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Characterization of a Bifunctional Cellulase and Its Structural Gene
THE cel GENE OF BACILLUS SP. D04 HAS EXO- AND ENDOGLUCANASE ACTIVITY*
(Received for publication, February 23, 1995, and in revised form, August 7, 1995)
Sang Jun Han, Yong Je Yoo‡, and Hyen Sam Kang§
From the Department of Microbiology, College of Natural Sciences and ‡Department of Chemical Engineering, College of
Engineering, Seoul National University, Kwanak-Gu, Seoul 151-742, Korea
Bacillus sp. D04 secreted a bifunctional cellulase that
had a molecular weight of 35,000. This cellulase de-
graded Cm-cellulose, cellotetraose, cellopentaose, p-ni-
trophenyl-
b
-D-cellobioside, and avicel PH101. Based on
the high performance liquid chromatography analysis
of the degradation products, this cellulase randomly
cleaved internal
b
-1,4-glycosidic bonds in cellotetraose
and cellopentaose as an endoglucanase. It also hydro-
lyzed the aglycosidic bond in p-nitrophenyl-
b
-D-cellobio-
side and cleaved avicel to cellobiose as an exoglucanase.
Cellobiose competitively inhibited the p-nitrophenyl-
b
-
D-cellobioside degrading activity but not Cm-cellulose
degrading activity. Ten mMp-chloromercuribenzoate in-
hibited p-nitrophenyl-
b
-D-cellobioside degrading activ-
ity completely, but Cm-cellulose degrading activity in-
completely. Cm-cellulose increased p-nitrophenyl-
b
-D-
cellobioside degrading activity, and vice versa, whereas
methylumbelliferyl-
b
-D-cellobiose strongly inhibited
p-nitrophenyl-
b
-D-cellobioside degrading activity. The
cellulase gene (cel gene), 1461 base pairs, of Bacillus sp.
D04 was cloned. The nucleotide sequence of the cel gene
was highly homologous to those of Bacillus subtilis DLG
and B. subtilis BSE616. The cel gene was overexpressed
in Escherichia coli, and its product was purified. The
substrate specificity and substrate competition pattern
of the purified recombinant cellulase were the same as
those of the purified cellulase from Bacillus sp. D04.
These results suggest that a single polypeptide cellulase
had both endo- and exoglucanase activities and each
activity exists in a separate site.
Cellulose is an unbranched glucose polymer composed of an
anhydro-
b
-1,4-glucose units linked by a
b
-1,4-D-glycosidic
bond. Cellulolytic enzymes degrade cellulose by cleaving this
glycosidic bond. Cellulases can be classified into three types:
endoglucanases (1,4-
b
-Dglucan 4-glucohydrolase, EC 3.2.1.4),
exoglucanases (
b
-1,4-D-glucan cellobiohydrolase), and
b
-gluco-
sidases (
b
-D-glucoside glucohydrolase, EC 3.2.1.21). Endoglu-
canases randomly hydrolyze internal
b
-1,4-glycosidic bonds in
cellulose. As a result, the polymer rapidly decreases in length,
but the concentration of the reducing sugar increases slowly
(1). Exoglucanases hydrolyze cellulose by removing the cellobi-
ose unit from the nonreducing end of cellulose; the reducing
sugars are rapidly increased, but the polymer length changes
little (1–3).
b
-Glucosidases cleave cellobiose and oligosaccha-
rides to glucose (1). Therefore, crystalline cellulose is efficiently
hydrolyzed by the synergistic action of all three types of
cellulases.
Cellulosic substrates hydrolyzed by only one type of cellulase
are catagorized as follows. Acid-swollen cellulose, Cm-cellulose,
cellulose azure, and trinitrophenyl Cm-cellulose are hydrolyzed
by endoglucanases (1). MUC
1
(4) and pNPC (5) are used as
substrates for the determination of exoglucanase activity, and
MUG (4) and pNPG (5) are cleaved by
b
-glucosidases. Filter
paper and avicel are efficiently hydrolyzed by the synergistic
action of endo- and exoglucanases, but not by either one alone
(6).
Some organisms (for example, Trichoderma sp.) (6–9, 11)
produce all three types of cellulases and efficiently degrade
cellulose by their synergistic effect. A cellulolytic hydrolase
with a considerable level of endo-, exoglucanase, and xylanase
activity has been described (3, 12–14). For example, Saul re-
ported a cellulase gene (cel B) of Caldocellum saccharolyticum
with a Cm-cellulose-degrading domain in the C-terminal region
and an MUC degrading domain in the N-terminal region (14).
