characterization of a gene encoding cellulase from uncultured soil bacteria

R E S E A R C H L E T T E R

Characterizationofa gene encoding cellulase fromunculturedsoil bacteriaSoo-Jin Kim1, Chang-Muk Lee1, Bo-Ram Han1, Min-Young Kim1, Yun-Soo Yeo1, Sang-Hong Yoon1,Bon-Sung Koo1 & Hong-Ki Jun2

1Microbial Genetics Division, National Institute of Agricultural Biotechnology, Rural Development Administration, Suwon, Korea; and 2Department of

Microbiology, Pusan National University, Busan, Korea

Correspondence: Hong-Ki Jun, Department

of Microbiology, Pusan National University,

Busan 609-735, Korea. Tel.: 182 51 510

2270; fax: 182 51 514 1778; e-mail:

[email protected]

Received 11 October 2007; accepted 17

January 2008.

First published online 18 March 2008.

DOI:10.1111/j.1574-6968.2008.01097.x

Editor: Clive Edwards

Keywords

cellulase; xylanase; uncultured bacteria;

metagenome.

Abstract

To detect cellulases encoded by uncultured microorganisms, we constructed

metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed

a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source.

Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2)

encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence

analysis with the deduced amino acid sequences of celM2 showed 36% sequence

identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity

to endo-1,4-b-xylanase A from Cellulomonas pachnodae. The highest enzymatic

CMC hydrolysis was observable at pH 4.0 and 45 1C with recombinant CelM2

protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no

substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-b-

cellobioside, pNP-b-glucoside, and pNP-b-xyloside. These results showed that

CelM2 is a novel endo-type cellulase.

Introduction

In an effort to explore the biotechnological potential bioca-

talysts from uncultured microorganisms, cultivation-

independent metagenome approaches have been widely

adopted (Rondon et al., 2000; Gillespie et al., 2002; Ellis

et al., 2003). In these approaches, environmental DNAs are

extracted directly from samples without individual cell

culture, and the whole genome is subjected to heterologous

gene expression after cloning into appropriate vectors. The

libraries can be further screened for novel enzymes (Cottrell

et al., 1999; Henne et al., 2000; Lorenz et al., 2002).

Cellulose, a linear polymer of b-linked glucose molecules,

present in plant cell walls, is the most abundant

biopolymer (Lynd et al., 2005). Prior studies for natural

cellulose hydrolysis have revealed many cellulolytic micro-

organisms and their complex cellulases (Lowe et al., 1987;

Ohmiya et al., 2003; Lynd et al., 2005). Bacterial and fungal

cellulases can be classified into three types: endoglucanases

(EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and b-glucosidases

(EC 3.2.1.21) (Han et al., 1995; Cho et al., 2006; Lee et al.,

2008). Xylans, the second most common natural biopolymer,

consist of mainly b-1,4-linked D-xylose heteropolysaccharide

units. Endo-1,4-b-xylanase (EC 3.2.1.8) and b-xylosidase (EC

3.2.1.37) can fully hydrolyze the backbone chain of xylan. In

addition, a-L-arabinofuranosidase (EC 3.2.1.55), acetyl ester-

ase (EC 3.1.1.6), and a-D-glucuronidase (EC 3.2.1.1) cleave

the side chain of xylan (Polizeli et al., 2005; Lee & Cho, 2006).

Often, cellulase and xylanase contain multiple enzyme units

that have a marked synergism against hemicellulosic residues

(Akila & Chandra, 2003; Pason et al., 2006).

Cellulases and xylanases are important enzymes used in

the bioconversion of renewable cellulosic biomass such as

biomass degradation and fuel production (Gawande &

Kamat, 1999; Fujita et al., 2002; Lynd et al., 2002; Ohmiya

et al., 2003). These enzymes are also involved in the textile

industry for biopolishing of fabrics (Ohmiya et al., 2003; Li

et al., 2005) as well as in agriculture for making digestible

animal feeds from cellulose (Zheng et al., 2000).

