August 2014⎪Vol. 24⎪No. 8

J. Microbiol. Biotechnol. (2014), 24(8), 1073–1080http://dx.doi.org/10.4014/jmb.1311.11051 Research Article jmbReview

Evaluation of β-1,4-Endoglucanases Produced by Bacilli Isolatedfrom Paper and Pulp Mill Effluents Irrigated SoilSangeeta Pandey, Rameshwar Tiwari, Surender Singh, Lata Nain, and Anil Kumar Saxena*

Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 110012, India

Introduction

Earth’s most abundant renewable organic material, the

lignocellulosic biomass, is the credible source of bioenergy

and commodity chemicals, which can be made economically

viable through identification of cellulase-producing bio-

agents having capability to accelerate the conversion of

cellulose to glucose.

Cellulose is a crystalline structure having tightly packed

bundles of microfibrils, which prevents penetration of

hydrolytic enzymes and its enzymatic conversion to

glucose. Owing to the physical nature of the substrate, the

enzymatic conversion of cellulose to glucose is slow and

economically unviable. The β-1,4 endoglucanase acts

randomly along the cellulose chains. It produces small

cellulose fragments and generates new sites on which

exoglucanases act to produce cellobiose or oligosaccharides.

The synergistic action of the enzymes endo-1,4-glucanase

(E.C. 3.2.1.4), exo-1,4-glucanase (E.C. 3.2.1.91), and 1,4-β-D

glucosidases (E.C. 3.2.1.21) is required for complete

hydrolysis of cellulose [8].

β-1,4-Endoglucanase has many applications in same

industries. It is used to remove color from denim to impart

a good stonewashing effect [11]. In detergents, β-1,4-

endoglucanase is used as an additive for color brightening,

softening, and removal of soil particulate matter [21].

Furthermore, β-1,4-endoglucanase decreases the viscosity

of β-glucan solution by random cleaving of the glycosidic

bond of β-glucan. This process is useful in beer brewing

and improving the quality of animal feed [8]. To date,

numerous cellulases have been isolated and characterized

from bacteria, fungi, and animal species [8].

The microbial origin cellulase genes have been cloned

and the nucleotide sequences of some of the endoglucanse

genes have been identified [25]. The study of cellulolytic

enzymes at the molecular level revealed that some of the

modular features of these enzymes are attributable to their

catalytic activity [17]. The sequence comparison indicated

that the catalytic cores of cellulase belong to a restricted

number of families with significant diversity in their

activities [2]. Cellulases produced by two Bacillus strains

isolated from hot springs of Zimbabwe exhibited 100%

homology with the endoglucanase of Bacillus subtilis and

belonged to GH 5 [9]. The endoglucanase of B. licheniformis

Received: November 14, 2013

Revised: April 8, 2014

Accepted: April 13, 2014

First published online

April 18, 2014

*Corresponding author

Phone: +91-11-25847649;

Fax: +91-11-25846420;

E-mail: saxena461@yahoo.com

upplementary data for this

paper are available on-line only at

http://jmb.or.kr.

pISSN 1017-7825, eISSN 1738-8872

Copyright© 2014 by

The Korean Society for Microbiology

and Biotechnology

A total of 10 cellulase-producing bacteria were isolated from soil samples irrigated with paper

and pulp mill effluents. The sequencing of 16S rRNA gene revealed that all isolates belonged

to different species of genus Bacillus. Among the different isolates, B. subtilis IARI-SP-1

exhibited a high degree of β-1,4-endoglucanase (2.5 IU/ml), β-1,4-exoglucanase (0.8 IU/ml),

and β-glucosidase (0.084 IU/ml) activity, followed by B. amyloliquefaciens IARI-SP-2. CMC was

found to be the best carbon source for production of endo/exoglucanase and β-glucosidase.

The β-1,4-endoglucanase gene was amplified from all isolates and their deduced amino acid

sequences belonged to glycosyl hydrolase family 5. Among the domains of different isolates,

the catalytic domains exhibited the highest homology of 93.7%, whereas the regions of signal,

leader, linker, and carbohydrate-binding domain indicated low homology (73-74%). These

variations in sequence homology are significant and could contribute to the structure and

function of the enzyme.

Keywords: Cellulase, β-1,4-endoglucanase, Bacillus, Paper and pulp mill effluent

S

S

1074 Pandey et al.

J. Microbiol. Biotechnol.

of the same family has also been reported [3].