A polysaccharide hydrolase of the rumen fungus Neocallima-
trix patriciarum has a multifunctional catalytic domain with
high endoglucanase, cellobiohydrolase, and xylanase activities
(12, 13).
Extensive recent studies on proteins (such as cellulase, pro-
tease, and amylase) secreted by Bacillus species (15) have
shown that the following Bacillus species produce cellulases:
Bacillus cereus (16), Bacillus licheniformis (17), Bacillus sub-
tilis (18), and Bacillus polymyxa (19). Because these strains did
not produce all three types of cellulase, they did not extensively
hydrolyze crystalline cellulose. We have investigated another
strain of this species, Bacillus sp. D04, having the ability to
degrade crystalline cellulose. We have determined that the
cellulase of Bacillus sp. D04 differed from that of other Bacillus
species in several respects. In particular it has both endo- and
exoglucanase activity. It degraded Cm-cellulose, cellotetraose,
and cellopentaose as an endoglucanase and cleaved aglycosidic
bonds in pNPC as an exoglucanase. It also cleaved avicel to
cellobiose. Substrate competition assays showed that the cel-
lulase of Bacillus sp. D04 had separate sites for endo- and
exoglucanase activity.
* This work was supported in part by grants from the Korea Ministry
of Education, Korea Institute of Energy and Resources, and the Re-
search Center for Molecular Microbiology, Seoul National University.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank
TM
/EMBL Data Bank with accession number(s) U27084
for cel gene cellulase.
§ To whom correspondence should be addressed. Tel.: 82-2-880-6701;
Fax: 82-2-876-4440; E-mail: sangjun@alliant.snu.ac.kr.
1
The abbreviations used are: MUC, methylumbelliferyl-
b
-D-cellobi-
ose; MUG, methylumbelliferyl-
b
-D-glycopyranoside; pNPC, p-nitro-
phenyl-
b
-D-cellobioside; pNPG, p-nitrophenyl-
b
-D-glycopyranoside;
pCMB, p-chloromercuribenzoate; PCR, polymerase chain reaction;
IPTG, isopropylthio-
b
-D-galactoside; PAGE, polyacrylamide gel
electrophoresis; HPLC, high performance liquid chromatography.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 43, Issue of October 27, pp. 26012–26019, 1995
© 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
26012
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EXPERIMENTAL PROCEDURES
Purification of Cellulase
Bacillus sp. D04 was cultured in 2 liters of medium (containing M9
minimal salts, 0.5% glucose and 0.5% avicel) at 45 °C for 13 h. After the
medium was centrifuged at 11,000 3gfor 10 min, the supernatant was
concentrated by ultrafiltration (10,000 nominal molecular weight cut-
off membrane was used). Ten mMpotassium phosphate buffer (pH 5.8)
was added to the concentrated sample, and the sample was reconcen-
trated to 100 ml. This sample was passed through Cm-Sepharose CL-6B
(7 350 mm) equilibrated with 10 mMpotassium phosphate buffer (pH
5.8) at a flow rate of 15 ml/h. The sample flow-through was loaded
directly onto hydroxylapatite (7 330 mm) equilibrated with 10 mM
potassium phosphate buffer (pH 5.8) and eluted with a 30-ml 10–250
mMpotassium phosphate salt gradient at a flow rate of 10 ml/h. The
concentration of protein was measured with Bradford solution
(Bio-Rad).
Cellulase Enzyme Assay
Cm-cellulose and Avicel Degrading Activity Assay—The Cm-cellulase
assay consisted of 800
m
l of 1% Cm-cellulose in 10 mMpotassium
phosphate buffer (pH 5.8) and 200
m
l of diluted enzyme solution, incu-
bated at 45 °C for 20 min. Avicel degrading activity was measured as
follows: 500
m
l of 10 mg/ml avicel in 10 mMpotassium phosphate buffer
(pH 5.8) was mixed with 500
m
l of suitably diluted enzyme and then
incubated for 72 h at 45 °C in a shaking incubator. The remaining avicel
was removed by centrifugation, and the amount of reducing sugar was
detected with 3,5-dinitrosalicylic reagent. One unit of Cm-cellulose and
avicel degrading activity was defined as the amount of enzyme required
for producing 1
m
mol of glucose/min.
pNPC Degrading Activity Assay—The reaction mixture, consisting of
800
m
l of pNPC at 1 mg/ml in 50 mMsodium acetate buffer (pH 5.8) and
200
m
l of suitably diluted enzyme, was incubated at 45 °C for 20 min.