Despite recent reports characterizing cellulases from

metagenome (Voget et al., 2006; Feng et al., 2007), the bio-

technological potential of novel cellulases from uncultured

soil metagenome has not been fully explored. In this study,

we have made metagenome libraries from environmental soil

samples to screen cellulolytic enzymes. From soil metagen-

ome, a novel cellulase gene was isolated and characterized.

FEMS Microbiol Lett 282 (2008) 44–51c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Materials and methods

Samples, strain, plasmid and metagenomiclibraries

Soil samples were collected from Upo wetland, Mujechi bog

and Daebudo sandbar in Korea (Kim et al., 2004). Soil DNA

was prepared by directed DNA extraction and purification as

described (Kim et al., 2007). Metagenomic fosmid libraries

from these samples were constructed by an adjusted protocol

as described previously (Yun et al., 2005). Briefly, we applied

two-step DNA purification with pulsed-field gel electrophor-

esis (PFGE) (CHEF, BioRad) to remove humic compounds

included in the soil DNAs. Crude soil DNAs were fractio-

nated by PFGE in 1% low melting point agarose under

4 V cm�1 electrical field at 14 1C for 12 h. A gel slice contain-

ing 100–190 kb of DNA was processed with agarase (1 U per

100 mg slice, Takara, Japan). After Sau3AI partial-digestion

(0.05 UmL�1 of DNA, 37 1C for 1 h), soil DNA was again

fractionated by PFGE into c. 40-kb lengths. The purified

DNA was ligated into BamHI-digested pSuperCosI vector for

packaging (MaxPLax, Epicentre). Escherichia coli DH5a was

used as a host cell for a routine library manipulation. The

plasmid pUC118/HincII/BAP purchased from Takara (Kyoto,

Japan) was used for the construction of shotgun libraries.

Library screening and sequence analysis

Metagenomic libraries were screened for carboxymethyl

cellulolytic activity by a Congo red overlay method (Teather

& Wood, 1982). The libraries were replicated onto 96-well

plates containing Luria–Bertani (LB) broth supplemented

with chloramphenicol (12.5 mg mL�1). After incubation at

37 1C for 24 h, the libraries were replicated onto LB agar

plates with 0.1% carboxymethyl cellulose (CMC, sodium

salt, Sigma) and chloramphenicol (12.5mg mL�1) using a

96-pin replicator. The reaction was further incubated at

28 1C for 7 days, followed by flooding with 0.1% aqueous

Congo red for 10 min and washing with excess 1 M NaCl

solution. Congo red interacts with (1 ! 4)-b-D-glucans,

(1 ! 3)-b-D-glucans and (1 ! 4)-b-D-xylans. A clearing

zone around the colonies shows CMC hydrolysis. DNA

fragments of these positive clones were then released by

sonication (Misonix Sonicator 3000, 10 s, 0.5 output).

Sheared DNAs were cloned into a pUC118/HincII vector

after end-repairing, and were screened again using the same

CMC hydrolysis test. DNA sequences were determined by a

PTC-200 Thermocycler (MJ Research) with an ABI PRISM

BigDye Terminator Cycle Sequencing Kit (Applied Biosys-

tems, version 3.1) according to the manufacturer’s instruc-

tions. The BLAST program at the National Center for

Biotechnology Information (NCBI) was used for database

searches and sequence comparisons. Amino acid sequences

were aligned using a CLUSTALW software package (MEGA 4.0).

Enzyme overexpression and purification

The putative cellulase gene was amplified from the CMC

positive clone (pCM2) using a sense primer (50-

TGGGGAGCTCATGCAAAACCCTTCAGTCA-30) with a

BamHI site and an antisense primer (50-GCCAAGCTTTCT-

GAGGGTGACGGTTCG-30) with a HindIII site. The ampli-

fied DNA was then ligated into BamHI and HindIII double-

digested pET21a(1) (Novagen), and the construct (pE-

CELM2) was transformed into E. coli BL21(DE3) cells.

