCGCAAAGCGTTCGATTTACTGCTGACCTGGACGAGCGACAATCTCGCCCAGGGCTCCCTGAGTC

AGCATCTGCCTGCCTGGTTGTGGGGGAAAAAGGATGCGGATACCTGGGCGGTGATCGACAAAAACTCT

GCGTCTGATGCGGATATCTGGATTGCCTGGTCGTTGCTGGAAGCGGGGCGTTTGTGGAAAGCGCCGCA

ATACACCGCCACCGGCAAAGCACTGCTAAAACGCATCGCCAGCGAAGAAGTGATCAAAGTGCCGGGT

TTAGGGCTGATGCTCCTGCCCGGCAACGTCGGTTTTACCGAGGAGAAAGCCTGGCGCTTTAACCCCAG

CTATCTCCCGCCGCAGCTGGCGAACTATTTCACCCGCTTTGGCGCGCCGTGGACCACGCTTCGCGAGAC

GAATCTGCGTTTACTGCTGGAAACCGCGCCAAAAGGATTTGCGCCCAACTGGGTGCAGTATCAGCAAA

AAAAAGGCTGGCAATTGCAGCCAGAAAAAACCTTTATCGGCAGTTACGACGCGATTCGCGTGTATCTC

TGGACGGGCATGATGCACGACCGCGATCCGCAAAAAGCCCGACTGCTGGCACGTTTTAAACCGATGGC

GACGCTCACAACAAAAAATGGCGTCCCGCCGGAGAAGGTCGATGTCGCAAGCGGTAAACCCACAGGC

GATGGCCCGGTCGGTTTCTCCGCCTCGCTGCTGCCTTTTTTACAGGACCGTGATGCACAAGCGGTGCAA

CGCCAGCGCGTCGCCGACCATTTTCCCGGCAATGACGCCTATTACAGCTACGTGCTGACCCTGTTCGGA

CAAGGATGGGATCAGCATCGTTTTCGCTTCACCGCAAAGGGTGAATTACACCCTGACTGGGGCCAGGA

ATGCGCAAGTTCTCATTAAACGTGCTGGATGTGTTGAATGCGCTGAAATGCGAGAACGTTCGCATTCT

GCTGACCGATTCTGTTTCAAGCGTGCAGATTGAAGATGCGGCTAGCCAAAGTGCAGCCTACGTCGTCA

TGCCAATGCGCCTCTAGTGGAAAATATCGGGCTATCTTACTTGCCATTTTCAACCTGGGCTGTGCTCGC

CCCTGTCACGTACTCCGTGTACGCTCCAGGGTCTGCGCGCAGTCCGCGTTGAAACTGGCTGCGCCGATT

ACGCCCTGGATCTCTGGATCAATGTACTGATATATGTCACTGACGCGCCTTCTCATCAAGGATCC

Insert Size 2,309bp

Coding region 1,083bp

Promoter

Cellulase gene coding region

Fig. 28 Restriction map of cellulase gene containing DNA cloned from Enterobacter cloacae strain JV

Fig. 29 Deduced amino acid sequence of cellulase gene cloned from Enterobacter cloacae strain JV

MVALVLAAANARAACSWPAWEQFKQDYISDGGRVIDPSDARKISTSEGQSYALFFALAANDRKAFDLLLT

WTSDNLAQGSLSQHLPAWLWGKKDADTWAVIDKNSASDADIWIAWSLLEAGRLWKAPQYTATGKALLK

RIASEEVIKVPGLGLMLLPGNVGFTEEKAWRFNPSYLPPQLANYFTRFGAPWTTLRETNLRLLLETAPKGFA

PNWVQYQQKKGWQLQPEKTFIGSYDAIRVYLWTGMMHDRDPQKARLLARFKPMATLTTKNGVPPEKVD

VASGKPTGDGPVGFSASLLPFLQDRDAQAVQRQRVADHFPGNDAYYSYVLTLFGQGWDQHRFRFTAKGE

LHPDWGQECASSH

Fig. 30 Restriction map of plasmid pET cel Ec having cellulase gene containing DNA cloned from Enterobacter cloacae strain JV

Fig. 31 Nucleotide sequence of insert DNA in pET cel Pf (HM235919) cloned from Pseudomonas fluorescence strain JV