The p-nitrophenol released from pNPC was detected at 420 nm after
adding 1 ml of 2% sodium carbonate. One unit of enzyme activity was
defined as the amount of enzyme required for producing 1
m
mol of
p-nitrophenol/min.
MUC Degrading Activity Assay—The reaction mixture, consisting of
800
m
l of 1 mg/ml MUC in 50 mMsodium acetate buffer (pH 5.8) and 200
m
l of suitably diluted enzyme, was incubated at 45 °C for 20 min. The
reaction was stopped by adding 3.5 ml of 0.5 Mglycine/NaOH buffer (pH
10.4). Fluorescence measurements were made on a Tasco FP-777 spec-
trofluorometer at 20 °C, with an excitation wave length of 365 nm and
detection at 450 nm. One unit of enzyme activity was defined as the
amount of enzyme required for producing 1
m
mol of 4-methylumbellif-
erone/min (9).
High Performance Liquid Chromatography Analysis of
Degradation Products
The reaction mixture, consisting of 80
m
l of oligosaccharides released
from cellulosic substrates, 20
m
l of 10% trichloroacetic acid, and 100
m
l
of 0.3% (w/v) ethanolic solution of dansyl hydrazine, was heated at
80 °C for 10 min, and then cooled (20–22). Samples were dried, dis-
solved in 78% acetonitrile solution, and analyzed with
m
-Bondapak
y
NH
2
column (3.9 3300 mm, Waters) (23). The dansyl hydrazone of
oligosaccharides were detected at 254 nm. The 78% acetonitrile solution
was used as an elution solvent, and the flow rate was 1.5 ml/min.
FIG.1.Purification and activity staining of cellulase from Ba-
cillus sp. D04. Panel A shows SDS-PAGE from various purification
steps of cellulase. Lane a was a sample obtained by ultrafiltration, lane
bwas a sample passed through Cm-Sepharose, and lane c was a puri-
fied sample from hydroxylapatite chromatography. Panel B shows the
activity staining of lane c in Panel A. Molecular weight markers were in
lane d:1, phosphorylase b(97,400); 2, glutamate dehydrogenase
(55,400); 3, lactate dehydrogenase (36,500); 4, trypsin inhibitor
(20,100). The arrowhead points to a zone in which Cm-cellulose was
degraded by the cellulase.
FIG.2.Hydroxylapatite chromatog-
raphy. Distribution of protein concentra-
tion (●), p-nitrophenyl-
b
-D-cellobiose (10
2
3unit/ml; E), and Cm-cellulose (unit/ml;
f) degrading activity after hydroxylapa-
tite chromatography (7 330 mm) eluted
with potassium phosphate salt gradient
(10–250 mM) at a flow rate of 10 ml/h.
TABLE I
The steps in purification of cellulase from Bacillus sp. D04
Purification steps Total Protein Total volume Total activity Specific activity -Fold
mg ml units units/mg
1. Cm-cellulose degrading activity
Ultrafiltration 35.40 100.0 122.10 3.44
Cm-Sepharose 2.44 120.0 18.00 7.22 2.1
Hydroxyapatite 0.07 2.4 9.00 128.00 37.2
2. pNPC degrading activity
Ultrafiltration 35.40 100.0 2.10 0.06
Cm-Sepharose 2.44 120.0 0.19 0.08 1.3
Hydroxyapatite 0.07 2.4 0.16 2.28 38.0
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Cloning and Determination of Nucleotide Sequence of the
Cellulase Gene
Chromosomal DNA from Bacillus sp. D04 was partially digested with
Sau3AI producing 3–5-kb DNA fragments. These were isolated by den-
sity gradient centrifugation in 10 to 40% sucrose using an SW 28 rotor
at 20,000 rpm for 20 h at 20 °C. Fragments were ligated with the
dephosphorylated BamHI site of pBluescript KS(1) and then trans-
ferred into an Escherichia coli DH5
a
strain. Transformants with Cm-
cellulose degrading activity were screened on an L-agar plates contain-
ing ampicillin at 100
m
g/ml, 0.5% Cm-cellulose, and trypan blue at 0.1
mg/ml. To screen MUC degrading activity, transformants were trans-
ferred onto L-agar plates containing ampicillin at 100
m
g/ml and MUC
at 50
m
g/ml. For the determination of the nucleotide sequence of the cel
gene, serial deletion of the gene was done by using Erase-a-Base
y
kit
(Promega). The sequence of cellulase gene was determined from both
strands by the dideoxynucleotide chain termination method using a
Sequenase
y
kit (U.S. Biochemical Corp.).
