Appl Environ Microbiol, July 1998, p. 2374-2379, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Analysis of Molecular Size Distributions of
Cellulose Molecules during Hydrolysis of Cellulose by
Recombinant Cellulomonas fimi
-1,4-Glucanases
Henrik
Stålbrand,1,*
Shawn D.
Mansfield,2
John N.
Saddler,2,3
Douglas G.
Kilburn,1,3
R. Antony
J.
Warren,1,3 and
Neil R.
Gilkes1,3
Department of Microbiology and
Immunology,1
Forest Product
Biotechnology, Department of Wood Science,2 and
Protein Engineering Network of Centers of
Excellence,3 University of British Columbia,
Vancouver, British Columbia Canada
Received 22 December 1997/Accepted 27 April 1998
 |
ABSTRACT |
Four 
-1,4-glucanases (cellulases) of the cellulolytic bacterium
Cellulomonas fimi were purified from Escherichia
coli cells transformed with recombinant plasmids. Previous
analyses using soluble substrates had suggested that CenA and CenC were
endoglucanases while CbhA and CbhB resembled the exo-acting
cellobiohydrolases produced by cellulolytic fungi. Analysis of
molecular size distributions during cellulose hydrolysis by the
individual enzymes confirmed these preliminary findings and provided
further evidence that endoglucanase CenC has a more processive
hydrolytic activity than CenA. The significant differences between the
size distributions obtained during hydrolysis of bacterial
microcrystalline cellulose and acid-swollen cellulose can be explained
in terms of the accessibility of -1,4-glucan chains to enzyme
attack. Endoglucanases and cellobiohydrolases were much more easily
distinguished when the acid-swollen substrate was used.
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INTRODUCTION |
Cellulose hydrolysis by aerobic
fungi, such as Trichoderma reesei, is usually explained in
terms of the synergistic activities of endo--1,4-glucanases and
exocellobiohydrolases. Models that describe the attack of cellulose at
susceptible regions by endoglucanases, followed by cellobiohydrolase
attack at the newly formed chain ends, continue to form the basis of
most discussions of enzymatic cellulose hydrolysis (2, 24).
Although the occurrence of endoglucanases and cellobiohydrolases in
fungi is firmly established, the extent to which the cellulase systems
of aerobic bacteria resemble those from fungi was unclear until
recently, because there was little evidence for the presence of
cellobiohydrolases in bacteria. However, it now appears that at least
some cellulolytic bacteria produce enzymes similar to the fungal
cellobiohydrolases. For example, Cellulomonas fimi produces
at least six -1,4-glucanases, of which four (CenA, CenB, CenC, and
CenD) are endoglucanases and two (CbhA and CbhB) appear to be
cellobiohydrolases that are the functional equivalents of T. reesei CBHI and CBHII (6, 15, 21, 22). Similar
cellobiohydrolases have been described for the actinomycete
Thermomonospora fusca (9).
C. fimi cellobiohydrolases.
The preferential
attack of cellulose at the ends of glucan chains by C. fimi
cellobiohydrolases CbhA and CbhB is strongly suggested by hydrolysis
experiments using cellooligosaccharides or carboxymethylcellulose (CMC)
(14, 15, 21, 22). However, we lack direct evidence for
exohydrolytic activity on cellulose itself. Accordingly, we have
examined the activities of CbhA and CbhB on cellulose by measurement of
molecular size distributions during hydrolysis. Analysis of CenA was
also included to allow comparison of cellobiohydrolase and
endoglucanase activities.
C. fimi CenC.
Previous studies have indicated that
CenA attacks susceptible linkages in soluble CMC in a relatively
nonprocessive manner (7, 14); i.e., the enzyme dissociates
from the substrate after each hydrolytic event. While CenB and CenD
attack CMC in a similar way (14, 23), C. fimi
CenC seems to act in a more processive fashion (16, 23).
Therefore, CenC activity was analyzed in order to determine if the
enzyme behaves in a similarly processive manner on cellulose.
Cellulose substrates.