Soils irrigated with untreated pulp and paper mill

effluent over the years contain high alkalinity and organic

matter. Irrigation with such effluents facilitates growth of

lignocellulose degraders in these soils. Hence, these soils

can be explored for mining of novel microbes and genes

involved in the hydrolysis of cellulose. Studies conducted

in the past have also reported a dominance of culturable

lignocellulose degraders in such habitats [24, 27]. Nonetheless,

the diversity of cellulolytic bacteria and endoglucanase has

not been explored extensively. The aim of this study was to

explore the diversity of cellulolytic bacteria and the allelic

variation in their endoglucanase genes. The sequences of

these genes have been compared with known cellulase

genes and their modular structures have been defined.

Additionally, characterization of the enzyme was carried

out for prediction of its protein based on sequences of genes

amplified from different bacteria.

Materials and Methods

Sample Collection

The Century Pulp and Paper Mill, Lalkuan, Uttarakhand

(79°E10’E longitude and 29°3’N latitude) is one of the largest

paper and pulp mills of India. The untreated effluent of this mill is

utilized for irrigation of nearby agricultural lands. The soil samples

were collected from these agricultural lands and transported

immediately to the laboratory for further processing.

Isolation and Screening of Cellulase-Producing Microorganisms

Carboxymethyl cellulase (CMCase)-producing bacteria were

isolated by using carboxymethyl cellulose (CMC) agar containing

0.5% CMC; 0.1% NaNO3; 0.1% K2HPO4; 0.1%, KCl; 0.05%

MgSO4·7H2O; 0.05% yeast extract; and 1.5% agar, pH 7.0. All

colonies were picked and spot inoculated in Reese’s minimal

medium (RMM) containing 1% CMC to confirm their cellulase

production potential, as described by Pandey et al. [16]. The

substrate specificity of the crude enzyme was determined by

performing the assay with different substrates; Avicel, CMC, and

rice straw. All experiments were conducted in triplicates (n = 3).

Molecular Identification of Bacterial Isolates

For molecular characterization of the isolates, 16S rRNA gene

sequencing was done. Isolation of genomic DNA was carried out

by a standard DNA extraction protocol [20]. The extracted DNA

was used as the template for PCR amplification of the 16S rRNA

gene using universal 16S rRNA gene primers pA (5’-AGAGTT

TGATCCTGGCTCAG-3’) and pH (5’-AAGGAGGTGATCCAG

CCGCA -3’) to obtain a product of approximately 1,500 bp [4].

Aliquots of purified 16S rRNA PCR products were digested

separately with three different restriction endonucleases (AluI,

MspI, and HaeIII) in a 25 µl reaction volume by using the

manufacturer’s recommended buffer and temperature. Restricted

DNA was analyzed on 2% agarose gels. Strong and clear bands

were scored for similarity and clustering analysis using the

NTSYS-2.02e software package, Exeter software (http://www.

exetersoftware.com/). Similarity among the isolates was calculated

by Jaccard’s coefficient and a dendrogram was constructed using

the UPGMA method [12]. The 16S rRNA gene was sequenced by

Xcelris Labs (Ahmedabad, India) using Sanger’s dideoxynucleotide

sequencing method. A similarity search for the sequence was

carried out using the BLAST program of the National Center of

Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

Hydrolytic Enzyme Production on Different Cellulosic Substrates

For enzyme production, cellulase-positive isolates were cultured

in RMM (KH2PO4 - 2 g, (NH4)2SO4 - 1.4 g, KNO3 - 1.4 g, MgSO4·7H2O

- 0.3 g, CaCl2·2H2O - 0.3 g, FeSO4·7H2O - 5 mg, MnSO4·2H2O - 1.6 mg,

ZnSO4·7H2O - 1.4 mg, CoCl2·6H2O - 2 mg, Agar - 20 g, Distilled

water - 1,000 ml) supplemented with CMC (1%), α-cellulose (1%),

Avicel PH101 (1%), Sigmacell 101 (1%), or rice straw (1%) as the

sole carbon source at 30°C and pH 7.0 ± 0.2. After 72h of

incubation, culture broths were centrifuged at 10,000 ×g for

15 min at 4ºC. The supernatant was collected and stored at 4ºC for

further enzyme assays. The substrate specificity of the crude

enzyme was determined by performing the assay with different

substrates; Avicel, CMC, and rice straw. All experiments were

conducted in triplicates (n = 3).