GGATCCCGGCCAGACGGTCTACGGGACCGACTTCATTGCCGATAAGGTGGATTATCTGGACACCAAG

GCACCAGGCGGGTCAAATCAGGAATAAGGGCACATTGCCCCGGCGTGAGTCGGGGCAATCCCGCAAG

GAGGGTATGATTAACCGCAGCGTGCTGAAAATTCCGGCGCTGGTGACCCCGCTGGTGCAGGCGCTGGT

GCTGGTGGGCGGCACCCTGGGCGTGGCGCAGGCGGAAGTGGGCAACCCGCGCGTGAACCAGCTGGGC

TATATTCCGAACGGCGATCGCATTGCGGTGTATAAAGCGAGCAACAACAGCGCGCAGACCTGGCAGCT

GACCCATAACGGCAGCCTGATTGCGAGCGGCCAGACCATTCCGAAAGGCAGCGATGCGAGCAGCGGC

GATAACATTCATCATATTGATCTGAGCAGCGTGACCGCGACCGGCAGCGGCTTTACCCTGACCGTGGG

CGGCGATAGCAGCTATCCGTTTAGCATTAGCAGCACCACCTTTAACGCGGCGTTTTATGATGCGCTGA

AATATTTTTATCATAACCGCAGCGGCATTGCGATTGAAACCCCGTATACCGGCGGCGGCCGCGGCAGC

TATGCGAGCCATAGCCGCTGGAGCCGCCCGGCGGGCCATCTGAACCAGGGCGCGAACAAAGGCGATA

TGAACGTGCCGTGCTGGAGCGGCACCTGCAACTATAGCCTGAACGTGACCAAAGGCTGGTATGATGCG

GGCGATCATGGCAAATATGTGGTGAACGGCGGCATTAGCGTGTGGACCCTGCTGAACCTGTATGAACG

CGCGCAGCATATTACCGGCAACCTGGCGGCGGTGGCGGATGGCAGCATGAACATTCCGGAAAGCGGC

AACGGCGTGGCGGATATTCTGGATGAAGCGCGCTGGCAGATGGAATTTATGCTGGCGATGCAGGTGCC

GCAGGGCCAGGCGAAAGCGGGCATGGCGCATCATAAAATTCATGATGTGGGCTGGACCGGCCTGCCG

CTGGCGCCGCATGAAGATCCGCAGCAGCGCGCGCTGGTGCCGCCGAGCACCGCGGCGACCCTGAACC

TGGCGGCGACCGCGGCGCAGGCGGCGCGCATTTGGAAAGATATTGATGCGGGCTTTGCGGCGCTGTGC

CTGACCGCGGCGGAACGCGCGTGGAACGCGGCGCAGGCGAACCCGAACGATATTTATAGCGGCAACT

ATGATAACGGCGGCGGCGGCTATGGCGATCGCTTTGTGGCGGATGAATTTTATTGGGCGGCGGCGGAA

CTGTATATTACCACCGGCGATAGCCGCTATCTGCCGACCATTAACAACTATACCCTGGAACGCACCGA

TTTTGGCTGGCCGGATACCGAACTGCTGGGCGTGATGAGCCTGGCGGTGGTGCCGGCGACCCATACCA

ACAGCCTGCGCATTGCGGCGCGCAACCATATTCAGACCATTGCGAGCACCCATCTGACCACCCAGAGC

GCGAGCGGCTATCCGGCGCCGCTGAGCAGCCTGGAATATTATTGGGGCAGCAACAGCGTGATTGCGA

ACAAACTGGTGCTGATGGGCCTGGCGTATGATTTTAGCGGCAACCAGAACTTTGCGCTGGGCGTGAGC

AAAGGCATTAACTATCTGTTTGGCATTAACGTGCTGCCGACCAGCTTTATTACCGGCCTGGGCACCAAC

ACCGTGGCGCAGCCGCATCATCGCTTTTGGGCGGGCGCGCTGAACAGCAACTATCCGTGGGCGCCGCC

GGGCGCGCTGAGCGGCGGCCCGAACGCGGGCCTGGAAGATAGCTTTAGCGCGAGCCGCCTGAGCGGC

TGCACCAGCCGCCCGGCGACCTGCTGGCTGGATAGCATTGATGCGTGGAGCACCAACGAAATTACCAT

TAACTGGAACGCGCCGCTGGCGTGGGTGCTGGGCTTTTATAACGATTTTGCGGCGACCCAGGGCGGCA

GCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCGTGCCGGTGAGCAGCAGCAGCAGCAGCAGCATTAT

TCCGAGCAGCAGCAGCAGCAGCATTCAGCCGAGCAGCAGCAGCAGCAGCATGCCGAGCAGCAGCAGC

AGCAGCAGCAGCGTGGTGGCGAGCAGCAGCAGCAGCGTGAGCGGCGGCCTGCGCTGCAACTGGTATG

GCACCCTGTATCCGCTGTGCGTGACCACCCAGAGCGGCTGGGGCTGGGAAAACAGCCAGAGCTGCATT

AGCGCGAGCACCTGCAGCGCGCAGCCGGCGCCGTATGGCATTGTGGGCGCGGCGAGCAGCAGCAGCC

AGGCGGCGAACCGCAGCCCGACCCTGCAGCTGAGCGCGAACGCGACCGGCTTTGAAGGCGGCAGCAT

