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Previous Article | Next Article 
Appl Environ Microbiol, March 1998, p. 1086-1090, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Characterization of EngF from Clostridium
cellulovorans and Identification of a Novel Cellulose
Binding Domain
Akihiko
Ichi-ishi,
Salah
Sheweita,
and
Roy H.
Doi*
Section of Molecular and Cellular Biology,
University of California, Davis, California 95616
Received 28 July 1997/Accepted 19 December 1997
 |
ABSTRACT |
The physical and enzymatic properties of noncellulosomal
endoglucanase F (EngF) from Clostridium cellulovorans were
studied. Binding studies revealed that the Kd
and the maximum amount of protein bound for acid-swollen cellulose were
1.8 µM and 7.1 µmol/g of cellulose, respectively. The presence of
cellobiose but not glucose or maltose could dissociate EngF from
cellulose. N- and C-terminally truncated enzymes showed that binding
activity was located at some site between amino acid residues 356 and
557 and that enzyme activity was still present when 20 amino acids but not 45 amino acids were removed from the N terminus and when 32 amino
acids were removed from the C terminus; when 57 amino acids were
removed from the C terminus, all activity was lost. EngF showed low
endoglucanase activity and could hydrolyze cellotetraose and
cellopentaose but not cellotriose. Activity studies suggested that EngF
plays a role as an endoglucanase during cellulose degradation. Comparative sequence analyses indicated strongly that the cellulose binding domain (CBD) is different from previously reported CBDs.
| |
INTRODUCTION |
The degradation of cellulose by
Clostridium cellulovorans is carried out by the action of
cellulosomal and noncellulosomal enzymes (2). The isolation
and the sequence of the gene for endoglucanase F (EngF), a
noncellulosomal enzyme, have been reported (19). The derived
sequence for this enzyme lacked the duplicated sequence that typically
occurs in cellulosomal enzymes and did not show the typical cellulose
binding domain (CBD) sequence reported for a number of endoglucanases
(22).
Further analysis of EngF was carried out to determine its biochemical
properties and its putative CBD. This was done by use of deletion
mutants that could define the sequence required for activity and for
binding to the substrate. These studies revealed not only the sequence
required for catalytic activity but also a most likely domain for
binding to the substrate. These results will be useful in future
studies of the possible synergistic action of cellulases produced by
C. cellulovorans.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
Escherichia coli
XL1-Blue (Stratagene, La Jolla, Calif.) was the host strain for all
cloning experiments. E. coli TB1 was used as the host strain
for the production of recombinant proteins, and E. coli
CJ236 was used as the host strain for the production of
uracil-containing single-stranded DNAs, which were used for site-directed mutagenesis.
pMAL-EngB was used for the expression of fusion proteins between
maltose binding protein (MBP) and EngB and was constructed from pMAL-c2
(New England BioLabs Inc., Beverly, Mass.) and the engB gene
from C. cellulovorans (4). pEQ52V was used for
the expression of EngD (12), and pEF4 was used for the
expression of the fusion protein between MBP and EngF (19).
pEF4 deletion mutants were constructed from pEF4 by site-directed
mutagenesis (13). For C-terminal deletion mutants, pEF4 was
mutagenized with an oligonucleotide which contained a stop codon and an
XbaI restriction site (Table
1). To construct N-terminal deletion
mutants, pEF14 was constructed from pEF4 by use of oligonucleotide AI
005 to make a restriction site (XmnI) between MBP and EngF. pEF14 was mutagenized with an oligonucleotide that included an SmaI restriction site (Table 1), and then the N-terminal
parts (XmnI-SmaI fragments) were removed by use
of XmnI and SmaI. pEF35 was constructed from
pEF32 with oligonucleotides AI 105 and AI 013.
General DNA procedures.
DNA was manipulated by standard
procedures (1, 18). Enzymatic treatments of DNA were carried
out as recommended by the manufacturers. DNA fragments were recovered
after electrophoresis with the QIAquick gel extraction kit (Qiagen
Inc., Chatsworth, Calif.).
Protein purification.