Transformed cells were grown in 500 mL of LB broth at

37 1C until an OD600 nm of 0.6 was reached. At this point,

isopropyl-b-D-thiogalactopyranoside (IPTG) was added to a

final concentration of 50mM, and the flasks were further

incubated at 30 1C for 5 h. To trap His-tag, the native soluble

extract was purified using nickel-nitroacetic acid (Ni-NTA)

agarose slurry according to the manufacturer’s instructions

(QIAgen, Germany). The purified CelM2 was separated on

an 8% sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis (SDS-PAGE) gel.

Determination of enzymatic activity

The cellulase activity was measured by incubating 25 mL of

enzyme (0.8 mg mL�1) with 1% (w/v) CMC in 100 mL of

100 mM sodium acetate buffer (pH 4.0) at 45 1C for 1 h. One

unit (U) of the CelM2 activity was defined as the amount of

enzyme releasing 1 mmol of reducing sugar per minute.

Specific activity was defined as the number of activity units

per microgram of protein. The reduced sugar was measured

by the 1% 3,5-dinitrosalicylic (DNS) reagent method (Mill-

er, 1959). The effects of pH and temperature on the cellulase

were further examined using purified recombinant enzyme

CelM2. To decide the optimal pH ranges, 100 mM sodium

acetate buffer (pH 3.5–6.0), 100 mM sodium phosphate

buffer (pH 6.0–8.0) and 100 mM Tris-HCl buffer (pH

8.0–9.0) were used. The pH stability was compared by

preincubating the enzyme overnight in 4 1C at various pH

as above, and then measuring the residual cellulase under

the same standard assay conditions. To find out the optimal

temperatures, the enzyme mixtures were incubated at tem-

peratures from 30 to 85 1C for 1 h. Thermostability data

were compared after preincubating the enzyme at various

temperatures from 35 to 65 1C, and then measuring the

residual activities. To investigate the substrate specificity,

20 mg of the CelM2 was tested in enzyme assays under

optimal conditions for 60 min by replacing CMC with 1%

(w/v) polysaccharide substrates such as barley glucan (Sigma),

oat spelt xylan (Sigma), birch wood xylan (Sigma), and

avicel PH101 (Fluka). The substrates p-nitrophenyl-

b-D-glucoside (p-NPG, Sigma), p-nitrophenyl-b-D-cellobio-

side (p-NPC, Sigma) and p-nitrophenyl-b-D-xyloside

(p-NPX, Sigma) were used as 0.1% in the test. The enzyme

assays were determined by the measurement of the

FEMS Microbiol Lett 282 (2008) 44–51 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

45Cellulase from uncultured bacteria

generated p-NP as described (Marques et al., 2003). The

specific activity was given in units per microgram of CelM2

proteins for the substrate specificity analysis.

Results and discussion

Screening of environmental metagenomiclibraries

Because screening of metagenome libraries usually requires

large inserts to study gene clusters, both fosmid and cosmid

vectors were used to construct a Korean soil metagenomic

library in E. coli. A preliminary search for clones expressing

cellulase activity on CMC identified a positive clone, pCM2,

from 70 000 clones screened. Unexpectedly, however, we

could isolate only one cellulase activity-expressing meta-

genome clone by initial screening. To identify genes respon-

sible for cellulase activity in pCM2, further subcloning

by sonication and screening using Congo red overlay

identified several positives (Fig. 1a). Among them, we

selected and sequenced one positive clone, pUCM2,

containing a 4024-bp DNA fragment. We observed higher

cellulase activity of pUCM2 than that of pCM2 (data not

shown).

With our screening approach, we made no attempt to

optimize transcription by T3, T7 and LacZ promoters. We

could only detect cellulase enzyme activity if the gene was

transcribed, translated and folded correctly in a heterolo-

gous host. Also, the detectable cellulase depends on the

enzyme localization either being secreted from the cell or cell

lysis allowing enough enzymes having activity with the

substrates used in the screen. This seems an important issue

when the cloned insert is far from the vector-derived

promoter sequences. It is also probable that the endogenous

gene expression was not successful for the ORFs gained from

bacterial species distantly related to E. coli because we made

no special attempt to optimize gene expression under their

native promoters (Rees et al., 2003). This may reflect our

observations that the clone pCM2 exhibited lower cellulase

than pUCM2.