GGTGTGCTGCACCCTGCATATTAACGGCGCGGCGAGCGATCCGGATGGCGATAACCTGACCTATAGCT

GGCAGGTGATTAGCGGCAACACCGTGGTGGCGAGCGGCAGCAGCAGCAGCGCGAGCATTCATGTGAG

CAACCAGCGCGGCTATGAAGTGAGCATGACCGTGAGCGATGGCCGCGGCGGCGTGGCGACCGAAACC

ACCTTTGTGAGCGTGTATTTTAGCGATTATTTTCCGGGCAGCAGCAGCAGCGCGAGCAACATTAACAG

CAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCGCGATTGTGAGCAGCAGCAGCAGCGTGGTGAGC

AGCAGCAGCAGCAGCGCGGCGAGCGGCGGCAACTGCCAGTATGTGGTGACCAACCAGTGGAACAACG

GCTTTACCGCGGTGATTCGCGTGCGCAACAACGGCAGCAGCGCGATTAACCGCTGGAGCGTGAACTGG

AGCTATAGCGATGGCAGCCGCATTACCAACAGCTGGAACGCGAACGTGACCGGCAACAACCCGTATG

CGGCGAGCGCGCTGGGCTGGAACGCGAACATTCAGCCGGGCCAGACCGCGGAATTTGGCTTTCAGGG

CACCAAAGGCGCGGGCAGCCGCCAGGTGCCGGCGGTGACCGGCAGCGTGTAAGTTTTCCGCCGAGGA

TGCCGAAACCATCGCAAGCCGCACCGTCATGCGTGCGCCCCGCGAAACCTTCCAGTCCGTCGGCTCGA

TGGTCCAGCAAGCTACGGCCAAGATCGAGCGCGACAGCGTGCAACTGGCTCCCCCTGCCCTGCCCGCG

CCATCGGCCGCCGTGGAGCGTTCGCGTCGTCTCGAACAGGAGGCGGCAGGTTTGGGGATCC

Insert Size 3,235bp

Coding region 2,883bp

Promoter

Cellulase gene coding sequence

Fig. 32 Restriction map of cellulase gene containing DNA cloned from Pseudomonas fluorescence strain JV

Fig. 33 Deduced amino acid sequence of cellulase gene cloned from Pseudomonas fluorescence

MINRSVLKIPALVTPLVQALVLVGGTLGVAQAEVGNPRVNQLGYIPNGDRIAVYKASNNSAQTWQLTHNG

SLIASGQTIPKGSDASSGDNIHHIDLSSVTATGSGFTLTVGGDSSYPFSISSTTFNAAFYDALKYFYHNRSGIAI

ETPYTGGGRGSYASHSRWSRPAGHLNQGANKGDMNVPCWSGTCNYSLNVTKGWYDAGDHGKYVVNGG

ISVWTLLNLYERAQHITGNLAAVADGSMNIPESGNGVADILDEARWQMEFMLAMQVPQGQAKAGMAHH

KIHDVGWTGLPLAPHEDPQQRALVPPSTAATLNLAATAAQAARIWKDIDAGFAALCLTAAERAWNAAQA

NPNDIYSGNYDNGGGGYGDRFVADEFYWAAAELYITTGDSRYLPTINNYTLERTDFGWPDTELLGVMSLA

VVPATHTNSLRIAARNHIQTIASTHLTTQSASGYPAPLSSLEYYWGSNSVIANKLVLMGLAYDFSGNQNFAL

GVSKGINYLFGINVLPTSFITGLGTNTVAQPHHRFWAGALNSNYPWAPPGALSGGPNAGLEDSFSASRLSGC

TSRPATCWLDSIDAWSTNEITINWNAPLAWVLGFYNDFAATQGGSSSSSSSSSSSVPVSSSSSSSIIPSSSSSSI

QPSSSSSSMPSSSSSSSSVVASSSSSVSGGLRCNWYGTLYPLCVTTQSGWGWENSQSCISASTCSAQPAPYGI

VGAASSSSQAANRSPTLQLSANATGFEGGSMVCCTLHINGAASDPDGDNLTYSWQVISGNTVVASGSSSSA

SIHVSNQRGYEVSMTVSDGRGGVATETTFVSVYFSDYFPGSSSSASNINSSSSSSSSSSSSAIVSSSSSVVSSSS

SSAASGGNCQYVVTNQWNNGFTAVIRVRNNGSSAINRWSVNWSYSDGSRITNSWNANVTGNNPYAASAL

GWNANIQPGQTAEFGFQGTKGAGSRQVPAVTGSV

Fig. 34 Restriction map of plasmid pET cel Pf having cellulase gene containing DNA cloned from Pseudomonas fluorescence strain JV

Fig. 35 Nucleotide sequence of insert DNA in pET cel Kp (HM235918) cloned from Klebsiella pneumoniae strain JV