Enzyme Assays

The β-1,4-endoglucanase (CMCase) and β-1,4-exoglucanase

(FPase) activity was determined by using CMC and Whatman no

1 filter paper, respectively, as substrate [5] and measuring the

amount of reducing sugar [12]. One unit (IU) of filter paper

activity or CMCase corresponded to 1 µmole of glucose formed

per minute during hydrolysis. The β-glucosidase activity was

assayed by measuring the amount of p-nitrophenol released from

p-nitrophenyl-β-D-glucopyranoside [26]. One unit (IU) of β-glucosidase

activity was defined as the amount of enzyme releasing 1 µmole

of p-nitrophenol per milliliter of crude enzyme per minute.

Designing of Primers and Amplification of β-1,4-Endoglucanase

Thirty-five sequences of bacterial β-1,4-endoglucanase were

retrieved from NCBI GenBank. The amino acid sequences of these

genes were aligned using T-Coffee alignment (http://www.ebi.ac.uk/

Tools/msa/tcoffee/). The alignment of the Bacillus sequences was

further used in the primer design for amino acid positions 1 to 5

(MKRSI) and 505 to 509 (GTEPN) to obtain a complete coding

region of β-1,4-endoglucanases. The degenerate primers endo-F

(5’-ATGAARMGIWSIATH-3’) and endo-R (5’-RTTIGGYTCIGTNCCC-

3’) amplified approximately 1,500 bp of the β-1,4-endoglucanase

gene. PCR conditions were optimized by gradient PCR by keeping

the reactions at different annealing temperatures ranging from

51°C to 56°C. The final reaction mixture (25 µl) contained each

Evaluation of β-1,4-Endoglucanases Produced by Bacilli 1075

August 2014⎪Vol. 24⎪No. 8

primer at a concentration of 0.5 mM, each dNTP mix at a

concentration of 200 µM, 2.5 U of Taq DNA polymerase, 1.5 mM

MgCl2, 20 ng of template DNA, and 2.5 µl of 10× PCR buffer. The

following thermal profile was used for the PCR: 94ºC, 2 min; 35

cycles of 94ºC, 1 min; 53ºC, 1 min; 72ºC, 2 min; 1 cycle of 72ºC, 10

min. The PCR product was purified using the Qiaquick PCR

purification kit (Qiagen, Valencia, CA, USA) and sequenced by

Xcleris Lab (India).

Sequence and Phylogenetic Analysis

Sequence fragments were assembled with the codon code

aligner program (http://www.codoncode.com/aligner/). The sequenced

gene was compared with available sequences from GenBank using

the BLASTX program (http://blast.ncbi.nlm.-nih.gov/Blast/). The

deduced amino acid sequence was analyzed with the EXPASY tool

(http://expasy.org/). The signal peptide sequence of protein was

predicted by the SignalP 3.0 server (http://www.cbs.dtu.dk/services/

SignalP/) and conserved domain analysis was conducted with

Pfam (http://pfam.wustl.edu/hmmsearch.shtml). The phylogenetic

tree was drawn with Mega 5.0 [22]. In order to statistically

evaluate the confidence of branching, bootstrapping was carried

out with data resampled 1,000 times. Possible open reading frames

(ORFs) were identified with the ORF finder at the NCBI database

(http://www. ncbi.nlm.nih.gov). Closely related protein sequences

in the databases for the candidate cellulases were identified with

BLASTN and BLASTP of NCBI. Module structures of the enzymes

were predicted by the simple modular architecture research tool

(SMART; http://smart.embl-heidelberg.de).

Nucleotide Sequence Accession Numbers

The sequences generated in this study were deposited in NCBI

GenBank. The 16S rRNA gene sequences retrieved from the cellulase-

producing cultures were assigned accession numbers KF204578-

KF204587. The accession numbers of β-1,4-endoglucanase genes

obtained from these cultures were KF240847-KF240856.

Results

Isolation and Screening of Cellulase-Producing Bacteria

A total of 200 bacterial strains, isolated from effluent

irrigated soil, were screened for the presence of cellulase-

producing activity. Congo red test revealed that, out of 200

isolates, 10 isolates (IARI-SP-1 to IARI-SP-10) produced a

clear halo zone around the colony on CMC agar plate and

were taken as positive for cellulase activity.