PCR Amplification of the cel Gene and Construction of pCO1
The cel gene in pBluescript KS(1) was amplified by PCR with 59-
CATATGAAACGGTCAATCTCT-39(ATG in the NdeI site (CATATG)
was a start codon of the cel gene) and M13 reverse primer. Amplification
was done by 30 cycles of PCR at standard reaction conditions: reaction
volume, 50
m
l; reaction composition, 10 mMTris-HCl (pH 8.3), 50 mM
KCl, 1.5 mMMgCl
2
,50
m
MdNTP, 2 fmol of template, 10 pmol primer,
and 2 units of Vent DNA
y
polymerase; cycle profile, 1 min at 95 °C, 1
min at 50 °C, 1.5 min at 72 °C. The PCR products were purified and
digested with HindIII. These fragments were ligated with pBluescript
KS(1) that had been digested with SmaI and HindIII. This recombi-
nant DNA was named pCO1.
Construction of pCO
2
and Transfer into E. coli BL21(DE) pLysS
The pCO1 vector was digested with EcoRV and then partially di-
gested with NdeI to generate the 1.5-kb cel gene. The overexpression
vector, pET-3a-d (Novagen, Inc.), was digested with BamHI, and then
end-filling was done with a Klenow fragment. This overexpression
vector was digested with NdeI and then ligated with the 1.5-kb cel gene.
This recombinant plasmid was named as pCO
2
. The pCO
2
vector was
transferred into E. coli BL21(DE) pLysS(F
2
ompT hadS
B
(r
B
2
m
B
2
) dcm
gal(DE) pLysS, Cm
r
) by electroporation (BTX electro cell manipulator
600; capacitance; 50 F, charging voltage; 1.0 kV, resistance; 129 ohms).
Overexpression of the Cellulase Gene
E. coli BL21(DE)pLysS with pCO
2
was cultured in Luria-Bertani
medium containing ampicillin at 50
m
g/ml and chloramphenicol at 30
m
g/ml for 12 h and then transferred into TBGM9 medium (tryptone at
10 mg/ml, NaCl at 5 mg/ml, M9 salt, 0.4% (w/v) glucose, and 1 mM
MgSO
4
) containing ampicillin at 50
m
g/ml. To obtain high levels of
transcription, these cells were grown to mid-log phase, IPTG was added
to a final concentration of 0.5 mM, and growth continued for3hat30°C.
FIG.3. HPLC analysis of degrada-
tion products. Panel A, HPLC analysis
of products released from cellotetraose.
The reaction mixture containing 10
m
lof
10 mMpotassium phosphate (pH 5.8), 40
m
l of the purified cellulase (0.1 mg/ml),
and 30
m
l of 10 mg/ml cellotetraose were
incubated for 0 (a), 60 (b), and 120 (c) min
at 45 °C. Panel B, HPLC analysis of prod-
ucts released from cellopentaose. 40
m
lof
10 mg/ml cellopentaose was used as a
substrate instead of cellotetraose. These
samples were incubated for 0 (a), 60 (b),
and 120 (c) min at 45 °C. Panel C, HPLC
analysis of products released from avicel.
800
m
l of 1% (w/v) avicel solution and 800
m
l of the purified cellulase (0.1 mg/ml)
were mixed and incubated for 72 h at
45 °C. (a) was a control that did not con-
tain cellulase, whereas (b) contained pu-
rified cellulase. These reaction products
were modified with dansyl hydrazine as
described under “Experimental Proce-
dures.” The absorbance was measured at
254 nm. The numbers 1–4 represent the
dansyl hydrazones of: 1, cellobiose; 2,
cellotriose; 3, cellotetraose; and 4,
cellopentaose.