Sequence analysis of a positive clone

The DNA sequence of pUCM2 revealed two tandem ORFs

with amino acid sequence homology to previous cellulases

and xylanases. The deduced amino acid sequence for the

first ORF showed a 59% identity with endo-1,4-b-xylanase

A from Cellulomonas pachnodae. The ORF1, designated

xynM2, consisted of 681 bp encoding a protein of 226 amino

acids, including an N-terminal signal peptide sequence (29

amino acids). Prediction of the signal peptide cleavage site

suggested that Gln30 was the N-terminal amino acid (Niel-

sen et al., 1997). The mature XynM2 consists of 197 amino

acids and has a theoretical molecular weight of 20.5 kDa. A

ribosomal binding site was located 7 bp upstream of the

ATG start codon, and potential � 35 and � 10 consensus

promoter sequences were recognized (Fig. 2a).

The other ORF showed 36% amino acids identity with

cellulase from the Synechococcus sp. CC9311. The ORF2,

designated celM2, consisted of 1989 bp encoding a protein of

662 amino acids, including a 30 amino acid N-terminal

signal peptide sequence. Prediction of a signal peptide

cleavage site suggested that Gln31 was the N-terminal amino

acid. The mature protein consists of 632 amino acids and

has a theoretical molecular weight of about 71.5 kDa.

Potential � 35 and � 10 consensus promoter sequences

were also recognized, explaining that the cellulase was

expressed by its own promoter in pUCM2 (Fig. 2a).

Multiple protein sequence alignments for these two ORFs

showed that both celM2 and xynM2 may have originated

from a currently unidentified bacterial source (Fig. 2b).

Recently, many studies have described that multienzyme

complexes produced by anaerobic bacteria are held together

into a complex to promote synergistic degradation of

cellulosic biomass. For these multienzyme complexes, cata-

lytic subunits such as endoglucanases, exoglucanases, and

xylanases are clustered together in its genome to permit

simultaneous xylanolytic-cellulolytic hydrolysis (Lynd et al.,

2002). For instance, a facultative anaerobic bacterium,

Paenibacillus curdlanolyticus also has a multienzyme that

degrades insoluble polysaccharides (Lynd et al., 2002; Pason

Fig. 1. Plate screening for CMC-hydrolyzing activity from a metagenomic library using a Congo red assay. (a) Isolation of shotgun clones by cellulase

assay. The most active clone was designated pUCM2. The same clone on either the CMC agar plate (b) or on xylan agar plate (c) confirmed cellulolytic

hydrolysis.


46 S.-J. Kim et al.

et al., 2006). Although arranging enzymes into a multi-

enzyme complex has obvious advantages over single-

enzyme, aerobic bacteria instead produce various extracel-

lular cellulolytic enzymes with individual binding modules.

Expression and purification of CelM2

We found that celM2 showed no significant sequence

homology to known cellulases. To characterize whether the

Fig. 2. Nucleotide sequence of the celM2/xynM2 gene and their flanking regions. (a) The deduced amino acid sequence is given in the one-letter code below

the nucleotide sequence. The translation termination codon is indicated by an asterisk. The presumptive promoter regions and RBS are indicated by an

underline and bold-type, respectively. The nucleotide sequence of pUCM2 has been assigned GenBank accession no. EF114228. (b) Multiple amino acid

sequence alignments of the celM2 and xynM2 by Neighbor-joining algorithm. Host strain and its GenBank accession number are marked in parenthesis.



celM2 gene product degrades cellulosic compounds, we

heterologously expressed celM2 in E. coli. The expressed

protein, CelM2, was purified to homogeneity from the

cellular extracts using Ni-NTA agarose slurry. However, the

final polypeptide product of CelM2 enzyme was smaller

(60 kDa) than the predicted molecular mass of 71.5 kDa

(Fig. 3). This inconsistency in recombinant protein size may

result from a similar proteolytic event reported previously

(Mawadza et al., 2000). In Bacillus subtilis, endoglucanases

such as CH43 are translated as a precursor protein and yield

an extracellular protein after removal of a peptide segment

from the carboxy-terminus. However, the biological

mechanism of CelM2 proteolysis in E. coli is not clear.