MBP fusion proteins were purified from
E. coli with an amylose column (New England BioLabs).
E. coli harboring a plasmid which encoded an MBP fusion
protein was grown in LB medium (18) supplemented with 100 µg of ampicillin per ml, and the culture was induced to produce an
MBP fusion protein by adding
isopropyl-
-D-thiogalactopyranoside (IPTG) to a final
concentration of 0.5 mM. The cells were broken by sonication, and the
crude cell extracts were poured into the amylose column. After the
column was washed with TES buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM
EDTA [pH 7.4]), the fusion protein was eluted with TES buffer
containing 10 mM maltose. To purify EngB and EngF, purified fusion
proteins were cleaved with factor Xa. After dialysis with sodium
phosphate buffer (pH 8.0), the cleavage mixture was passed over a Q
Sepharose FF column (Pharmacia Biotech Inc., Piscataway, N.J.). EngB
and EngF were then eluted with phosphate buffer (pH 8) and an NaCl
gradient from 0 to 500 mM. EngD was purified from the periplasmic
fraction of E. coli TB1 harboring pEQ52V according to the
method of Hamamoto et al. (11).
Cellulose binding assay.
For quantitative analysis, the
cellulose binding assay mixture contained 1 mg of Avicel PH101,
acid-swollen cellulose (ASC), or xylan and an appropriate amount of
enzyme (50 µg/ml to 1 mg/ml [total protein]) in a final volume of 1 ml of 50 mM sodium phosphate buffer (pH 8.0). The ASC was prepared from
Avicel as described previously (24). The mixture was
incubated at 4°C for 1 h with slow vertical rotation. After
1 h of incubation, cellulose was removed by centrifugation and the
free protein concentration ([P], micromolar units) of the
supernatants was measured by the MicroBCA method (Pierce, Rockford,
Ill.). The bound protein concentration ([PC], micromoles per gram of
cellulose) was determined by subtracting [P] from the total protein
concentration. All assays were done in triplicate. Adsorption
parameters were obtained by use of the equation of Sakoda and Hiromi
(17), [PC] = [P][PC]max/Kd + [P], where
Kd (micromolar units) and [PC]max
(micromoles per gram of cellulose) are the equilibrium dissociation
constant and the maximum amount of protein bound, respectively.
For qualitative analysis, assay tubes contained 0.5 mg of ASC and 25 µg of enzyme in 0.5 ml of PSM buffer (50 mM sodium phosphate [pH
8.0], 50 mM NaCl, 10 mM maltose). After incubation at 4°C for 1 h with vertical rotation, assay tubes were spun in a microcentrifuge to
sediment the cellulose. After the PSM buffer was removed, the pellets
were washed three times with 1 ml of PSM buffer. The pellets were then
resuspended in 50 µl of sodium dodecyl sulfate (SDS) sample buffer
and analyzed by SDS-12.5% polyacrylamide gel electrophoresis (PAGE).
Enzyme assay.
Cellulase activity on Avicel PH101, ASC, and
carboxymethyl cellulose (CMC, low viscosity, 50 to 200 cps; Sigma) was
assayed by measuring liberated reducing sugars as D-glucose
equivalents by the bicinchoninic acid method (9). Activity
on Avicel was assayed after incubation (16 h at 37°C) of 1.8 µg of
EngB or EngD or 6.1 µg of EngF with 5 mg of Avicel in 1 ml of 50 mM
acetate buffer (pH 5.0). Carboxymethyl cellulase (CMCase) activity was assayed after incubation (30 min at 37°C) of 0.09 µg of EngB, 0.18 µg of EngD, or 3.1 µg of EngF with 1 ml of 0.5% (wt/vol) CMC in 50 mM acetate buffer (pH 5.0). Activity on ASC was assayed after
incubation (1 h at 37°C) of 0.36 µg of EngB or EngD or 6.1 µg of
EngF with 5 mg of ASC in 1 ml of 50 mM acetate buffer (pH 5.0).
The CMCase activity of the EngF deletion mutants was assayed on 0.5%
CMC agar (1.5%) plates. Enzyme solutions were spotted on CMC plates,
and the plates were incubated overnight at 37°C. Then, the agar
plates were stained with 0.1% (wt/vol) Congo red and destained with 1 M NaCl to visualize the halo formed by enzymatic activity.
Viscometric assay.
Viscometric activity was measured by use
of a Brookfield DV-III rheometer according to the method of Garcia et
al. (9). Eight milliliters of a 0.4% CMC solution was
weighed in a chamber for small sample volumes and preequilibrated at
40°C. After 15 min of preequilibration, 80 µl of CMC was removed
and 80 µl of enzyme was added to the remaining CMC solution. The
sample was mixed for 1 min by moving the chamber up and down. The
spindle was started at 60 rpm, and the data readings were taken at
1-min intervals. High-viscosity CMC (1,500 to 3,000 cps; Sigma) was used for the viscometric assay.