GGATCCTGAAGGGACATGCTGCTGTCGCGCGGAAACAACTACACGCGGCTGGCCACTGCCCCCCTAT

AACATTCCTTCTTTAAAAAATCCGGCCGGCGCCGGAGGCTTTCGCACGCCTGTTGGCCGGCTACTACGT

ATATATAAGCTCTGTATGGGATCCCGACTCGGATCTGTGGATAGCCTGGGCGCTGCTGGATGAAAGTA

CTTTGTGGCGCGGTGCTCTCCGCGCTACTGCTGGCGGCGGGTCCGGTGAGCGCCGCCTGCCAGTGGCC

CGCCTGGGAGCAGTTCAAAAAAGCGTACGTCAGCCCTGAGGGACGGGTGATTGACCCCAGCGATGCG

CGCAAAATCTCCACCTCGGAAGGACAGAGCTATGGCCTGTTTTTCGCCCTGGCCGCCAACGATCGCGC

AGGGTTCGACAAACTGCTTACCTGGACGCAGAACAACCTGGCGGAAGGCGACCTGCGGCAGCATCTG

CCGGGCTGGCTGTGGGGCAAAAAGGACGATGAGCAGTGGACGTTGCTGGACAGCAACTCGGCCTCCG

ACTCGGATCTGTGGATAGCCTGGGCGCTGCTGGAGGCGGGGCGCCTGTGGCAGCAGCCGCAGTACACC

GAGACCGGAAAAGCGCTGCTGGCGCGGATTGTCGAGGAAGAGACGTGGCGGTACCCGGCCTTGGCAC

GATGCTGCTGCCGGGAAAAGTCGGTTTTGCCGATGACAGCGGCTGGCGGTTTAACCCCAGCTATCTTC

CGCCGCAGCTGGCCACCTACTTTGTGCGTTTTGGCGCGCCGTGGCCTGCCCTGCGCGACAGCAACCTGC

GCCTGCTGCTGGAGACCGCGCCGAAAGGCTTTACCCCGGACTGGGTGCGCTATGAGAAAGGGAAGGG

CTGGCAGCTGAAAACCGAAAAGCCGCTGATCGGCAGCTATGACGCGATTCGCGTCTATCTGTGGGTGG

GCATGCTCCATGATGGCGATAAGCAGAAAGCGCGCCTGCTGCAACGCTTCGCGCCGATGGCGGCGCA

GACCACGGAGCAGGGGGTGCCGCCGGAGAAAGTGAATATCGCCACCGGCAAAACCAGCGGCCAGGG

GCCCGTGGGCTTCTCCGCGGTGATGCTACCGTTTTTACAGGACGACGAGGCCCGGTCAGTGCAACGCC

AGCGCGTCGCCGATAACTATCCCGGCGCGGATGCCTACTACAGTGCAGTTCTGACGCTGTTCGGCCAG

GGTTGGGATCAACACCGTTTTCGTTTCACTGCGGGTGGCGAATTACAACCTGACTGGAACCAGGAATG

CGCAAGCTCTCACTAAGCGTCTATCTGTGGGTGGGCATGCTCCATGATGGCGAGGATCCATTTGCCTAT

TAAAACGGGATCC

Insert Size 1,339bp

Coding region 1,172bp

Promoter

Cellulase gene coding sequence

Fig. 36 Restriction map of cellulase gene containing DNA cloned from Klebsiella pneumoniae strain JV

Fig. 37 Deduced amino acid sequence of cellulase gene cloned from Klebsiella pneumoniae strain JV

MGSRLGSVDSLGAAGKYFVARCSPRYCWRRVRAPPASGPPGSSSKKRTSALRDGLTPAMRAKSPPRKDRA

MACFSPWPPTIAQGSTNCLPGRRTTWRKATCGSICRAGCGAKRTMSSGRCWTATRPPTRICGPGRCWRRG

ACGSSRSTPRPEKRCWRGLSRKRRWRYPALARCCCREKSVLPMTAAGGLTPAIFRRSWPPTLCVLARRGLP

CATATCACCWRPRRKALPRTGCAMRKGRAGSKPKSRSAAMTRFASICGWACSMMAISRKRACCNASRR

WRRRPRSRGCRRRKISPPAKPAARGPWASPRCYRFYRTTRPGQCNASASPITIPARMPTTVQFRCSARVGIN

TVFVSLRVANYNLTGTRNAQALTKRLSVGGHAPWRGSI

Fig. 38 Restriction map of plasmid pET cel Kp cellulase gene containing DNA cloned from Klebsiella pneumoniae strain JV

Fig. 39 Nucleotide sequence of insert DNA in pET cel Pa (GQ872426) cloned from Pseudomonas aeruginosa strain JV