Characterization of CelM2

The recombinant protein CelM2 with smaller size than the

expected polypeptide size showed cellulolytic hydrolysis on

CMC plates. The maximum activity was observed at pH 4.0

and 45 1C (Fig. 4a and b). The pH stability of CelM2 with

substrate CMC was monitored between pH 3.5 and 9.0 in

100 mM buffer at 4 1C for 24 h. The protein was stable in the

pH ranges from 3.5 to 4.0. No residual activity was detected

between pH 8.0 and 9.0 (Fig. 4c).

Besides, the protein was active over a broad range of

temperatures under acidic conditions (Fig. 4b). After in-

cubation in sodium acetate buffer (100 mM, pH 4.0),

maximum activity was observed at 45 1C. The temperature

stability of CelM2 was determined by measuring residual

CMC hydrolysis at various temperatures (Fig. 4d). Thermo-

stability data showed that, even after 60 min of incubation at

55 1C, about 50% cellulolytic activity was retained. However,

CelM2 is rapidly deactivated at 65 1C in less than 10 min.

Substrate specificity

The recombinant protein CelM2 shows endoglucanase-like

hydrolysis. Under optimum conditions, CelM2 favors var-

ious celluloses as its physiological substrates (Table 1).

CelM2 exhibited the highest activity toward barley glucan

(140.99� 0.79 U per microgram of protein, Umg�1) fol-

lowed by CMC (104.99� 2.06 U mg�1). Activities toward

birch wood xylan (51.72� 0.79 U mg�1) and oat spelt xylan

(20.20� 1.32 Umg�1) were moderate. In addition, CelM2

Fig. 2. Continued.

Fig. 3. SDS-PAGE analysis of purified recombinant CelM2 protein. M, broad

range marker; T, cell lysate; S, cell free supernatant; 1, eluates in 100 mM

imidazole solution; 2, eluates in 150mM imidazole solution; 3, eluates in

200mM imidazole solution; 4, eluates in 250mM imidazole solution.


48 S.-J. Kim et al.

could also hydrolyze avicel to a lesser extent (11.17�0.86 Umg�1). No significant substrate hydrolysis was observed

on oligosaccharides such as pNP-b-cellobioside, pNP-b-xyloside

and pNP-b-glucoside under the given assay conditions.

Compared to CMC, the activity of CelM2 was signifi-

cantly higher toward barley glucan with the b-1,3/4-linkage.

The substrate preference was observable even towards

microcrystalline cellulose materials such as avicel, which is

almost completely resistant to hydrolysis by most endoglu-

canases. A previous study described exoglucanase as being

able to hydrolyze microcrystalline cellulose by peeling off the

terminal cellulose chains from microcrystalline structure

(Lynd et al., 2002). Natural microcrystalline is structurally

heterogeneous, with amorphous regions in particular near

the crystal surfaces. However, some endoglucanases from

Bacillus sp. and fungi exhibit avicelase activity in addition to

the CMCase activity with the help of exo-activity resident

(Kim, 1995). The avicel hydrolysis by CelM2 also suggests

that the enzyme may have exoglucanase activity on micro-

crystalline celluloses (Teeri, 1997).