TLC analysis.
Purified enzymes (EngB, EngD, and EngF) were
incubated with oligosaccharide (cellotriose, cellotetraose, and
cellopentaose) solutions for 16 h at 37°C. The reaction products
were separated on thin-layer chromatography (TLC) plates (Merck) with a
solvent system containing 1-butanol-ethanol-water (5:5:2.5). For
detection of the products, the plates were sprayed with staining
reagent (5% H2SO4 in methanol) and baked for
10 min at 100°C.
| |
RESULTS AND DISCUSSION |
Adsorption assay.
We had shown previously that EngF of
C. cellulovorans would bind very weakly to Avicel
(19). In order to quantify its binding ability, we measured
the adsorption of recombinant EngF to Avicel, ASC, and xylan. A time
course experiment showed that 1 h was sufficient for equilibrium
binding of the enzyme to the cellulose preparations (data not shown).
Furthermore, the presence of 10 mg of Avicel did not result in greater
binding of the enzyme (data not shown). Figure
1 shows a typical equilibrium adsorption
isotherm for the adsorption of EngF to ASC. The
Kd and [PC]max for ASC were
estimated to be 1.8 µM and 7.1 µmol/g of cellulose, respectively.
On the other hand, an insignificant amount of EngF bound to Avicel and xylan. These results indicated that the CBD of the noncellulosomal enzyme EngF had a high affinity for amorphous cellulose. On the other
hand, the CBD of C. cellulovorans CbpA, the scaffolding protein of the cellulosome, showed a high affinity for crystalline cellulose (10).
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FIG. 1.
Equilibrium adsorption isotherm for the adsorption of
EngF to ASC. Adsorption assays were done at 4°C for 1 h. After
1 h of incubation, free protein concentration was measured with
the MicroBCA reagent. Bound protein concentration ([PC]) was
determined by subtracting free protein concentration ([P]) from total
protein concentration. Each data point is the mean for three replicate
samples.
|
|
The effects of soluble carbohydrates on the adsorption of EngF to ASC
were examined. The EngF bound to ASC was washed with PS buffer (50 mM
phosphate, 50 mM NaCl [pH 8]) that included 10 mM glucose,
cellobiose, or maltose. Glucose and maltose did not cause a
dissociation of EngF from ASC, but EngF was dissociated from ASC when
it was washed with buffer containing 10 mM cellobiose (Fig.
2A).
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FIG. 2.
Effect of soluble sugars and deletions on cellulose
binding by EngF. (A) Effects of soluble sugars on EngF binding ability.
After incubation of EngF and ASC, ASC was washed with phosphate buffer
(pH 8.0) that contained no sugar (lane 1), glucose (lane 2), cellobiose
(lane 3), or maltose (lane 4). The ASC pellets were suspended in SDS
sample buffer, and bound proteins were analyzed by SDS-12.5% PAGE.
Lane M, molecular weight standards (in thousands). (B) Cellulose
binding by EngF deletion mutants. EngF (lanes 1 and 2), deletion mutant
pEF7 (lanes 3 and 4), and deletion mutant pEF32 (lanes 5 and 6) (see
Fig. 3 for structures of mutants) were incubated with ASC. After
incubation, ASC was washed with buffer containing no maltose (lanes 1, 3, and 5) or 10 mM maltose (lanes 2, 4, and 6). The ASC pellets were
suspended in SDS sample buffer, and bound proteins were analyzed by
SDS-12.5% PAGE.
|
|
Determination of the CBD and catalytic domain of EngF.
Since
EngF did bind to ASC, we decided to determine the location of the CBD
of EngF by constructing a series of EngF deletion mutants. EngF
deletion mutants were made from pEF4, which carried genes encoding the
MBP-EngF fusion proteins. All deletion mutants were fused to MBP,
located at the N-terminal end (Fig. 3).
MBP itself showed very weak binding to ASC. All MBP-EngF deletion mutants also showed weak binding to ASC. We eliminated the weak binding
effect of MBP by using a buffer that included maltose and sodium
chloride (50 mM sodium phosphate buffer [pH 8.0], 10 mM maltose, 50 mM NaCl). This buffer did not affect the binding of EngF to ASC (Fig.