Previous determinations of molecular
size distribution during hydrolysis have shown that the choice of
substrate is an important consideration (10). In this study
we used two forms of cellulose: bacterial microcrystalline cellulose
(BMCC) and phosphoric acid-swollen cellulose (PASC). These celluloses
were chosen in order to simplify the interpretation of data by avoiding
complications due to low surface/volume ratios and substrate
heterogeneity, which are associated with the use of substrates like
cotton or pulp fibers (24). Both BMCC and PASC have a high
surface/volume ratio (17). BMCC is a highly crystalline form
of cellulose I prepared from cellulose produced by Acetobacter
xylinum. PASC is produced by swelling microcrystalline cellulose
in concentrated phosphoric acid; although often described as amorphous,
it is probably a low-crystallinity form of cellulose II (1).
Recent data suggest that cellulose I and cellulose II contain glucan
chains arranged in parallel orientation (12).
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MATERIALS AND METHODS |
Enzymes and cellulose preparations.
The genes encoding CenA
(8), CenC (23), CbhA (15), and CbhB
(21) were expressed in Escherichia coli, and the
corresponding enzymes were purified by cellulose affinity
chromatography and anion-exchange chromatography, as previously
described. BMCC was prepared from A. xylinum (ATCC 23769)
(5). PASC was prepared from Avicel PH101 by using 88%
(wt/vol) phosphoric acid (27).
Enzymatic hydrolysis conditions.
Reaction mixtures contained
100 µg of bovine serum albumin per ml and 1 mg of BMCC per ml or 10 mg of PASC per ml in 50 mM citrate buffer (pH 7.0)-0.02%
NaN3. The enzyme concentrations used for BMCC hydrolysis
were 4 nmol of CenA, 4 nmol of CenC, 2 nmol of CbhA, and 2 nmol of CbhB
per ml of reaction mix; for PASC hydrolysis the concentrations used
were 10 nmol of CenA, 10 nmol of CenC, 40 nmol of CbhA, and 40 nmol of
CbhB per ml of reaction mix. Control reactions contained no enzyme.
Reaction mixtures were incubated at 37°C with gentle shaking for
96 h, and samples were withdrawn for analysis at appropriate
intervals. Samples were centrifuged at 5,000 × g for
15 min, and supernatants were removed for analysis of soluble products.
The cellulose pellet was washed three times with ice-cold distilled
water and dried at 60°C prior to analysis of molecular size
distribution.
Analysis of soluble products.
Glucose and soluble
cellooligosaccharide in the supernatants were quantified by
anion-exchange chromatography on a CarboPac PA-1 column using a DX-500
high-pressure liquid chromatography system equipped with a pulsed
amperometric detector (Dionex, Sunnyvale, Calif.). Samples were run in
triplicate. Glucose and cellooligosaccharide standards with a degree of
polymerization (DP) of 2 to 5 were from Sigma Chemical Co., St. Louis,
Mo., and Seikagaku America, Inc., Rockville, Md. The percent
solubilization of cellulose was calculated from the total sugars
released.
Analysis of size distribution of insoluble products.
The
size distributions of cellulose molecules were determined by gel
permeation chromatography of their tricarbanyl derivatives. Cellulose
pellets were carbanilated as previously described (20). Derivatized samples were recovered by evaporation of the reactants (26), redissolved in iso-octane and evaporated to dryness.
Samples were then redissolved in tetrahydrofuran to a concentration of about 0.2 mg/ml and filtered through a Teflon membrane (0.45-µm pore
size) prior to analysis. A Waters 625 liquid chromatography system
(Millipore Corp., Milford, Mass.) equipped with four TSK-GEL columns
(Varian, Sunnyvale, Calif.) was used for gel permeation chromatography.
The columns (G1000 HXL, G3000 HXL, G4000 HXL, and G6000 HXL) were
connected in series and had nominal molecular weight cutoffs of 1 × 103, 6 × 104, 4 × 105, and 4 × 107, respectively. Samples
were eluted with tetrahydrofuran at a flow rate of 1 ml/min. The column
series was calibrated by using polystyrene standards (25).
Cellulose tricarbanilates were quantified by their absorption at 254 nm
by using a Waters 486 UV spectrophotometer. The DP of cellulose was
calculated by dividing the molecular weight of the tricarbanilated
derivative by the molecular weight of tricarbanilated anhydroglucose
(i.e., DP = Mw/519). Number-average and
weight-average DPs were calculated as previously described
(28).