GGATCCTCCCTTATAAATATTATTTGTCCTGCTGGGCGGTATTCGCTGGTTGTTTTCAGCATATTAATAA

TAAAACCGCCTCCTCCTCGATGACGCCGTTTTCAACCAGAGGGATCGGCCTTTGAGCGAGCCATTATCA

ATATAAAAATGTGGTGACGAGGGAGACAGTGAGTTTGGTTTTGAGCGGGCTTGACGGAGTCTTTGCCCT

GTTCAGAAGCGGCGACCGCTATGTTGTTGCGGTCATTTCACGTCTGTTGTAAAGCCAAGTTTATTCTTTA

GCAACTTGTGCCTTGGCAGTCACCATAATAATTGAGGATTGAATAATGGGACATGTTACATCTCCCTCT

AAACGCTATCCTGCCTCTTTTAAGCGGGCAGGTAGTATTCTTGGCGTGAGTATTGCGCTTGCTGCATTTT

CCAATGTGGCAGCAGCGGGTTGTGAGTATGTGGTTACCAATAGCTGGGGGTCGGGATTTACCGCAGCAA

TTCGTATTACTAACTCAACATCTTCTGTAATCAATGGTTGGAACGTCAGCTGGCAATATAACAGCAACC

GTGTTACCAACTTATGGAATCCCAACCTTTCCGGTAGCAATCCCTATACGGCGTCCAACCTGAGTTGGA

ATGGCACTATTCAACCTGGGCAAACGGTAGAGTTTGGTTTCCAGGGGGTTACCAATAGCGGCACTGTCG

AGAGTCCAACAGTGAATGGTGCTGCATGTACTGGCGGAACCAGTTCTTCGGTGAGTTCTTCCAGTGTTG

TGAGTTCAAGCTCGTCATCGCGTAGCAGTGTGTCTTCAAGCTCTGTTGTGTCCTCAAGCTCCAGCGTAGT

GAGUTCATCGTCTTCCTCTGTGGTCAGCGGCGGCGGCCAGTGTAATTGGTATGGAACCCTGTATCCGTT

GTGTGTGAGCACAACCTCTGGTTGGGGTTACGAAAACAACAGAAGCTGTATCTCTCCATCAACCTGTTC

GGCCCAGCCGGCGCCTTATGGGATTGTCGGGGGCTCGAGCTCGCCCAGCTCAATTTCCAGTTCAAGCGT

CCGCTCCAGCAGCTCGTCTTCTGTAGTACCGCCTAGCAGTAGCTCATCTTCCAGTGTTCCATCAAGCAGC

TGTTCCAGTGTTAGCTCCAGCTCGGTTGTCTCTTCCAGTTCATCTTCTGTGAGTGTGCCCGGAACCGGTG

TGTTCCGTGTTAATACTAAGGGTAACCTGACGAAAGACGGTCAACTGCTGCCTGCGCGTTGCGGGAACT

GGTTTGGTTTGGAGGGGCGTCATGAGCCATCAAATGATGCCGATAACCCCAGCGGTGCGCCAATGGAGT

TGTATGCGGGTAACATGTGGTGGGTGAATAACAGCCAAGGTTCCGGTCGCACCATTCAGCAGACTATGA

CTGAGTTGAAGCAGCAGGGTATTACTATGTTGCGTCTGCCAATTGCACCGCAAACCCTGGATGCAAATG

ACCCGCAAGGTCGCAGTCCAAACCTCAAAAACCATCAATCCATTCGTCAATCCAACGCGCGTCAAGCAT

TGGAGGATTTCATCAAACTGGCTGATCAAAATGACATCCAGATCTTTATTGATATCCACTCCTGCTCTAA

TTACGTTGGTTGGCGGGCCGGTCGTTTGGATGCCCGTCCGCCCTATGTGGATGCGAATCGCGTTGGTTAC

GACTTCACTCGTGAAGAGTATTCCTGTTCUGCTACCAATAACCCCAGTTCTGTTACCAGGTTCCATGCTT

ACGATAAGCAGAAGTGGTTGGCAAACCTGCGTGAAATCGCCGGACTATCCGCCAAGCTGGGGGTAAGT

AACCTGATTGGTATTGATGTCTTCAATGAGCCTTATGATTACACTTGGGCAGAATGGCAGGGTATGGTT

GAAGAGGCCTATCAGGCGATCAATGAAGTTAACCCCAATATGCTTATTATCGTTGAAGGTATTTCCGCC

AATGCTAATACGCAAGATGGAACACCTGACACATCCGTACCTGTGCCACACGGTAGCACCGACTTGAAT

CCAAACTGGGGTGAAAACCTCTACGAAGCGGGTGCTAACCCACCCAACATTCCCAAGGATCGCCTGTTG

TTCTCTCCACACACTTATGGTCCGTCCGTGTTTGTTCAAAGACAATTCATGGAACCGGCGCAGACAGAG

TGTGCAGGGCTGGAAGGTGATGAAGCAGCTCAGGCCAGGTGCGGTATTGTGATTAATCCGACCGTGCTT

GAGCAAGGTTGGGAAGAGCACTTTGGCTATCTGCGTGAATTGGGTTACGGTATTTTGATTGGTGAATTT

GGCGGTAATATGGATTGGCCTGGTGCCAAGTCGAGCCAGGCTGACCGTAATGCCTGGAGCCATATCACC

ACCAACGTTGACCAGCAATGGCAACAGGCGGCGGCAAGCTATTTCAAGAGGAAAGGGATAAATGCTTG

CTACTGGTCGATGAACCCTGAATCAGCAGATACCATGGGTTGGTATTTAACTCCCTGGGATTCAGTGAC

TGCCAACGATATGTGGGGTCAGTGGACAGGTTTCGATCCTCGTAAAACCCAGCTGTTGCACAATATGTG

GGGTTTGTAATTTACCCGCGATTGTGACTTAGCATAAAAAAACCGGGGCTTTTCAGGCCCCGGTTTTTTT

ATGGTTTTGGGTAGCGATATTTATTTCAGATGGCCAAGGTTTTTTTGCACGATTTGCAGGATAGGTTTAA

ACACCTTGGGTGAACCACAGACGACATGGCCAGTATCCAAAAAGGATCC

Insert Size 2,247bp

Coding region 2,749bp

Promoter

Cellulase gene coding sequence

Fig. 40 Restriction map of cellulase gene containing DNA cloned from Pseudomonas aeruginosa strain JV

Fig. 41 Deduced amino acid sequence of cellulase gene cloned from Pseudomonas aeruginosa strain JV