CelM2 also significantly hydrolyses nonglucosidic poly-

mers such as birchwood xylan and oat-spelt xylan. No

significant substrate hydrolysis was observed on pNP-

b-xyloside. The hydrolysis of both xylan and cellulose

suggests an overlapping substrate specificity of CelM2. This

mode of action is consistent with previous reports on

100

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3 4 5 6 7 8 9 10

pH

100

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100

75

25

0

50

3 4 5 6 7 8 9 10

pH

Rel

ativ

e ac

tivity

(%

)R

elat

ive

activ

ity (

%)

Rel

ativ

e ac

tivity

(%

)R

elat

ive

activ

ity (

%)

100

90

80

70

60

50

40

20 30 40 50 60 70

20100 30 40 50 60 70

80 90

Temperature (°C)

Time (min)

35°C45°C55°C65°C

(a) (b)

(c) (d)

Fig. 4. Biochemical characterization of CelM2 enzyme. (a) CMC hydrolysis by CelM2 was assessed in sodium acetate buffer (�), sodium phosphate

buffer (m), and Tris-HCl buffer (’) at 45 1C for 60 min. The relative CelM2 activities were measured at the indicated pH. (b) Temperature effect on the

activity of CelM2. Enzyme activity was assayed in 100 mM sodium acetate buffer (pH 4.0) for 60 min at the given temperatures. (c) CelM2 stability under

various pH conditions was measured as described in ‘Materials and methods’. (d) Stability of CelM2 activity by temperature variation. The residual

activity disappeared in 20 min at 65 1C. The average of triplicate experiments is presented.

Table 1. Substrate specificity of CelM2

Substrate� Specific activityw (U mg�1)

Barley glucan (b-1,3/4-glucan) 140.99�0.79

CMC (b-1,4-glucan) 104.99�2.06

Xylan from birch wood (b-1,4-xylan) 51.72�0.79

Xylan from oat spelt (b-1,4-xylan) 20.20�1.32

Avicel PH101 (b-1,4-glucan) 11.17�0.86

Cellobiose o 0.01

p-Nitrophenyl-D-cellobioside 0.16�0.37

p-Nitrophenyl-D-glucoside o 0.01

p-Nitrophenyl-D-xyloside 0.69�0.35

�Ten microliters of purified CelM2 (2 mg mL�1) was added to 100mL

sodium acetate buffer (pH 4.0) containing 1% (w/v) substrate. CMC,

carboxymethyl celluose.wThe specific activity was given in units per microgram of protein. One

unit (U) of the CelM2 activity was defined as the amount of enzyme

releasing 1mmol of reducing sugar per minute. No significant activity was

detected for oligosaccharides. SE of mean was calculated from triplicate.



various cellulases (Shepherd et al., 1981; Han et al., 1995;

Cho et al., 2006). For instance, the cellulase from Thermo-

ascus aurantiacus also degraded xylan. Likewise, it

was proposed that xylanase activity occurred on cellulase

(Shepherd et al., 1981).

Currently, significant metabolic efforts have been made to

increase cellulase and xylanase production using micro-

organisms originating from natural environments

(Ponce-Noyola & de la Torre, 1995; Pason et al., 2006; Lee

et al., 2008). One industrial strategy is to find hyperproducer

strains by screening and selection of induced mutants.

Another approach includes assembling a bifunctional cellu-

lase-xylanase by end-to-end fusion (Hong et al., 2006).

Therefore, our results suggest that celM2 may be a useful

novel gene source for endo-cellulose biomass degradation.

Acknowledgements

This work was supported by a grant from the National Institute

of Agricultural Biotechnology Number 05-4-11-16-3

and 07-4-11-16-5. Additional funds were provided from

the Technology Development Program for Agriculture and

Forestry of the Korean Ministry of Agriculture and Forestry.

Authors’contribution

S.J.K. and C.M.L. contributed equally to this work.

References

Akila G & Chandra TS (2003) A novel cold-tolerant Clostridium

strain PXYL1 isolated from a psychrophilic cattle manure

digester that secretes thermolabile xylanase and cellulase.

FEMS Microbiol Lett 219: 63–67.

