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(Received for publication, February 23, 1995; and in revised form, August 7, 1995) From the
Bacillus sp. D04 secreted a bifunctional cellulase that
had a molecular weight of 35,000. This cellulase degraded Cm-cellulose,
cellotetraose, cellopentaose, p-nitrophenyl-
Cellulose is an unbranched glucose polymer composed of an
anhydro- 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 ( Some organisms (for example, Trichoderma sp.)(6, 7, 8, 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, 13, 14) .
For example, Saul reported 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 Neocallimatrix patriciarum has a multifunctional
catalytic domain with high endoglucanase, cellobiohydrolase, and
xylanase activities(12, 13) . Extensive recent
studies on proteins (such as cellulase, protease, and amylase) secreted
by Bacillus species (15) have shown that the following Bacillus species produce cellulases: Bacillus
cereus(16) , Bacillus licheniformis(17) , Bacillus subtilis(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 cellulase of Bacillus sp.
D04 had separate sites for endo- and exoglucanase activity.
Figure 1:
Purification and activity staining of
cellulase from Bacillus sp. D04. Panel A shows
SDS-PAGE from various purification steps of cellulase. Lane a was a sample obtained by ultrafiltration, lane b was a
sample passed through Cm-Sepharose, and lane c was a purified
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.
Figure 2:
Hydroxylapatite chromatography.
Distribution of protein concentration (
Figure 3:
HPLC analysis of degradation products. Panel A, HPLC analysis of products released from
cellotetraose. The reaction mixture containing 10 µl of 10 mM potassium phosphate (pH 5.8), 40 µl of the purified cellulase
(0.1 mg/ml), and 30 µl of 10 mg/ml cellotetraose were incubated for
0 (a), 60 (b), and 120 (c) min at 45 °C. Panel B, HPLC analysis of products released from
cellopentaose. 40 µl of 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
µl of 1% (w/v) avicel solution and 800 µl 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 contain cellulase, whereas (b) contained purified cellulase. These reaction products were
modified with dansyl hydrazine as described under ``Experimental
Procedures.'' 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.
Figure 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 (
Figure 5:
The substrate competition of pNPC and
Cm-cellulose degrading activity. The various concentrations of pNPC
(0.005-0.05%, w/v) and Cm-cellulose (0.1-0.5%, w/v) were
mixed and incubated with the purified cellulase for 1 h at 45 °C.
The pNPC degrading activity in the presence of various concentrations
of Cm-cellulose (
Figure 6:
Nucleotide sequence of the cel gene and homology between cellulase genes from Bacillus
subtilis. The potential promoter region (-35 (TAGACAA),
-10 (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 sequence is a typical
Figure 7:
The overexpression of cellulase gene from E. coli BL21(DE) pLysS with pCO
Figure 8:
Purification and activity staining of the
overexpressed cellulase. Panel A, SDS-PAGE of purified
overexpressed cellulase. Lane b was a resuspended ammonium
sulfate pellet. Lane c was a purified cellulase by
Cm-Sepharose. In Panel B, lanes d and e show
the activity staining of lanes b and c in Panel
A, respectively. Molecular weight markers were in lane a: 1,
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 hydrolyze Cm-cellulose, swollen cellulose, cellotetraose,
and cellopentaose, but not pNPC(1) . The exoglucanase of Ruminococcus 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 2). Moreover, this
cellulase cleaved only the aglycosidic bond in pNPC as does an
exoglucanase of Trichoderma viride and Sporotrichum
pulveralentum(5) , and randomly cleaved internal
To determine
whether the active site of endo- and exoglucanase are separately
existed, we studied differential effects of compounds that specifically
inhibited one type of cellulase activity. The cellobiose competitively
inhibited pNPC degrading activity, but did not inhibit Cm-cellulose
degrading activity (Table 3). However, since the K 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 sequence from the cel gene. The 29 amino
acids (from Met (1) to Ala(28) , Fig. 6) in the
N terminus was a typical
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s) U27084 [GenBank]for cel gene cellulase.
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 26012-26019
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
THE cel GENE OF BACILLUS SP. D04 HAS EXO- AND
ENDOGLUCANASE ACTIVITY (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-D-cellobioside, and avicel PH101.
Based on the high performance liquid chromatography analysis of the
degradation products, this cellulase randomly cleaved internal
-1,4-glycosidic bonds in cellotetraose and cellopentaose as an
endoglucanase. It also hydrolyzed the aglycosidic bond in p-nitrophenyl--D-cellobioside and cleaved avicel
to cellobiose as an exoglucanase. Cellobiose competitively inhibited
the p-nitrophenyl--D-cellobioside degrading
activity but not Cm-cellulose degrading activity. Ten mMp-chloromercuribenzoate inhibited p-nitrophenyl--D-cellobioside degrading activity
completely, but Cm-cellulose degrading activity incompletely.