MGHVTSPSKRYPASFKRAGSILGVSIALAAFSNVAAAGCEYVVTNSWGSGFTAAIRITNSTSSVINGWNVS

WQYNSNRVTNLWNPNLSGSNPYTASNLSWNGTIQPGQTVEFGFQGVTNSGTVESPTVNGAACTGGTSSSV

SSSSVVSSSSSSRSSVSSSSVVSSSSSVVSSSSSSVVSGGGQCNWYGTLYPLCVSTTSGWGYENNRSCISPSTC

SAQPAPYGIVGGSSSPSSISSSSVRSSSSSSVVPPSSSSSSSVPSSSCSSVSSSSVVSSSSSSVSVPGTGVFRVNTK

GNLTKDGQLLPARCGNWFGLEGRHEPSNDADNPSGAPMELYAGNMWWVNNSQGSGRTIQQTMTELKQQ

GITMLRLI

Fig. 42 Restriction map of plasmid pET cel Pa having cellulase gene containing DNA cloned from Pseudomonas aeruginosa strain JV

Fig. 43 Nucleotide sequence of insert DNA in pET cel Pm (HM235922) cloned from Proteus mirabilis strain JV

GGATCCCTGGACGGCAATGTCGCTGTATCGTGAGTTTATACCCTGATGGTCATCTTGCTGATTATAACT

TAACGAGCGCATATTAATAATAAAACCGCCTCCTCCTCGATGACGCCGTTTTCAACCAGAGCGGGCTC

CAGGTATGATCTCGATTGACTCATCGACACGGCAATGTCGCTGTATCGTATGTCCATAAGACAGTTATC

TCGATTGACTCATCGACTTGGTATTTTGCTAACACTAAGTGTGATGTTAATGATATCCCCAATGACACA

AGCGACAGAAACCCAGGCAGGATGGCAACAATTTAAAGCGCGTTACATTACTCCCGAGGGGCGTGTC

ATTGACAGTGCTAATCAAAATATTTCACACTCAGAAGGCCAAGGATATGGCATGTTAATGGCGGTTAT

GAGTGACGATCGCCAAACCTTTGCACAATTATGGCACTGGACGGCAATGTCGCTGTATCGTGGTGATC

TTGGTTTATTTAAATGGCGTTATGAACCCGAAAATAACCAACATACACCCGATCCTAATAATGCAACA

GATGGTGATATTTTAATTGCTTGGGCATTATTAAAGGCTGGGGAAAAATGGCAGGACGAAAGCTATCT

TTCAGCGTCAGATTCTATTCAGCATGCGATCCTCGAACACACTTTAGTGAAGACAGAAAACTACAGTG

TACTCTTACCGGGAATTAATGGCTTTAAAACTCCCGAAGAAATTATTATTAACCCCTCCTATTTTATCTT

TCCGGCATGGAAAGATTTCTATCGAGTTAGTCATGATAGCCGCTGGAAAAACTTAATTAACGATAGCC

AATCCTTATTAAGAAAAATGCGTTTTGGTAAATATAAATTACCAAGTGATTGGGTAAGTTTATACCCTG

ATGGTCATCTTGCCCCGAGTGAAAAATGGCCAGCACGATTCAGTTTTGATGCTATTCGCATTCCTCTTT

ATTTAGCATGGGCACAAGATAAACTCGCATTACAACCTTTTGTGAATTATTGGCAACAATTCGATCGTG

ATAAGACGCCTGCGTGGATCAGTATTGATGGTAAAGAGCGCGCTGATTATAACTTAACGCCAGGTATG

ATGGCGGTAAGAGATCTGACAATGAAAACAGTAATTGAAAATGTTGATTTAACCAAGGATACGGATT

ATTACTCTTCAGCCTTACATTTATTAGCCGCCTTTGCGCAGAATAATCACAACGACTATTAACGACAGA

AACCCAGGCAGGATGGCAACTCTTTCCGGCATGGAAAGATTTCTATCGAGTTGATTATAACTTAACGC

CAGGTATGATTGGCTTTAAAAGCTATTCGCATTCCTCTTTATTTATATGGCACTGGACGGCAAGGATCC

Insert Size 1,356bp

Coding region 1,038bp

Promote

Cellulase gene coding sequence

Fig. 44 Restriction map of cellulase gene containing DNA cloned from Proteus mirabilis strain JV

Fig. 45 Deduced amino acid sequence of cellulase gene cloned from Proteus mirabilis strain JV

MSLYRMSIRQLSRLTHRLGILLTLSVMLMISPMTQATETQAGWQQFKARYITPEGRVIDSANQNISHSEGQG

YGMLMAVMSDDRQTFAQLWHWTAMSLYRGDLGLFKWRYEPENNQHTPDPNNATDGDILIAWALLKAG

EKWQDESYLSASDSIQHAILEHTLVKTENYSVLLPGINGFKTPEEIIINPSYFIFPAWKDFYRVSHDSRWKNLI

NDSQSLLRKMRFGKYKLPSDWVSLYPDGHLAPSEKWPARFSFDAIRIPLYLAWAQDKLALQPFVNYWQQF

DRDKTPAWISIDGKERADYNLTPGMMAVRDLTMKTVIENVDLTKDTDYYSSALHLLAAFAQNNHNDY

Fig. 46 Restriction map of plasmid pET cel Pm having cellulase gene containing DNA cloned from Proteus mirabilis strain JV