2). The binding of complete EngF and EngF from deletion mutants (Fig.
2B) indicated that complete EngF and the truncated protein produced by
pEF32 (Fig. 3) were able to bind to cellulose in the presence of
maltose, whereas the protein produced by pEF7 (Fig. 3) was unable to
bind to cellulose in the presence of maltose. These results supported
our conclusion that pEF7 lacked the CBD.
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FIG. 3.
Cellulose binding by and enzyme activity of truncated
EngF. MBP (shaded boxes) was fused to EngF deletion mutants (open
boxes). Binding ability was measured by a cellulose binding assay and
SDS-PAGE, and enzyme activity was detected with CMC agar plates (see
Materials and Methods). +, truncated protein had binding ability or
CMCase activity;  , protein had no binding ability or CMCase activity.
The numbers over the open boxes indicate the amino acid positions in
EngF.
|
|
A summary of adsorption assays with a series of EngF deletion mutants
is shown in Fig. 3. N-terminal deletion mutants were still able to bind
to ASC when 355 amino acids had been removed. When 400 amino acids were
deleted from the N-terminal end of EngF, the protein produced by this
deletion mutant (pEF33) lost its ability to bind to ASC. On the other
hand, when we deleted only 32 amino acids from the C terminus (pEF15
and pEF35), the deletion protein could not bind to ASC. The ability of
the protein produced by pEF32 to bind to cellulose strongly indicated
that the CBD of EngF existed at the C-terminal end of the molecule,
between residues 356 and 557.
We also determined the catalytic domain of EngF by using a series of N-
and C-terminal deletion mutants and CMC plate assays (Fig. 3). When 20 amino acids were deleted from the N terminus of EngF (pEF21), there was
a slight drop in enzyme activity. Deletion of 45 amino acids from the N
terminus (pEF22) resulted in a total loss of enzyme activity. When 32 or 57 amino acids were deleted from the C terminus (pEF15 or pEF8),
there was a slight drop in enzyme activity. Deletion of 109 amino acids
from the C terminus (pEF13) resulted in a total loss of enzyme
activity, indicating that some portion of the enzyme between 57 and 109 amino acids from the C terminus was required for enzyme activity.
Therefore, the catalytic site was found between amino acids 50 and 500.
Proteins produced by pEF8 and pEF15 had enzyme activity but did not
have binding ability. The protein produced by pEF22 retained binding
ability but did not have CMCase activity. These results indicated that
the CBD and the catalytic domain did not exist in the same part of
EngF. When we deleted up to 448 or 400 amino acids from the C-terminal
end of EngF, CMCase activity was lost. Some other enzymes partially
homologous in sequence to EngF, including CelF from Bacillus
sp. strain 1139 and endoglucanase from Bacillus sp. strain
KSM-635, could have deletions of up to 404 or 584 amino acids from the
C-terminal end, respectively (6, 15, 16), without a loss of
activity. These amino acids correspond to amino acid 389 of EngF (Fig.
4).
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FIG. 4.
Comparison of the amino acid sequence of the CBD region
of EngF from C. cellulovorans with putative CBD regions of
endoglucanases from other organisms. Identical amino acids are
indicated with an asterisk, and similar amino acids are indicated with
a plus sign (both above and below sequences); gaps are indicated with a
dash. The numbers on the left indicate the amino acid positions in the
proteins. Vertical arrows and letters indicate termini of the following
proteins: A, C-terminal ends of truncated CelF (Bacillus sp.
strain 1139; L-404) and endoglucanase (Bacillus sp. strain
KSM-635; V-584); B, N-terminal end (G-401) of pEF33; and C, C-terminal
end (I-525) of pEF35. The search for homologous sequences was done with
a BLAST search of GenBank. Retrieved sequences were aligned with the
multiple-alignment program Clustal W (21). Alignment was
then edited manually for maximal fit. CC, EngF from C. cellulovorans (GenBank accession no. U37056) (19); CJ,
CelA from C. josui (D85526); 22-28, endoglucanase from
Bacillus sp. strain 22-28 (D85236); N-4, CelC from
Bacillus sp. strain N-4 (M25500) (8); 1139, CelF
from Bacillus sp. strain 1139 (D00066 and N00066)
(7); KSM-64, endoglucanase from Bacillus sp.
strain KSM-64 (M84963) (20); KSM-635, endoglucanase from
Bacillus sp. strain KSM-635 (M27420) (16).
|
|
Comparison of EngF activity to the activities of other C. cellulovorans glucanases.