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RESULTS |
Cellulose preparations (BMCC or PASC) were incubated with C. fimi endoglucanase (CenA or CenC) or cellobiohydrolase (CbhA or
CbhB) for up to 96 h. Aliquots of each reaction mixture were removed at various times, and the molecular size distributions of the
insoluble cellulose fractions were analyzed by size-exclusion chromatography of their tricarbanilate derivatives. The hydrolysis of
cellulose was assessed by quantitative analysis of soluble cellooligosaccharides released at corresponding incubation times (Table
1); this allowed the comparison of size
distributions at equivalent levels of degradation by the four enzymes.
Analysis of soluble products.
Incubation of BMCC with CbhA for
96 h resulted in 67% solubilization (Table 1). The soluble
products consisted largely of cellobiose (0.69 mg/ml). Low levels of
cellotriose (
0.01 mg/ml) were detected after incubation for up to
48 h (Fig. 1).
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FIG. 1.
Release of soluble sugars during hydrolysis of BMCC by
CenA, CenC, CbhA, and CbhB for up to 96 h. Glucose (G1,  ) and
cellotriose (G3,  ) (left-hand y axis) and cellobiose (G2,
 ) (right-hand y axis) were analyzed by anion-exchange
chromatography, as described in Materials and Methods.
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Significant solubilization of BMCC by CenA (25%) and CbhB (18%) was
also observed (Table 1). The soluble products were largely cellobiose
(0.24 and 0.16 mg/ml, respectively) (Fig. 1). A trace of cellotriose
(<0.01 mg/ml) was seen after only 5 min of incubation with CenA, but
this rapidly fell to undetectable levels. With CbhB, the cellotriose
level reached 0.02 mg/ml and then remained relatively constant. CenC
solubilized BMCC only to a very low degree (3%) (Table 1; Fig. 1).
All enzymes efficiently hydrolyzed PASC. The total extents of
solubilization after incubation with CenA, CenC, CbhA, and CbhB were
61, 45, 50, and 27%, respectively (Table 1). Cellobiose was the major
product for all enzymes (5.7, 4.3, 4.9, and 2.2 mg/ml, respectively)
(Fig. 2). Cellotriose was detected
following incubation with CenA, CbhA, and CbhB. Oligosaccharides with
DPs higher than that of cellotriose were not observed.
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FIG. 2.
Release of soluble sugars during hydrolysis of PASC by
CenA, CenC, CbhA, and CbhB. Glucose (G1, ) and cellotriose (G3, )
(left-hand y axis) and cellobiose (G2, ) (right-hand
y axis) were analyzed by anion-exchange chromatography, as
described in Materials and Methods.
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Analysis of molecular size distributions of insoluble cellulose
during hydrolysis.
The size distribution curves for untreated BMCC
and PASC showed two maxima (Fig. 3).
Major and minor peaks occurred at DPs of ~250 and ~18 in both
cellulose preparations. The size distribution for BMCC was broader than
that for PASC, as reflected in the calculated polydispersity values
(weight-average DP/number-average DP) of 4.3 and 2.9, respectively. The
BMCC curve also shows the presence of a small fraction of material with
a DP of <7. Cellooligosaccharides of this size are normally water
soluble (18). Their occurrence in the insoluble fraction
suggests that they are either adsorbed to the surface of BMCC particles
or occluded within them.
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FIG. 3.
Molecular size distributions of untreated BMCC and PASC.
DP is presented on a logarithmic scale.
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The size distribution curves for BMCC during hydrolysis are shown in
Fig. 4. For all enzymes, hydrolysis was
accompanied by a slight increase in the DP of the major peak maximum
(Table 1). For CenA, this occurred between 6 and 12 h. More
substantial shifts in the positions of the major peak maximum were seen
with CenC and CbhB. The increase occurred after only a few minutes of
incubation with CenC, although the total extent of substrate
solubilization at this time was very low. The largest shift in the
position of the major peak was seen with CbhA. No significant shift in
the position of the minor peak in BMCC was observed with any of the C. fimi enzymes (Fig. 4).
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FIG. 4.
Molecular size distributions of BMCC during hydrolysis
by CenA, CenC, CbhA, and CbhB for up to 96 h. The incubation time
(hours) corresponding to each curve is indicated. DP is presented on a
logarithmic scale.
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Corresponding size distribution curves during the hydrolysis of PASC
are shown in Fig. 5. In contrast to the
patterns observed with BMCC, a major decrease in the position of the
major peak maximum occurred during incubation with CenA or CenC (Fig.