2.4 Discussion

Cellulase enzyme has a wide range of applications in food, animal feed, textile, fuel and

chemical industries. Other areas of application include the paper and pulp industry, waste

management, medical/pharmaceutical industry, plant protoplast production and in the treatment

of pollutants (Mandels, 1985; Beguin and Aubert, 1993; Coughlan, 1985). Bacterial cellulase

enzyme is easy to be expressed and purified from bacterial system (E. coli). Cellulase is a

complex enzyme system having exo, endo and β glucocidase. Using gene cloning strategies, it is

possible to clone genes and get their products for various applications. Utilizing them for

digesting cellulosic materials for ethanol fermentation is one of the several uses of cellulase. In

order to express the bacterial cellulase gene in ethanol fermenting bacteria, five different

cellulase genes were cloned from cellulolytic bacteria such as, E. cloacae, P. mirabilis,

P. fluorescens, P. aeruginosa and K. pneumoniae. The cellulase gene from cellulolytic bacteria

has been cloned in E. coli using pET20b(+) plasmid vector having T7 inducible promoter

(Gunasekera and Kemp, 1999). Cellulase gene clones from different cellulolytic bacteria were

screened by congored overlay method (Teather and Wood, 1982). It is a relatively straight

forward technique to screen and select a small fraction of recombinant clones which express and

secrete cellulase enzyme. With this technique cellulase gene clones were screened by overlaying

agar the medium supplemented with CMC and then incubating the plates, usually at 37°C for 24

hr. During this time, the CMC molecules that are present in the immediate vicinity of a cellulase-

producing colony are digested. To visualize the digestion of CMC the petri plate was flooded

with a solution of Congored and then with sodium chloride. The bacterial colony producing

cellulase will be surrounded by a yellow colored halo, whereas, the background will be red. The

Congo Red-carboxymethylcellulose procedure has permitted researchers to isolate

endoglucanase genes that are expressed in E. coli from clone banks of Streptomyces lividians

(Shareck et al., 1987), Clostridium thermocellum (Millet et al., 1985; Beguin et al., 1987),

C. cellulolyticum (Faure et al., 1988), Thermoanaerobacter cellulolyticus (Honda et al., 1987),

Thermomonospora fusca (Hu and Wilson, 1988), Erwinia chtysanthemi (Boyer et al., 1987),

Pseudomonas fluorescens var. cellulosa (Gilbert et al., 1987; Lejeune et al., 1986), Cellvibrio

mixtus (Wynne and Pemberton, 1986), Ruminococcus albus (Ohmiya et al., 1988), Cellulomonas

uda (Nakamura et al., 1986), Bacteroides succinogenes (Crosby et al., 1984) and Bacillus

subtilis(Koide et al., 1986).

For this strategy to be effective the cloned endoglucanase gene must be expressed in the

heterologous host cell (E. coli) and secreted either into the growth medium or to the host cell

periplasm so that the substrate can be utilized. In additions to the Congo Red-overlay technique,

other methods have been employed to directly select the expression of endoglucanase gene

clones (Glick and Pasternak, 1989).

Totally more than 50 to 120 transformants were selected from each cellulolytic

bacterium by zone formation. Among the high cellulase expressing clones, pET-cel-Ec and pET-

cel-Pa, showed higher cellulase activity. The cellulase gene cloned from E. cloacae contained an

insert size 0f 2.25 kb BamHI. Sequencing of this insert revealed the presence of a 1083-bp open

reading frame (GQ368735) potentially encoding a protein with a molecular mass of 40. 33KDa.

The predicted amino acid sequence was 99–100% identical to that of the endo-1, 4-D-glucanase

of Citrobacter rodentium ICC168 (FN543502.1) and Enterobacter sp. (CP000653.1). The

cellulase gene cloned from P. fluorescens contained an insert size of 2.7 kb BamHI fragment.

Sequencing of this cloned DNA showed the presence of a 2247-bp open reading frame

(HM235919) that encoded a protein with a molecular mass of 80.160 kDa. The predicted amino

acid sequence of the gene was 99% identical to that of the cellulase of Cellvibrio japonicus celC

gene for cellodextrinase C (X61299.1). The cellulase gene cloned from K. pneumoniae

contained in an insert 1.3-kb BamHI fragment. Sequencing of this insert DNA revealed the

presence of a 1107-bp open reading frame (HM174251) that potentially encoded a protein with

a molecular mass of 40.88 kDa The predicted amino acid sequence of this gene was 100%

identical to that of the cellulase of K. pneumoniae cellulase gene (HM235918.1). The cellulase

gene cloned from P. aeruginosa contained in an insert of 3 kb BamHI fragment. Sequencing of

this insert showed that the presence of a 2883-bp open reading frame (GQ872426) that

potentially encoded a protein with a molecular mass of 100.07 kDa. The predicted amino acid

sequence was 99–100% identical to that of the glycosyl hydrolase family of Teredinibacter

turnerae T7901 (CP001614.2). The recombinant cellulase producing E. coli harbored a Proteus

mirabilis derived 1.7-kb BamHI fragment. Sequencing of this insert revealed the presence of a