EngF had very weak activity on
Avicel, and the activity on ASC of EngF was 100 times lower than that
of EngB or EngD. The specific activities of EngF on Avicel, ASC, and
CMC were 0.08, 1.7, and 9.6 µmol of glucose/min/µmol of protein,
respectively. The CMCase activity of EngF was 150 times lower than that
of EngB (3) or 30 times lower than that of EngD
(11), but viscometric analyses of CMC hydrolysis indicated
that EngF was a true endoglucanase (Fig.
5). EngB and EngD had similar activities
on ASC. The activity on Avicel of EngD was higher than that of EngB,
but the CMCase activity of EngD was lower than that of EngB. These
results suggested that EngF played a secondary role in cellulose
degradation.
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FIG. 5.
Viscometric analysis of CMC hydrolysis by C. cellulovorans cellulases. Viscosity is plotted versus the release
of reducing sugar upon hydrolysis of CMC by EngB (square), EngD
(triangle), and EngF (circle). Reaction mixtures contained 10 ng of
EngB per ml, 36 ng of EngD per ml, or 1 µg of EngF per ml and 0.4%
(wt/vol) CMC in 50 mM acetate buffer (pH 5.0).
|
|
Analysis of cellodextrin hydrolysis by TLC.
EngF could not
hydrolyze cellotriose but did hydrolyze cellotetraose and cellopentaose
(Table 2). EngF hydrolyzed cellopentaose faster than cellotetraose, as indicated by product yields at given time
points (data not shown). EngB and EngD were much more active on
cellodextrins than EngF, according to product yields and the total
hydrolysis of cellotetraose and cellopentaose. EngD produced glucose
from all three substrates, but EngB and EngF did not produce glucose
from any of the substrates. This result may have been due to
transglycosylation, which has been reported for a homologous enzyme
from a Bacillus species (7). Thus, EngF differed
from EngB and EngD in specific activities, in substrates degraded, and
in the products that were formed.
Homology of EngF with other glucanases.
The amino acid
sequence of EngF showed high homology with those of CelA from
Clostridium josui (5), endoglucanase from Bacillus sp. strain 22-28 (14), CelC from
Bacillus sp. strain N-4 (8), CelF from
Bacillus sp. strain 1139 (7), endoglucanase from
Bacillus sp. strain KSM-64 (20), and
endoglucanase from Bacillus sp. strain KSM-635
(16). Alignments of the C-terminal regions of these enzymes
are shown in Fig. 4. These enzymes are members of family 5, and EngF
may be a retaining enzyme, since six enzymes of family 5 screened for
their stereochemistry were retaining enzymes (23).
CBDs, which have been found not only in cellulases but also in
xylanases, have been grouped into 10 families, families I to X, on the
basis of amino acid sequence homology (22). Previously reported enzymes, such as CelC from Bacillus sp. strain N-4
(8), CelF from Bacillus sp. strain 1139 (7), endoglucanase from Bacillus sp. strain
KSM-64 (20), and endoglucanase from Bacillus sp.
strain KSM-635 (16), had not been included in those
families, nor was the existence of CBDs analyzed or reported for these
enzymes. We suggest that these enzymes, which have high homology to
EngF in their C-terminal regions, contain CBDs and that some or all of
the highly conserved C-terminal sequences comprise a new type of CBD
family.
| |
ACKNOWLEDGMENTS |
We thank David Johnston and Sharon P. Shoemaker for assistance in
the viscometric assays, Jae-Seon Park for the pMAL-EngB construct, and
Laercio M. Malburg, Jr., for useful discussions and suggestions. We
thank Seikagaku America, Inc., for providing us with cellopentaose.
This work was supported in part by grant 94-37308-0399(I) from the U.S.
Department of Agriculture and by grant DE-FG03-92ER20069 from the U.S.
Department of Energy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Molecular and Cellular Biology, University of California, Davis, CA
95616-8535. Phone: (916) 752-3191. Fax: (916) 752-3085. E-mail:rhdoi{at}ucdavis.edu.
Present address: Faculty of Life Science, Toyo University, Gunma
374-01, Japan.
Present address: Department of Bioscience and Technology,
Alexandria University, Alexandria, Egypt.
| |
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Abstract
Introduction