5; Table 1). A decrease in the position of the major peak maximum was
also seen with CbhA and CbhB, but these shifts were significantly smaller than those with the endoglucanases. The minor peak maximum remained visible after prolonged incubation with CbhA or CbhB but was
no longer evident in the CenA and CenB incubations, probably as a
result of the accumulation of large quantities of low-DP material.
Similar peaks of low-DP material have been observed in cellulose
preparations from various sources, but their significance remains
unclear (10, 11).
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FIG. 5.
Molecular size distributions of PASC during hydrolysis
by CenA, CenC, CbhA, and CbhB for up to 96 h. The incubation time
(hours) corresponding to each curve is indicated. DP is presented on a
logarithmic scale.
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Comparisons of the cellulose molecular size distributions at
approximately equivalent levels of enzymatic solubilization were made
(Fig. 6). The data are the same as those
shown in the corresponding curves in Fig. 4 and 5, but the DP axis is
scaled linearly in Fig. 6. Data for hydrolysis of BMCC by CenC is
excluded because the extent of solubilization after 96 h was only
3%.
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FIG. 6.
Molecular size distributions of BMCC and PASC after
approximately equal levels of solubilization. For BMCC, the extents of
solubilization by CenA, CbhA, and CbhB were 25, 31, and 18%,
respectively. For PASC, the extents of solubilization by CenA,
CenC, CbhA, and CbhB were 28, 25, 29, and 27%, respectively. Further
details are contained in Table 1. DP is presented on a linear scale.
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The data in Fig. 6 correspond to 25, 31, and 18% solubilization of
BMCC by CenA, CbhA, and CbhB, respectively. The overall shapes of DP
distribution profiles were similar before and after solubilization,
although hydrolysis was accompanied by a slight increase in the DP of
the major peak maximum with all enzymes.
Solubilization of PASC by CenA, CenC, CbhA, and CbhB (28, 25, 29, and
27%, respectively) resulted in the loss of high-DP material and the
accumulation of lower-DP material in all cases. However, the DP
distribution profiles were not similar. The downward shift in the
distribution profiles was much more pronounced for the endoglucanases
CenA and CenC than for CbhA or CbhB (Fig. 6).
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DISCUSSION |
The cellulose-degrading system of C. fimi contains at
least six cellulases. Four of these enzymes (CenA, CenB, CenC, and
CenD) were designated as endoglucanases and two (CbhA and CbhB) were designated as cellobiohydrolases, based largely on analysis of CMC
hydrolysis (6, 15, 21, 22, 24). CenC appeared to show a more
processive action, relative to the other endoglucanases (23). In the present investigation we have compared the
activities of CenA, CenC, CbhA, and CbhB by determining the molecular
size distribution products obtained by hydrolysis of two types of
insoluble cellulose preparations.
The analysis of soluble products showed that all enzymes gave
cellobiose as the major product (Fig. 1 and 2), as expected for
cellobiohydrolases but not necessarily for endoglucanases. In addition,
low cellotriose concentrations were observed during PASC hydrolysis by
CbhA and CbhB. Cellotriose is not hydrolyzed by CbhB (21);
consequently, it accumulates during the incubation period. The
endoglucanase CenA, which hydrolyzes cellotriose (4), produces initially higher levels of cellotriose which subsequently decline, most likely due to further hydrolysis. The results emphasize the difficulty of distinguishing the activities of the various enzymes
from analysis of soluble products only.
The DP distribution of the insoluble products after BMCC hydrolysis
showed a moderate shift towards higher DP for all four enzyme
treatments (Fig. 4; Table 1). A similarly small upward shift in the
position of the major peak maximum was also observed during hydrolysis
of BMCC by T. reesei CBHII (11). The DP
distribution profiles for BMCC during separate treatment by the four
enzymes in the present work show a striking similarity, and
consequently the modes of action of the different enzymes were not
distinguished when BMCC was used as the substrate. In the PASC
incubations, however, a different pattern was seen (Fig. 5; Table 1).
During the incubation with endoglucanases, a major decrease of the DP position of the major peak maximum was observed. A significantly smaller downward shift was observed for CbhA and CbhB.
Differences in the behaviors of the four enzymes on BMCC and PASC are
particularly evident in Fig. 6, where the cellulose DP distributions
are compared at approximately equal levels of solubilization. For all
enzymes, it is evident that substantial solubilization of BMCC occurred
without major changes in the overall shapes of distribution profiles.