Effects of Cellulosic Sources on the Production of Cellulases

All the 10 isolates positive for β-1,4-endoglucanase were

selected for quantitative estimation of cellulases. IARI-SP-1

was the best cellulase producer with the highest β-1,4-

endoglucanase (2.5 IU/ml), β-1,4-exoglucanase (0.8 IU/ml),

and β-glucosidase (0.084 IU/ml) activity, followed by IARI-

SP-2 (Fig. 1). Among different carbon sources tested, CMC,

followed by rice straw, was found to be the best carbon

source for the production of endo/exoglucanase and β-

glucosidase by all isolates. The results also indicated that

all the isolates invariably produced less β-glucosidase on

all cellulosic substrates. The crude cellulase preparation of

all 10 positive isolates was tested using various cellulosic

substrates in order to provide a better understanding of the

cellulase enzyme (Table 1). All isolates recorded higher

enzyme activity with CMC as the substrate in comparison

with Avicel.

Molecular Identification

The RFLP analysis of the 16S rRNA gene of all 10

cellulase-producing morphotypes with three different

restriction endonucleases (AluI, MspI, and HaeIII) indicated

Fig. 1. Effects of different cellulosic sources on the production

of (A) CMCase (B) FPase, and (C) β-glucosidase by different

bacterial isolates.

1076 Pandey et al.

J. Microbiol. Biotechnol.

variations in the profile. A combined dendrogram constructed

based on similarity percentage among the isolates indicated

that all 10 cellulase-producing isolates belonged to different

clusters (Fig. 2). A partial 16S rRNA gene sequence of about

1,500 bp of all the endoglucanase-positive bacterial isolates

was analyzed by BLAST and the sequences were deposited

in NCBI GenBank. DNA sequencing and phylogenetic

analysis revealed that all positive isolates showed 95-100%

similarity with the known sequences in GenBank and

belonged to different species of genus Bacillus (Fig. 3).

Phylogenetic Analysis of β-1,4-Endoglucanase Gene

The nucleotide sequences of the cloned β-1,4-endoglucanase

genes were compared with those of the cellulase genes

registered in the NCBI database, using the BLASTN

program. The nucleotide sequences of IARI-SP-1 and IARI-

SP-2 indicated 99% and that of IARI-SP-3 showed 98%

similarity with β-1,4-endonuclease of B. subtilis strain BS-

02. The nucleotide sequences of IARI-SP-4, IARI-SP-8, and

IARI-SP-10 indicated 98%, 96%, and 97% similarity,

respectively, with B. subtilis strain shu-3. IARI-SP-5, IARI-

SP-6, IARI-SP-7, and IARI-SP-9 indicated 98%, 98%, 98%,

and 99% identity with B. subtilis strain AH18, B. subtilis

strain DR, Uncultured bacterium clone celWS14, and

B. megaterium strain AP25, respectively (Table 2). Based on

amino acid sequences of the 10 identified ORFs, it was

observed that all sequences are closely related to glycosyl

hydrolase family 5 (GH5). The comparison of deduced

amino acid sequences of the 10 bacterial isolates and

B. subtilis strain BS-02 (GenBank Accession No. JF965375)

reveals a close evolutionary relationship among the isolates

(Fig. 4). The analysis of the deduced amino acid sequences

of the cellulases from our 10 isolates using the pfam

program of Expasy revealed a modular enzyme composed

of two discrete domains in the following order: catalytic

domain (CD) (Q-48 through S-301) of the glucosyl hydrolase

family 5/A2 (endoglucanase, E.C. 3.2.1.4), and carbohydrate-

binding domain (CBD) (V-356 through G-437) of family

IIIa. Similar to the modular organization of many Bacillus

endoglucanases, the CDs of these enzymes were located in

the N-terminal region and the CBDs in the C-terminal

region. There were 7 to 8 amino acid residue substitutions

in the signal peptide region (1 through 29), and 22 amino

acid residues were conserved among all of the cellulases,

resulting in 79% homology. The leader region (30 through

47) consisted of 18 residues, and 16 amino acid residues

Table 1. Substrate specificity of the cellulases from different

Bacillus strains towards different substrates.