1053-bp open reading frame (HM235922) that potentially encoded a protein with a molecular

mass of 40.54 kDa. The predicted amino acid sequence was 100% identical to that of the putative

cellulase family of P. mirabilis (AM942759.1). The E. coli cells containing the cellulase genes

from different cellulolytic bacteria exhibited cellulase activity comparable to that of wild type

cellulolytic bacteria from which the cellulase genes were isolated. Clearing zone around the

colonies containing cellulase gene was larger than the clearing zone found in wild type colonies

of wild type of cellulolytic bacteria. High copy number (~40) of pET20b(+) might be the reason

for the higher expression of the cellulase in E. coli clones (Gunasekera and Kemp, 1999).

There are reports of cloning and sequencing of cellulase genes for various purposes. A

gene encoding cellulase was cloned from Bacillus sp. into E. coli and the nucleotide sequence

was determined. The cellulase gene, designated as celS, was composed of 1,497 base pairs and

the nucleotide sequence of the celS gene was found highly homologous to those of other

B. subtilis cellulase genes. The enzyme encoded by celS was highly active on CMC (Jung et al.,

1996). The sequence coding for CMCase was isolated from the Salmonella typhimurium.

Comparison between the deduced amino acid sequence of CMCase (368 amino acid residues)

and that of the previously published CMCase revealed that this enzyme belongs to the cellulase

family. The protein was overproduced in E. coli using T7 expression system. The CelC protein

was able to degrade cellulosic substrates, such as CMC (Yoo et al., 2004).

A genomic library of Bacillus subtilis CD4 was constructed in E. coli JM83. A clone

designated as E. coli pBcelR was identified which formed blue colony in the presence of 5-

bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) and hydrolysed carboxymethyl cellulose

(CMC). The clone E. coli (pBcelR) expressed both cellobiase and endoglucanase activities and

contained an insert of 1.2 kb. E. coli pBcelR encoded a protein of 12.9 kDa which was endowed

with bifunctional (endoglucanase and cellobiase) activities (Srivastava et al., 1999).

The maximum exoglucanase, endoglucanase and cellobiase activity of pET-cel-Ec was

observed (8.5 CMC/ml, 0.171 FPU/ml and 2.7 CB/ml) when compared to others. A similar kind

of bifunctional cellulase was described from Caldocellum sacchrolyticum (Saul et al., 1990)

which contained one catalytic domain for hydrolysis of CMC and another for 4,

methylumbelliferyl-Umbelliferyl β-D cellobioside (MUC). The bi- and multi-functional

polysaccharide hydrolases have been characterized from few microorganisms possessing

considerable levels of activities of endoglucanase, exoglucanase and/or xylanase (Hamamoto et

al., 1990; Gilkes et al., 1991). These hydrolases have been shown to possess either single or

multi-functional properties (Foong et al., 1991; Xue et al., 1992) or mono-functional catalytic

domains (Saul et al., 1990; Gilbert et al., 1990). A bifunctional enzyme from Clostridium

cellulovorans has also been reported (Foong et al., 1991) of which endoglucanase and xylanase

activities were located on the same region of the gene. The multi-functional polysaccharide

hydrolases are ideally suited for genetic manipulation, as simultaneous expression of

endoglucanase and cellobiase activities should alleviate the problem of catabolic repression

which is a rate limiting step in the economics of cellulose hydrolysis. In the construction of

cellulolytic E. coli strains, the transfer of genetic information in the form of bi-functional enzyme

possessing exo, endoglucanase and cellobiase activities could overcome these problems of

coordinate expression of these three activities.

Most of the cloned genes expressed detectable cellulase activity in E. coli, and in many

cases, expression was independent from vector promoters. However, fusion of the genes with

promoters and ribosomal initiation sites from highly expressed genes in E. coli generally resulted

in considerably higher expression (Schwarz et al., 1988). Besides E. coli, cellulase genes from

Streptomyces lividans, Clostridium thermocellum Cellulomonas fimi, E. cloacae, P. fluorescens

var. cellulosa and Trichoderma reesei have also been cloned and introduced into a variety of

other hosts, such as B. subtilis (Ghangas and Wilson, 1987), Bacillus megaterium (Lee et al.,

1988), Bacillus stearothermophilus (Soutschek-Bauer and Staudenbauer, 1987), Brevibacterium

lactoJermentum (Paradis et al., 1987), Streptomyces lividans (Ghangas and Wilson, 1988),

Z. mobilis (Vasan et al., 2011; Lejeune, 1986) and S. cerevisiae (Shoemaker et al., 1984). The

choice of an alternate host was motivated by the ability to secrete proteins. Z. mobilis are

organisms that efficiently ferment simple sugars such as glucose into ethanol. They could be

used as hosts for the expression of cloned cellulase genes. The expression of active cellulase

genes would enable these organisms to directly digest cellulosic substrates such lignocellulosic

biomass to ethanol. In the present study Z. mobilis was used to express the cloned cellulase gene

from five different cellulolytic bacteria.