Loss of high-DP cellulose was slightly more pronounced for CenA than
for CbhA and CbhB, as expected for a randomly acting endoglucanase, but
the activity of CenA is not easily distinguished from the activities of
the two cellobiohydrolases when BMCC is used as the substrate.
In contrast, differences between the activities of the various enzymes
were clearly seen when PASC was used as the substrate (Fig. 6).
Solubilization of PASC by CenA, CenC, CbhA, and CbhB to equal levels
resulted in the loss of high-DP material and the accumulation of
lower-DP material in all cases, but the shifts in the distribution
profiles were much more pronounced for the endoglucanases CenA and CenC
than for CbhA or CbhB. The relatively small decreases in DP observed
with CbhA and CbhB on PASC provide evidence that both are predominantly
exo-acting enzymes, confirming a similar conclusion based on analyses
of CMC hydrolysis using viscometric measurement of hydrolysis (14,
15, 21, 22). The data support our earlier suggestion that
C. fimi CbhA and CbhB correspond to similar pairs of
cellobiohydrolases seen in fungal cellulase systems and, more
generally, that aerobic fungi and bacteria have similar types of
cellulase systems (6, 22).
The differences between the molecular size distribution data obtained
with BMCC and PASC can be explained by the model presented in Fig.
7. BMCC is shown as a highly crystalline
substrate in which only cellulose chains on the surface are susceptible
to enzyme attack. Internal glucan chains become accessible to
endoglucanases and cellobiohydrolases only after extensive
solubilization of overlying molecules. Consequently, differences in
mode of attack by the two types of enzyme are not easily observed.
However, a much larger proportion of the substrate is accessible to
simultaneous attack in the case of PASC; as a result, differences in
the modes of attack by the various enzymes are much more evident in
size distribution analyses. PASC is clearly a better substrate than BMCC to distinguish between endo- and exo-acting cellulases, and the
data show that designation of exoglucanase activity on the basis of
molecular size distribution data obtained with BMCC alone (11) should be viewed with caution.
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FIG. 7.
Models of PASC and BMCC, before and after 25%
solubilization by C. fimi CenA and CbhB, based on molecular
size distribution data. Solid lines represent individual -1,4-glucan
chains with a DP of ~250, equal to the major peak maximum of
untreated substrates. Broken or shorter lines represent shorter chains.
In both forms of cellulose, the chains are shown in parallel
orientation, with reducing ends towards the right. CenA and CbhB
molecules are represented as filled and unfilled ovals, respectively.
CenA is shown as a nonprocessive endoglucanase that attacks accessible
glucan chains randomly (4); CbhB is shown as a processive
exoglucanase that attacks accessible glucan chains from the reducing
end (6). In PASC, most glucan chains are accessible as
result of swelling and disruption of crystallinity; in contrast, only
those chains on the surface of BMCC are accessible to enzymes.
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In addition, the data in Fig. 6 also provide support for the previous
suggestion that CenC is a relatively processive endoglucanase, showing
a tendency to make several successive attacks before dissociating from
the substrate to renew its attack elsewhere (23). It is anticipated that the molecular size distribution profiles for a
processive endoglucanase would show characteristics that are intermediate between those of a nonprocessive endoglucanase and those
of an exo-acting cellobiohydrolase. Examination of the PASC data in
Fig. 6 shows that this is indeed the case for CenC. Evidence for two
other relatively processive -1,4-glucanases in bacteria was reported
recently (3, 9). The role of these enzymes in the attack of
cellulosic substrates is currently unknown, but it is noteworthy that
starch-degrading systems include 
-amylases with a high degree of
processivity (13, 19).
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ACKNOWLEDGMENTS |
We thank Emily Kwan for expert technical assistance.
H.S. acknowledges the Svenska Institutet for travel support.
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FOOTNOTES |
*
Corresponding author. Present address: Department of
Biochemistry, Center for Chemistry and Chemical Engineering, Lund
University, P.O. Box 124, S-221 00, Lund, Sweden. Phone: 46-46-222 82 02. Fax: 46-46-222 45 34. E-mail:
henrik.stalbrand{at}biokem.lu.se.
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Appl Environ Microbiol, July 1998, p. 2374-2379, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