Bacterial

isolates

β-1,4-Endoglucanase (IU/ml)

CMC Avicel Rice straw

IARI-SP-1 9.5 ± 1.0 2.6 ± 0.9 7.5 ± 0.9

IARI-SP-2 8.0 ± 0.9 2.3 ± 0.8 6.8 ± 0.6

IARI-SP-3 4.5 ± 0.6 1.8 ± 0.7 4.5 ± 0.6

IARI-SP-4 6.8 ± 0.7 1.5 ± 0.6 5.3 ± 0.5

IARI-SP-5 6.3 ± 0.6 1.2 ± 0.8 4.5 ± 0.7

IARI-SP-6 5.5 ± 0.6 1.4 ± 0.6 4.0 ± 0.5

IARI-SP-7 4.3 ± 0.5 1.0 ± 0.7 2.8 ± 0.6

IARI-SP-8 4.5 ± 0.6 0.9 ± 0.8 2.5 ± 0.7

IARI-SP-9 2.5 ± 0.4 0.8 ± 0.7 1.5 ± 0.6

IARI-SP-10 1.1 ± 0.5 0.6 ± 0.8 0.9 ± 0.6

The results are presented as the mean ± SD (standard deviation), n = 3.

Fig. 2. Dendrogram showing clustering of 10 isolates generated from RFLP analysis of the 16S rRNA gene by three restriction

endonucleases (AluI, MspI, and HaeIII), using the UPGMA algorithim and Jaccard’s coefficient.

Evaluation of β-1,4-Endoglucanases Produced by Bacilli 1077

August 2014⎪Vol. 24⎪No. 8

among them were conserved, resulting in 89% homology.

The CD consisted of 254 residues (48 through 301), and 238

amino acid residues were conserved among the cellulases,

resulting in 93.7% homology. In the linker region (302

through 355) consisting of 54 residues, 40 amino acid

residues were conserved among the cellulases, resulting in

74% homology. In the CBD (356 through 437) consisting of

83 residues, 57 amino acid residues were conserved among

the cellulases, resulting in 68.6% homology. The CD

showed the highest 93.7% homology, whereas the regions

of signal, leader, linker, and CBD regions showed low

homology. The β-1,4-endoglucanase gene encoded a signal

peptide at the N-terminal end of the protein. The active site

131-H, 169-E, and 257-E are conserved among all isolates

except IARI-SP-4, in which Q is present at position 257.

IARI-SP-1 and IARI-SP-2, being the highest cellulase producers

found in this study, were found to be 100% homologous for

the β-1,4-endoglucanase gene.

Fig. 3. Phylogenetic tree of culturable bacteria based on the 16S rRNA gene sequence isolated from soil irrigated with effluents of

century paper mill.

Table 2. Phylogenetic affiliations of the β-1,4-endoglucanase gene, isolated from different Bacillus sp., with their nearest neighbor.

Bacterial Isolate Accession number Nearest neighbor of β-1,4-endoglucanase gene Homology (% )

B. subtilis IARI-SP-1 KF240847 B. subtilis strain BS-02 99

B. amyloliquefaciens IARI-SP-2 KF240848 B. subtilis strain BS-02 99

B. licheniformis IARI-SP-3 KF240849 B. subtilis strain BS-02 98

Bacillus sp. IARI-SP-4 KF240850 B. subtilis strain shu-3 98

B. pumilus IARI-SP-5 KF240851 B. subtilis strain AH18 98

B. thuringiensis IARI-SP-6 KF240852 B. subtilis strain DR 98

B. cereus IARI-SP-7 KF240853 Uncultured bacterium clone celWS14 98

B. mycoides IARI-SP-8 KF240854 B. subtilis strain shu-3 96

B. megaterium IARI-SP-9 KF240855 B. megaterium strain AP25 99

B. oceanisediminis IARI-SP-10 KF240856 B. subtilis strain shu-3 97

1078 Pandey et al.

J. Microbiol. Biotechnol.

Discussion

Soils perpetually irrigated with paper and pulp effluent

represents a unique niche with a predominance of cellulose-

degrading microorganisms, owing to their high organic

matter content. The predominance of Bacillus and Bacillus-

derived genera as dominant cellulase producers in the

present study confirms the earlier reports from such

environments [6, 18]. Genus Bacillus, being endospore

formers, are tolerant to the harsh conditions (high pH)

prevailing in such soils, which makes them the major

microflora of such environments [15].

