(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--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.
Cellulose is an unbranched glucose polymer composed of an
anhydro--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.
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 ()(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) .
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 (), 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.
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 (), 5 (
), 10 (
), and 40 (
)
mM cellobiose (Panel A) and 0 (
), 1 (
), 2
(
), and 4 (
) mM pCMB (Panel B). The V
and K
of 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.
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 (, 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.
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 -glucanase
signal peptide of Bacillus species. Lines 3 and 4 indicate nucleotide sequences of the cellulase genes of Bacillus subtilis BSE616 and DLG,
respectively.
Figure 7:
The overexpression of cellulase gene from E. coli BL21(DE) pLysS with pCO. 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.
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, -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.
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
-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.
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 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.
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 -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.
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.