Cm-cellulose increased p-nitrophenyl--D-cellobioside degrading
activity, and vice versa, whereas
methylumbelliferyl--D-cellobiose strongly inhibited p-nitrophenyl--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.
-1,4-glucose units linked by a
-1,4-D-glycosidic bond. Cellulolytic enzymes degrade
cellulose by cleaving this glycosidic bond. Cellulases can be
classified into three types: endoglucanases (1,4--Dglucan
4-glucohydrolase, EC 3.2.1.4), exoglucanases
(-1,4-D-glucan cellobiohydrolase), and -glucosidases
(-D-glucoside glucohydrolase, EC 3.2.1.21).
Endoglucanases randomly hydrolyze internal -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 cellobiose unit from
the nonreducing end of cellulose; the reducing sugars are rapidly
increased, but the polymer length changes
little(1, 2, 3) . -Glucosidases cleave
cellobiose and oligosaccharides to glucose(1) . Therefore,
crystalline cellulose is efficiently hydrolyzed by the synergistic
action of all three types of cellulases.
)(4) and pNPC (5) are
used as substrates for the determination of exoglucanase activity, and
MUG (4) and pNPG (5) are cleaved by -glucosidases.
Filter paper and avicel are efficiently hydrolyzed by the synergistic
action of endo- and exoglucanases, but not by either one
alone(6) .
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 
g for 10 min, the supernatant was concentrated by
ultrafiltration (10,000 nominal molecular weight cut-off membrane was
used). Ten mM potassium phosphate buffer (pH 5.8) was added to
the concentrated sample, and the sample was reconcentrated to 100 ml.
This sample was passed through Cm-Sepharose CL-6B (7 50 mm)
equilibrated with 10 mM potassium phosphate buffer (pH 5.8) at
a flow rate of 15 ml/h. The sample flow-through was loaded directly
onto hydroxylapatite (7 30 mm) equilibrated with 10 mM potassium phosphate buffer (pH 5.8) and eluted with a 30-ml
10-250 mM potassium 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 µl of 1%
Cm-cellulose in 10 mM potassium phosphate buffer (pH 5.8) and
200 µl of diluted enzyme solution, incubated at 45 °C for 20
min. Avicel degrading activity was measured as follows: 500 µl of
10 mg/ml avicel in 10 mM potassium phosphate buffer (pH 5.8)
was mixed with 500 µl 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 µmol of glucose/min.pNPC Degrading Activity Assay
The reaction
mixture, consisting of 800 µl of pNPC at 1 mg/ml in 50 mM sodium acetate buffer (pH 5.8) and 200 µl 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 µmol of p-nitrophenol/min.MUC Degrading Activity Assay
The reaction mixture,
consisting of 800 µl of 1 mg/ml MUC in 50 mM sodium
acetate buffer (pH 5.8) and 200 µl of suitably diluted enzyme, was
incubated at 45 °C for 20 min. The reaction was stopped by adding
3.5 ml of 0.5 M glycine/NaOH buffer (pH 10.4). Fluorescence
measurements were made on a Tasco FP-777 spectrofluorometer 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 µmol of
4-methylumbelliferone/min(9) .High Performance Liquid Chromatography Analysis of
Degradation Products
The reaction mixture, consisting of 80 µl of
oligosaccharides released from cellulosic substrates, 20 µl of 10%
trichloroacetic acid, and 100 µl of 0.3% (w/v) ethanolic solution
of dansyl hydrazine, was heated at 80 °C for 10 min, and then
cooled(20, 21, 22) . Samples were dried,
dissolved in 78% acetonitrile solution, and analyzed with
µ-Bondapak NH
column (3.9 300
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.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 density 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(+) and then transferred into an Escherichia
coli DH5
strain. Transformants with Cm-cellulose degrading
activity were screened on an L-agar plates containing ampicillin at 100
µg/ml, 0.5% Cm-cellulose, and trypan blue at 0.1 mg/ml. To screen
MUC degrading activity, transformants were transferred onto L-agar
plates containing ampicillin at 100 µg/ml and MUC at 50 µg/ml.
For the determination of the nucleotide sequence of the cel gene, serial deletion of the gene was done by using
Erase-a-Base kit (Promega). The sequence of cellulase
gene was determined from both strands by the dideoxynucleotide chain
termination method using a Sequenase
kit (U.S.