TABLE II
The substrate specificity of cellulase
Substrate Concentration
a
Specific activity
b
Purified cellulase
from Bacillus
sp. D04
Purified recombinant
cellulase from
E. coli
mg/ml units/mg
Cm-cellulose 10 128.0 101.9
pNPC 1 2.28 2.25
MUC 1 53.7 45.5
pNPG 1 0 0
MUG 1 0 0
Avicel 1 0.014 0.01
a
Concentration indicates weight concentration of each substrate.
b
The unit of each substrate is described under “Experimental
Procedures.”
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Purification of Overexpressed Cellulase
The overexpressed recombinant cellulase from E. coli
BL21(DE)pLysS was partially purified by ammonium sulfate fraction-
ation as previously described (24). This partially purified cellulase was
dialyzed in 20 mMsodium acetate buffer (pH 4.8) and then loaded onto
Cm-Sepharose CL-6B (15 350 mm) equilibrated with dialysis buffer.
The proteins were eluted with a 80-ml 20–500 mMsodium acetate (pH
4.8) salt gradient at a flow rate of 20 ml/h.
Activity Staining of Cellulase
The protein sample was mixed with protein loading dye and then
incubated at 68 °C for 1 h. These samples were loaded onto a 10%
polyacrylamide gel containing 0.1% Cm-cellulose, then subjected to
electrophoresis. After SDS-PAGE, one of the gels was stained with
Coomassie Blue R250. Another was soaked and gently shaken in 50 mM
phosphate buffer (pH 6.8) containing 25% isopropanol for 30 min. It was
transferred to 50 mMphosphate buffer (pH 6.8) and shaken for 30 min.
The buffer was removed, and the gel was incubated for 20 min at 37 °C.
This gel was stained with 1% Congo Red solution for 5 min and
destained with 1 MNaCl/NaOH solution.
RESULTS
Purification of Cellulase—Because Bacillus sp. D04 secreted
cellulase into medium, concentrated medium was used as start-
ing material for enzyme purification. Many other proteins were
removed by passage through the Cm-Sepharose CL-6B (Fig.
1A). The sample that eluted at 180 mMpotassium phosphate
from hydroxylapatite had both Cm-cellulose and pNPC degrad-
ing activities (Fig. 2). SDS-PAGE of this sample revealed only
a 35,000-Da single polypeptide (Fig. 1A). The molecular weight
of the native form of this cellulase, determined by gel perme-
ation chromatography (Superose 12, Pharmacia Biotech Inc.),
was also about 35,000. Activity staining showed that this pu-
rified protein had Cm-cellulose degrading activity (Fig. 1B).
The steps in purification of this protein are given in Table I.
Substrate Specificity of the Purified Cellulase—The activity
of the purified cellulase was assayed with various cellulosic
substrates. This cellulase degraded Cm-cellulose, pNPC, MUC,
and avicel PH101 (Table II). However, the specific activity
toward avicel was much lower than that of the soluble sub-
strates. Neither MUG nor pNPG was hydrolyzed (Table II).
HPLC Analysis of Oligosaccharides from Cellulosic Sub-
strates—HPLC analysis showed that a single peak was de-
tected at 280 nm as a reaction product on hydrolysis of pNPC
by this cellulase (data not shown). The retention time of it was
the same as that of p-nitrophenol. Therefore we identified it as
p-nitrophenol. This means that enzyme cleaved only aglyco-
sidic bond in pNPC, producing cellobiose and p-nitrophenol.
The enzyme cleaved cellulosic substrates (such as cellotetraose
and cellopentaose) to glucose, cellobiose, and other oligosaccha-
rides. Since these compounds are not detected at any wave
length, we modified them with dansyl hydrazine because sugar
dansyl hydrazones could be detected at 254 nm. On the basis of
HPLC analysis, the purified cellulase cleaved cellotetraose to
cellobiose and cellotriose (Fig. 3A). It also produced cellobiose,
cellotriose, and cellotetraose from cellopentaose (Fig. 3B).
These results indicate that the purified cellulase randomly
cleaved internal
b
-1,4-glycosidic bonds in these cellulosic sub-
strates as an endoglucanase. Based on the above result, it
would seem that the smallest substrate recognized by the en-
doglucanase of Bacillus sp. D04 is a cellotetraose. Both endo-
and exoglucanase activities were detected by using cellote-
traose and cellopentaose as substrates. But pNPC is not a
substrate for endoglucanase of Bacillus sp. D04 because it is
shorter than cellotetraose. Therefore only exoglucanase activ-
ity was detected by using pNPC as a substrate. The enzyme
also produced cellobiose from avicel as an exoglucanase (Fig.