Cho KM, Hong SY, Lee SM, Kim YH, Kahng GG, Kim H & Yun

HD (2006) A cel44C-man26A gene of endophytic Paenibacillus

polymyxa GS01 has multi-glycosyl hydrolases in two catalytic

domains. Appl Microbiol Biotechnol 73: 618–630.

Cottrell MT, Moore JA & Kirchman DL (1999) Chitinases from

uncultured marine microorganisms. Appl Environ Microbiol

65: 2553–2557.

Ellis RJ, Morgan P, Weightman AJ & Fry JC (2003) Cultivation-

dependent and -independent approaches for determining

bacterial diversity in heavy-metal-contaminated soil. Appl

Environ Microbiol 69: 3223–3230.

Feng Y, Duan CJ, Pang H et al. (2007) Cloning and identification

of novel cellulase genes from uncultured microorganisms in

rabbit cecum and characterization of the expressed cellulases.

Appl Microbiol Biotechnol 75: 319–328.

Fujita Y, Takahashi S, Ueda M et al. (2002) Direct and efficient

production of ethanol from cellulosic material with a yeast

strain displaying cellulolytic enzymes. Appl Environ Microbiol

68: 5136–5141.

Gawande PV & Kamat MY (1999) Production of Aspergillus

xylanase by lignocellulosic waste fermentation and its

application. J Appl Microbiol 87: 511–519.

Gillespie DE, Brady SF, Bettermann AD et al. (2002) Isolation of

antibiotics turbomycin A and B from a metagenomic library of

soil microbial DNA. Appl Environ Microbiol 68: 4301–4306.

Han SJ, Yoo YJ & Kang HS (1995) Characterization of a

bifunctional cellulase and its structural gene. The cell gene of

Bacillus sp. D04 has exo- and endoglucanase activity. J Biol

Chem 270: 26012–26019.

Henne A, Schmitz RA, Bomeke M, Gottschalk G & Daniel R

(2000) Screening of environmental DNA libraries for the

presence of genes conferring lipolytic activity on Escherichia

coli. Appl Environ Microbiol 66: 3113–3116.

Hong SY, Lee JS, Cho KM et al. (2006) Assembling a novel

bifunctional cellulase-xylanase from Thermotoga maritima by

end-to-end fusion. Biotechnol Lett 28: 1857–1862.

Kim CH (1995) Characterization and substrate specificity of an

endo-beta-1,4-D-glucanase I (Avicelase I) from an extracellular

multienzyme complex of Bacillus circulans. Appl Environ

Microbiol 61: 959–965.

Kim SJ, Koo BS, Park IC, Yoon SH, Yun SS, Sa TM & Yeo YS

(2004) Evaluation of DNA recovery from soil and sediment

samples. Agric Chem Biotechnol 47: 194–198.

Kim SJ, Lee CM, Kim MY, Yeo YS, Yoon SH, Kang HC & Koo BS

(2007) Screening and characterization of an enzyme with beta-

glucosidase activity from environmental DNA. J Microbiol

Biotechnol 17: 905–912.

Lee JH & Cho SH (2006) Xylanase production by Bacillus sp. A-6

isolated from Rice Bran. J Microbiol Biotechnol 16: 1856–1861.

Lee YJ, Kim BK, Lee BH et al. (2008) Purification and

characterization of cellulase produced by Bacillus

amyoliquefaciens DL-3 utilizing rice hull. Bioresour Technol

99: 378–386.

Li XT, Jiang ZQ, Li LT, Yang SQ, Feng WY, Fan JY & Kusakabe I

(2005) Characterization of a cellulase-free, neutral xylanase

from Thermomyces lanuginosus CBS 288.54 and its

biobleaching effect on wheat straw pulp. Bioresour Technol 96:

1370–1379.

Lorenz P, Liebeton K, Niehaus F & Eck J (2002) Screening for

novel enzymes for biocatalytic processes: accessing the

metagenome as a resource of novel functional sequence space.

Curr Opin Biotechnol 13: 572–577.