Biochemical Corp.).
PCR Amplification of the cel Gene and Construction of
pCO1
The cel gene in pBluescript KS(+) was amplified
by PCR with 5`-CATATGAAACGGTCAATCTCT-3` (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 µl; reaction composition, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 50 µM dNTP, 2 fmol of template, 10
pmol primer, and 2 units of Vent DNA 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(+) that had been digested with SmaI and HindIII. This recombinant DNA was named pCO1.
Construction of pCO
The pCO1 vector was digested with EcoRV and then
partially digested 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 pCOand Transfer into E.
coli BL21(DE) pLysS. The pCO vector was transferred into E. coli BL21(DE)
pLysS(FompT hadS
(rm
)
dcm gal(DE) pLysS, Cm
) 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 was
cultured in Luria-Bertani medium containing ampicillin at 50 µg/ml
and chloramphenicol at 30 µg/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
) containing
ampicillin at 50 µg/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 for 3 h at 30
°C.Purification of Overexpressed Cellulase
The overexpressed recombinant cellulase from E. coli BL21(DE)pLysS was partially purified by ammonium sulfate
fractionation as previously described(24) . This partially
purified cellulase was dialyzed in 20 mM sodium acetate buffer
(pH 4.8) and then loaded onto Cm-Sepharose CL-6B (15 50 mm)
equilibrated with dialysis buffer. The proteins were eluted with a
80-ml 20-500 mM sodium 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 mM phosphate 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 M NaCl/NaOH solution.
Purification of Cellulase
Because Bacillus sp. D04 secreted cellulase into medium, concentrated medium was
used as starting 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 mM potassium phosphate from hydroxylapatite had both Cm-cellulose and
pNPC degrading 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 permeation chromatography (Superose 12, Pharmacia Biotech Inc.),
was also about 35,000. Activity staining showed that this purified
protein had Cm-cellulose degrading activity (Fig. 1B).
The steps in purification of this protein are given in Table 1.
), p-nitrophenyl--D-cellobiose (10 unit/ml; 
), and Cm-cellulose (unit/ml; )
degrading activity after hydroxylapatite chromatography (7
30
mm) eluted with potassium phosphate salt gradient (10-250
mM) at a flow rate of 10 ml/h.
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 2). However, the
specific activity toward avicel was much lower than that of the soluble
substrates. Neither MUG nor pNPG was hydrolyzed (Table 2).
HPLC Analysis of Oligosaccharides from Cellulosic
Substrates
HPLC analysis showed that a single peak was detected
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 aglycosidic
bond in pNPC, producing cellobiose and p-nitrophenol. The
enzyme cleaved cellulosic substrates (such as cellotetraose and
cellopentaose) to glucose, cellobiose, and other oligosaccharides.
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
-1,4-glycosidic bonds in these cellulosic substrates as an
endoglucanase. Based on the above result, it would seem that the
smallest substrate recognized by the endoglucanase of Bacillus sp. D04 is a cellotetraose. Both endo- and exoglucanase activities
were detected by using cellotetraose 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 activity 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 Inhibitors
The Cm-cellulose and pNPC degrading
activities of this cellulase were differentially inhibited by several
inhibitors. pCMB at 10 mM completely inhibited the pNPC
degrading activity but inhibited Cm-cellulose degrading activity by
67%. In 40 mM cellobiose, Cm-cellulose degrading activity was
not inhibited, but the pNPC degrading activity was inhibited by 57.8% (Table 3). Cellobiose changed the K
but not
the V
of pNPC degrading activity as a
competitive inhibitor (Fig. 4A), whereas both the V and K of pNPC degrading
activity were changed by pCMB as a mixed-type inhibitor (Fig. 4B). Both Cm-cellulose and pNPC degrading
activity required Ca, but were strongly inhibited by
Zn
. Mg
slightly increased pNPC
degrading activity and weakly inhibited Cm-cellulose degrading activity (Table 3).
), 5 (), 10 (), and 40 (
)
mM cellobiose (Panel A) and 0 (
), 1 (), 2
(), and 4 (
) mM pCMB (Panel B). The V
and Kof pNPC
degrading activity was 214 µmol/min and 5.29 mM,
respectively. Cellobiose did not change V
but K was changed to 8.62 and 12.62 mM in the presence of 10 and 40 mM cellobiose, respectively.
The V
and K were
changed to 96 µmol/min and to 6.75 mM by 4 mM pCMB.
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 degrading activity was not
inhibited, but was increased by Cm-cellulose (Fig. 5A).