3C).
Differential Inhibition of Cellulase Activity with Various In-
hibitors—The Cm-cellulose and pNPC degrading activities of
FIG.4.1/V versus 1/[S] plot of pNPC degrading activity in the
presence of cellobiose and pCMB. The various concentrations of
pNPC were incubated with purified cellulase in the presence of 0 (●), 5
(E), 10 (f), and 40 ()m
Mcellobiose (Panel A)and0(●),1(E),2(f), and
4()m
MpCMB (Panel B). The V
max
and K
m
of pNPC degrading activity
was 214
m
mol/min and 5.29 mM, respectively. Cellobiose did not change
V
max
but K
m
was changed to 8.62 and 12.62 mMin the presence of 10
and 40 mMcellobiose, respectively. The V
max
and K
m
were changed to 96
m
mol/min and to 6.75 mMby4mMpCMB.
TABLE III
The differential effects of various reagents on cellulase activity
Reagents Concentration Control cellulase activity
a
Cm-cellulose degrading
activity pNPC degrading
activity
mM%
PMSF
b
1 100 100
10 100 100
pCMB 1 76.2 51.1
10 33.1 0
Cellobiose 10 100 67.2
40 100 42.2
Ca
21
5 140.9 110.8
Zn
21
5 37.4 48.0
Mg
21
5 91.5 113.8
a
The control celluase activity was measured without reagents.
b
Phenylmethylsulfonyl fluoride.
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this cellulase were differentially inhibited by several inhibi-
tors. pCMB at 10 mMcompletely inhibited the pNPC degrading
activity but inhibited Cm-cellulose degrading activity by 67%.
In 40 mMcellobiose, Cm-cellulose degrading activity was not
inhibited, but the pNPC degrading activity was inhibited by
57.8% (Table III). Cellobiose changed the K
m
but not the V
max
of pNPC degrading activity as a competitive inhibitor (Fig. 4A),
whereas both the V
max
and K
m
of pNPC degrading activity
were changed by pCMB as a mixed-type inhibitor (Fig. 4B).
Both Cm-cellulose and pNPC degrading activity required Ca
21
,
but were strongly inhibited by Zn
21
.Mg
21
slightly increased
pNPC degrading activity and weakly inhibited Cm-cellulose
degrading activity (Table III).
Substrate Competition—To investigate whether a purified
cellulase contains each endo- and exoglucanase active site, we
performed substrate competition assays. At a high ratio of
Cm-cellulose (0.5%, w/v) to pNPC (0.005%, w/v), pNPC degrad-
ing activity was not inhibited, but was increased by Cm-cellu-
lose (Fig. 5A). Cm-cellulose degrading activity was not inhib-
ited, but was slightly increased in the presence of various
concentrations of pNPC (Fig. 5B). But 60% of the pNPC de-
grading activity was inhibited by MUC even at a low ratio of
MUC (0.01%, w/v) to pNPC (0.005%, w/v) (Fig. 5C).
Cloning and Nucleotide Sequence of the cel Gene—L-agar
plates containing Cm-cellulose and trypan blue were used to
clone the gene for Cm-cellulose degrading activity from the
genomic library in pBluescript KS(1), which was described
under “Experimental Procedures.” Cm-cellulose was stained
with trypan blue, but the hydrolyzed Cm-cellulose was not. As
a result, a halo formed around the colony with the Cm-cellulose
degrading activity. Eleven colonies having Cm-cellulose de-
grading activity were obtained (data not shown). To determine
the MUC degrading activity of these colonies, they were trans-
ferred onto an L-agar plate containing MUC. The colony with
exoglucanase activity cleaved MUC to cellobiose and methyl-
umbelliferone which emitted fluorescence when it was exposed
to UV light. All colonies with Cm-cellulose degrading activity
emitted fluorescence under the UV light after incubating for 12
h at 37 °C on L-agar plates containing MUC (data not shown).