Enzyme assays indicated that all isolates invariably

produced less β-glucosidase on different cellulosic substrates

tested. However, all isolates had greater catalytic activity

for CMC in comparison with other cellulosic substrates,

which revealed that enzymes produced by all isolates

could be categorized as endoglucanase with significant

Fig. 4. Alignment of deduced amino acid sequences of endoglucanases of the 10 isolates with B. subtilis strain shu-3.

Region 1-29, signal peptide; Region 48-301, cellulase catalytic domain (glucosyl hydrolase family 5/A2); Region 302-355, linker region; Region

356-437, cellulose-binding domain (CBD family IIIa); Sites 131-H, 169-E, and 257-E, active sites (proton donor or nucleophile).

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August 2014⎪Vol. 24⎪No. 8

amount of exoglucanase activity. Many reports suggest

that the cellulase systems from bacilli are incomplete and

could not act on microcrystalline cellulose [1], but others

do confirm the exoglucanase activity in certain bacilli [9].

The exoglucanase activity of the crude enzymes might be

due to the presence of a CBD in the β-1,4-endoglucanase.

Earlier studies have indicated that the CBD plays crucial

roles in crystalline cellulose hydrolysis, by binding to the

cellulose surface and enhancing the sacchrification ability

of endoglucanses [14, 23]. In the present study, rice straw

was found to be a good carbon source for production of

cellulase and hence can be evaluated as a substrate for the

large-scale production of cellulase, replacing costly substrates

like CMC. There are several studies on cellulose degradation

by Bacillus sp., and large variations in enzyme activity

under both optimized and non-optimized conditions have

been reported [3, 7, 9, 18]. For example, Li et al. [7] have

reported that in a Bacillus sp., maximum cellulase activity

(0.26 U/ml) was detected when the culture was grown in

Luria broth supplemented with 1% CMC at 37°C for 24 h.

Our data on the β-1,4-exoglucanase, β-1,4-endoglucanase,

and β-glucosidase activities of all 10 isolates were generated

from crude culture supernatants, but were encouraging

as all 10 bacterial isolates exhibited considerably higher

endoglucanase activities (ranging from 1.1 to 2.5 IU/ml)

than earlier reported [18, 19, 28]. The maximum CMCase

activity in the alkaline range may be due to the alkaline

condition prevailing in the soil. The nucleotide sequencing

and BLAST search of β-1,4-endoglucanase revealed limited

variations, indicating a close evolutionary relationship

among the isolates. The high proportion of cellulase enzyme

belonging to the GH5 family among bacilli found in our

study is in agreement with earlier reports [3, 9]. The CDs of

these enzymes were located in the N-terminal region and

the CBDs in the C-terminal region, like for endoglucanases

of many other Bacillus species earlier reported. Currently, the

CDs of polysaccharides are grouped into at least 15 of the

more than 80 known glycosyl hydrolase families, whereas

CBDs fall into at least 13 families. Two basic residues at

positions 2 and 3 (lysine and arginine) in the hydrophilic

leader region were followed by a hydrophobic core of 18

amino acid residues rich in leucine and isoleucine, which is

consistent with the findings of Mezes et al. [10]. The β-1,4-

endoglucanase gene encoded a signal peptide at the N-

terminal end of the protein and was characterized by a

short, hydrophilic, basic region along with a subsequent

long, hydrophobic region. The presence of a signal peptide

suggests higher extracellular cellulase activity observed for

various strains screened during the present study.

The soils irrigated with pulp and paper mill effluent

specifically enrich cellulolytic bacilli that exhibit both endo-

and exoglucanase activities. The analysis of the β-1,4-

endoglucanase gene isolated from Bacillus sp. provides a

new insight into the potential capacity of the microbial

community to hydrolyze the lignocellulosic biomass.

Although all sequences found in the present study belonged

to the GH5 family, sequence analysis revealed significant

variations among them. These variations need to be further

exploited to correlate the efficiency of the enzyme, and its

ability to hydrolyze crystalline cellulose and also to be

secreted out of the cell in large amounts. Research is under

way to clone and overexpress the endoglucanase gene in a

suitable expression vector so that the product could be

utilized in the biofuel program being carried out at the

Institute.

Acknowledgments

The authors acknowledge funding from NAIP (70-24)

and ICAR National Fund for Basic, Strategic and Frontier

Application Research in Agriculture and sponsoring the

research project. The first author thanks the Indian Agricultural

Research Institute for providing fellowship during the

doctoral program.

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