Lowe SE, Theodorou MK & Trinci AP (1987) Cellulases and

xylanase of an anaerobic rumen fungus grown on wheat straw,

wheat straw holocellulose, cellulose, and xylan. Appl Environ

Microbiol 53: 1216–1223.

Lynd LR, Weimer PJ, van Zyl WH & Pretorius IS (2002)

Microbial cellulose utilization: fundamentals and

biotechnology. Microbiol Mol Biol Rev 66: 506–577.

Lynd LR, van Zyl WH, McBride JE & Laser M (2005)

Consolidated bioprocessing of cellulosic biomass: an update.

Curr Opin Biotechnol 16: 577–583.

Marques AR, Coutinho PM, Videira P, Fialho AM & Sa-Correia I

(2003) Sphingomonas paucimobilis beta-glucosidase Bgl1: a


50 S.-J. Kim et al.

member of a new bacterial subfamily in glycoside hydrolase

family 1. Biochem J 370: 793–804.

Mawadza C, Hatti-Kaul R, Zvauya R & Mattiasson B (2000)

Purification and characterization of cellulases produced by

two Bacillus strains. J Biotechnol 83: 177–187.

Miller GL (1959) Use of dinitrosalicylic acid reagent for

determination of reducing sugar. Anal Chem 31:

426–428.

Nielsen H, Engelbrecht J, Brunak S & von Heijne G (1997)

Identification of prokaryotic and eukaryotic signal peptides

and prediction of their cleavage sites. Protein Eng 10: 1–6.

Ohmiya K, Sakka K, Kimura T & Morimoto K (2003) Application

of microbial genes to recalcitrant biomass utilization and

environmental conservation. J Biosci Bioeng 95: 549–561.

Pason P, Kyu KL & Ratanakhanokchai K (2006) Paenibacillus

curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme

system that degrades insoluble polysaccharides. Appl Environ

Microbiol 72: 2483–2490.

Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA &

Amorim DS (2005) Xylanases from fungi: properties and

industrial applications. Appl Microbiol Biotechnol 67: 577–591.

Ponce-Noyola T & de la Torre M (1995) Isolation of a high-

specific-growth-rate mutant of Cellulomonas flavigena on

sugar-cane. Appl Microbiol Biotechnol 42: 709–712.

Rees HC, Grant S, Jones B, Grant WD & Heaphy S (2003)

Detecting cellulase and esterase enzyme activities encoded by

novel genes present in environmental DNA libraries.

Extremophiles 7: 415–421.

Rondon MR, August PR, Bettermann AD et al. (2000) Cloning

the soil metagenome: a strategy for accessing the genetic and

functional diversity of uncultured microorganisms. Appl

Environ Microbiol 66: 2541–2547.

Shepherd MG, Tong CC & Cole AL (1981) Substrate specificity

and mode of action of the cellulases from the thermophilic

fungus Thermoascus aurantiacus. Biochem J 193: 67–74.

Teather RM & Wood PJ (1982) Use of Congo red-polysaccharide

interactions in enumeration and characterization of

cellulolytic bacteria from the bovine rumen. Appl Environ

Microbiol 43: 777–780.

Teeri TT (1997) Crystalline cellulose degradation: new insight

into the function of cellobiohydrolases. Trends Biotechnol 15:

160–167.

Voget S, Steele HL & Streit WR (2006) Characterization of a

metagenome-derived halotolerant cellulase. J Biotechnol 126:

26–36.

Yun S, Lee J, Kim S et al. (2005) Screening and isolation of a gene

encoding 4-hydroxyphenylpyruvate dioxygenase from a

metagenomics library of soil DNA. J Korean Soc Appl Biol

Chem 48: 345–351.

Zheng W, Schingoethe DJ, Stegeman GA, Hippen AR & Treachert

RJ (2000) Determination of when during the lactation cycle to

start feeding a cellulase and xylanase enzyme mixture to dairy

cows. J Dairy Sci 83: 2319–2325.



characterization of a gene encoding cellulase from uncultured soil bacteria

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