Therefore, 11 colonies had a cellulase gene with both Cm-
cellulose and MUC degrading activities. The nucleotide se-
quence of this gene (Fig. 6) showed one open reading frame of
1461 base pairs was a possible gene encoding the cellulase. A
potential promoter (235 (TAGACAAT) and 210 (TACAAT))
and the Shine-Dalgarno sequence (ribosomal binding site) were
identified in the upstream region. Based on the nucleotide
sequence homology with other cellulase genes, the cellulase
gene of Bacillus sp. D04 has a high homology with those of B.
subtilis DLG (26) and B. subtilis BSE616 (27) (Fig. 6).
Overexpression of Recombinant Cellulase Gene and Purifica-
tion of Recombinant Cellulase—E. coli BL21(DE)pLysS with
pCO
2
overexpressed a 55,000-Da protein after IPTG was added
(Fig. 7). Activity staining showed that 55,000 and 35,000-Da
proteins had Cm-cellulose degrading activity (Fig. 8B). The
35,000-Da protein with Cm-cellulose degrading activity was
purified by Cm-Sepharose CL-6B chromatography (Fig. 8A).
Characteristics of Recombinant Cellulase—The purified re-
combinant cellulase degraded Cm-cellulose, pNPC, MUC, and
FIG.5.The substrate competition of
pNPC and Cm-cellulose degrading
activity. The various concentrations of
pNPC (0.005–0.05%, w/v) and Cm-cellu-
lose (0.1–0.5%, w/v) were mixed and incu-
bated with the purified cellulase for1hat
45 °C. The pNPC degrading activity in
the presence of various concentrations of
Cm-cellulose (●, 0%; E, 0.1%; f, 0.25%; ,
0.5%, w/v) is shown in Panel A. Panel B
indicates Cm-cellulose degrading activity
in the presence of various concentrations
of pNPC (●, 0%; E, 0.005%; f, 0.01%; ,
0.05%, w/v). The pNPC degrading activity
in the presence of various concentrations
of MUC (●, 0%; E, 0.01%; f, 0.05%; ,
0.25%, w/v) is shown in Panel C. Panels D,
E, and Fshow the substrate competition
of purified recombinant cellulase. The
substrate concentrations and reaction
times were the same as for the purified
cellulase. Panel D and Eshow pNPC and
Cm-cellulose degrading activity in the
presence of Cm-cellulose and pNPC, re-
spectively. Panel F shows pNPC degrad-
ing activity in MUC.
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FIG.6.Nucleotide sequence of the
cel gene and homology between cel-
lulase genes from Bacillus subtilis.
The potential promoter region (235 (TA-
GACAA), 210 (TACAAT) region), the
Shine-Dalgarno sequence (AAGGAGG)
are underlined. The stop codon is marked
as ***. The nucleotide sequence of the cel
gene is shown as line 1. Line 2 indicates
amino acid sequence deduced from the cel
gene and the underlined amino acid se-
quence is a typical
b
-glucanase signal
peptide of Bacillus species. Lines 3 and 4
indicate nucleotide sequences of the cellu-
lase genes of Bacillus subtilis BSE616
and DLG, respectively.
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avicel (Table II), but proteins extracted from E. coli DH5
a
strain did not degrade these cellulosic substrates. Cm-cellulose
slightly increased pNPC degrading activity, and vice versa (Fig.
5, Dand E). The pNPC degrading activity was strongly inhib-
ited by MUC (Fig. 5F).
DISCUSSION
The following results suggest that the purified 35,000-Da
cellulase secreted by Bacillus sp. D04 has both endo- and
exoglucanase activity. The endoglucanase of Clostridium
themocellum,Cellulomona fimi, and other Bacillus species hy-
drolyze Cm-cellulose, swollen cellulose, cellotetraose, and cel-
lopentaose, but not pNPC (1). The exoglucanase of Ruminococ-
cus flavafaciens FD-1 (2) and Aspergillus fumigatis (4)
hydrolyzed pNPC, MUC, and filter paper, but not Cm-cellulose.
However, the cellulase of Bacillus sp. D04 hydrolyzed Cm-
cellulose, pNPC, and MUC (Table II). Moreover, this cellulase
cleaved only the aglycosidic bond in pNPC as does an exoglu-
canase of Trichoderma viride and Sporotrichum pulveralentum
(5), and randomly cleaved internal
b
-1,4-glycosidic bonds in
cellotetraose and cellopentaose (Fig. 3, Aand B) as an endo-
glucanase. These results imply that cellulase of Bacillus sp.