Cm-cellulose degrading activity was not inhibited, but was slightly
increased in the presence of various concentrations of pNPC (Fig. 5B). But 60% of the pNPC degrading activity was
inhibited by MUC even at a low ratio of MUC (0.01%, w/v) to pNPC
(0.005%, w/v) (Fig. 5C).
, 0%; , 0.1%; , 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%; , 0.005%; , 0.01%;
, 0.05%, w/v). The
pNPC degrading activity in the presence of various concentrations of
MUC (
, 0%; , 0.01%; , 0.05%;
, 0.25%, w/v) is
shown in Panel C. Panels D, E, and F show
the substrate competition of purified recombinant cellulase. The
substrate concentrations and reaction times were the same as for the
purified cellulase. Panel D and E show pNPC and
Cm-cellulose degrading activity in the presence of Cm-cellulose and
pNPC, respectively. Panel F shows pNPC degrading activity in
MUC.
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(+), 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 degrading activity were obtained
(data not shown). To determine the MUC degrading activity of these
colonies, they were transferred onto an L-agar plate containing MUC.
The colony with exoglucanase activity cleaved MUC to cellobiose and
methylumbelliferone 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 sequence of this gene (Fig. 6) showed
one open reading frame of 1461 base pairs was a possible gene encoding
the cellulase. A potential promoter (-35 (TAGACAAT) and -10
(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).
-glucanase
signal peptide of Bacillus species. Lines 3 and 4 indicate nucleotide sequences of the cellulase genes of Bacillus subtilis BSE616 and DLG,
respectively.
Overexpression of Recombinant Cellulase Gene and
Purification of Recombinant Cellulase
E. coli BL21(DE)pLysS with pCO 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).
. Lane b showed proteins which were extracted before IPTG was added. Lanes c, d, and e showed proteins which were
extracted at 1-h intervals after IPTG was added. Molecular weight
markers were in lane a: 1, -galactosidase
(118,000); 2, bovine serum albumin (78,000); 3,
ovalbumin (47,100); 4, carbonic anhydroase (31,400); 5, soybean trypsin inhibitor (25,000); and 6,
lysozyme (18,800). The arrowhead points to overexpressed
products.
-galactosidase (118,000); 2, bovine serum
albumin (78,000); 3 ovalbumin (47,100); 4, carbonic
anhydroase (31,400); 5, soybean trypsin inhibitor (25,000);
and 6, lysozyme (18,800). The arrowheads point to
zones in which Cm-cellulose was degraded by
cellulase.
Characteristics of Recombinant Cellulase
The
purified recombinant cellulase degraded Cm-cellulose, pNPC, MUC, and
avicel (Table 2), but proteins extracted from E. coli DH5 strain did not degrade these cellulosic substrates.
Cm-cellulose slightly increased pNPC degrading activity, and vice
versa (Fig. 5, D and E). The pNPC
degrading activity was strongly inhibited by MUC (Fig. 5F).
-1,4-glycosidic bonds in cellotetraose and cellopentaose (Fig. 3, A and B) as an endoglucanase. 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.
of cellobiose was 35.4 mM (Fig. 4A),
cellobiose was not a strong inhibitor in pNPC degrading activity. pCMB,
a thiol protease inhibitor, inhibited pNPC degrading activity
completely and Cm-cellulose degrading activity incompletely (Table 3). Therefore endo- and exoglucanase activities were
differently inhibited by cellobiose and pCMB. Xue et al.(13) showed that the polysaccharide hydrolase from N.
patriciarum has a multifunctional catalytic domain that contains
endoglucanase, cellobiohydrolase, and xylanase activities. On the basis
of the substrate competition assays of this enzyme, Cm-cellulose and
xylan strongly inhibited 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 Bacillus sp. D04 was different from those of N. patriciarum. At a 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 activity 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.-glucanase signal peptide of Bacillus species(28) . As an estimated molecular 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 activity. But a 35,000-Da protein
with this activity was also detected, 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 cellulase, we are
attempting to develop mutant which has only one type of glucanase
activity.
)-D-cellobiose; MUG,
methylumbelliferyl--D-glycopyranoside; pNPC, p-nitrophenyl--D-cellobioside; pNPG, p-nitrophenyl--D-glycopyranoside; pCMB, p-chloromercuribenzoate; PCR, polymerase chain reaction; IPTG,
isopropylthio--D-galactoside; PAGE, polyacrylamide gel
electrophoresis; HPLC, high performance liquid chromatography.
We thank Dr. Jong-Il Kim for his valuable comments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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