D04 has both endo- and exoglucanase activity. The presence of
both activities in the purified cellulase is confirmed by the fact
that this cellulase also degraded crystalline cellulose (Fig. 3C),
even though the hydrolysis efficiency of avicel was less than
that of soluble cellulosic substrates. Probably, this was due to
the low affinity of the purified cellulase against a crystalline
cellulose.
To determine whether the active site of endo- and exoglu-
canase are separately existed, we studied differential effects of
compounds that specifically inhibited one type of cellulase ac-
tivity. The cellobiose competitively inhibited pNPC degrading
activity, but did not inhibit Cm-cellulose degrading activity
(Table III). However, since the K
i
of cellobiose was 35.4 mM
(Fig. 4A), cellobiose was not a strong inhibitor in pNPC degrad-
ing activity. pCMB, a thiol protease inhibitor, inhibited pNPC
degrading activity completely and Cm-cellulose degrading ac-
tivity incompletely (Table III). Therefore endo- and exoglu-
canase activities were differently inhibited by cellobiose and
pCMB. Xue et al. (13) showed that the polysaccharide hydro-
lase from N. patriciarum has a multifunctional catalytic do-
main that contains endoglucanase, cellobiohydrolase, and xy-
lanase activities. On the basis of the substrate competition
assays of this enzyme, Cm-cellulose and xylan strongly inhib-
ited hydrolysis of MUC (13). Thus, they clearly demonstrated
that only one active site has three types of enzyme activities.
But the substrate competition pattern of the cellulase of Bacil-
lus sp. D04 was different from those of N. patriciarum.Ata
high ratio of Cm-cellulose to pNPC or vice versa, one substrate
did not inhibit hydrolysis of the another substrate (Fig. 5, A
and B). But as MUC and pNPC were common substrates for
exoglucanase, MUC strongly inhibited pNPC degrading activ-
ity even if the ratio MUC (0.01%, w/v) to pNPC (0.005%, w/v)
was low (Fig. 5C). Thus, above results imply that the purified
cellulase has separate sites of endo- and exoglucanase activity.
In order to rule out the possibility that enzymatic activity of
either the endo- or the exoglucanase in the purified cellulase
from Bacillus sp. D04 is due to a minor contaminating protein,
we overexpressed the cel gene from a pET family vector in E.
coli and compared its characteristics to those of the purified
cellulase from Bacillus sp. D04. We deduced amino acid se-
quence from the cel gene. The 29 amino acids (from Met (1) to
Ala (28), Fig. 6) in the N terminus was a typical
b
-glucanase
signal peptide of Bacillus species (28). As an estimated molec-
ular weight based upon amino acid composition of the cel gene
was about 55,000. E. coli BL21(DE) pLysS with this gene
produced 55,000-Da protein with Cm-cellulose degrading activ-
ity. But a 35,000-Da protein with this activity was also de-
tected, which is the molecular mass of the cellulase purified
from Bacillus sp. D04. These results indicate that the cellulase
was produced as a precursor form from the cel gene and then
processed (such as elimination of signal peptide, etc.) to its
mature form. The purified 35,000-Da protein with cellulase
activity was used as a recombinant cellulase. The substrate
specificity and competition pattern of recombinant cellulase
were the same as those of a purified cellulase from Bacillus sp.
D04. These results clearly eliminate the possibility that the
purified cellulase from Bacillus sp. D04 might have a minor
contaminating protein involved in catalyzing either the endo-
or the exoglucanase activity. Therefore a single polypeptide
cellulase of Bacillus sp. D04 has both two kinds of activity. To
localize each endo- and exoglucanase activity site in the cellu-
lase, we are attempting to develop mutant which has only one
type of glucanase activity.
Acknowledgment—We thank Dr. Jong-Il Kim for his valuable
comments.
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FIG.7. The overexpression of cellulase gene from E. coli
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Bifunctional Cellulase from Bacillus sp. D04 26019
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Kang
Sang Jun Han, Yong Je Yoo and Hyen Sam
AND ENDOGLUCANASE ACTIVITY
GENE OF BACILLUS SP. D04 HAS EXO-
Cellulase and Its Structural Gene: THE cel
Characterization of a Bifunctional
Enzymology:
doi: 10.1074/jbc.270.43.26012
1995, 270:26012-26019.J. Biol. Chem.
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