Quantitative estimating the effect of cellulase
components in degradation of cotton fibers
Lu-Shang Wang, Yu-Zhong Zhang,and Pei-ji Gao*
State Key Laboratory of Microbial Technology, Shandong University,
Jinan ,250100,China
Yang Hong
Department of Mathematics, Shandong University, Jinan,250100 China
*
Corresponding author(gaopj@sdu.edu.cn)
Keywords: cotton cellulose degradation, cellulase, multivariate regression analysis.
A comprehensive mechanistic
fibers
has
been
model for enzymatic degradation of cotton
established
experiments and combination
that
based
on
by multivariate
a
complete
factorial
stepwise regression
analysis. The model was derived from two key reactions : cellulose
solubility and glucose formation. The proposed model can be applied to
quantitative estimating the effect of three cellulase components,
cellobiohydrolase I, endoglucanase I and one -glucanase, alone and in
combination in cellulose degradation. Utilization of all experimental
data in statistical parameter values of the model leads to the
conclusion that enzymatic degradation of cotton cellulose is a serial
and heterogeneous process that included, at least, both sequentially
occurred and then paralleling progressed process:cellulose fibers were
de-polymerized
and
solubilized into soluble
oligo-saccharides, in
which the main effect only by the synergism between cellobiohydrolase I
and endoglucanase I, and then the oligomers were hydrolyzed into
glucose by the randomly reaction of -glucanase. The validity of the
proposed model has been checked with the filter paper activity assay,
and
its
applicability
for
practical
process
of
other
cellulose
/cellulase system was also discussed.
1
INTRODUCTION
Cellulose is the most abundant renewable polysaccharide in nature. Its
biodegradation by microorganisms is one of the major steps of the
carbon cycle on Earth. Therefore, the efficient utilization of this
process could provide a significant contribution to solve the problems
present in environment and ecology. However, based on the present
technology, cellulose utilization through enzymatic hydrolysis process
does not appears economical for the production of sugar syrups, or
alcohol
fuels.
Since
the
complete
conversion
of
the
cellulosic
substrates to glucose by the cellulase could not be easy achieved,even
though a suitable reaction condition and a long incubation time were
given(1-3).Despite the significant technological advancements achieved
in recent years in this field, the enzyme cost is also the most
expensive part in these processes. This cost was primarily due to the
high concentration of enzymes used and a long incubation time that
would be all required to obtained a complete hydrolysis of cellulose.
A mechanistic kinetic model for enzymatic hydrolysis of cellulose
is
needed not only for understanding its mechanism,but also for developing
a practical process of optimal usage of cellulase. In
deriving a mechanism
general, for
model, the structure feature of substrate and the
mode action of reaction system are necessary to be investigated in
detail.
However,
for
cellulose/cellulase
system,
because
of
the
structural heterogeneity of cellulose and the complexity of cellulase
system, especially for its act synergistically, make it difficulty to
2
describe
quantitatively
its
rate
and
mechanism
by
using
the
traditional kinetic techniques(4,5). With this background, we then
tried to resolved these difficulties by following the approach of
Solomon
and
Erickson(6) .
Which
is
based
on
the
utilization
of
statistics and experimental design for data collection and analysis in
fed-bath
fermentation
process.
In
our
previous
studies(7),
a
preliminary experiment was performed based on the factorial experiment
design and combination of the multivariate
regression analysis that
can be used for quantitatively estimating the effect of individual
cellulase component in reducing sugars formation during cellulose
degradation. In present work, we further developing this method to
obtained
a
comprehensive
and
mechanistic
picture
of
enzymatic
hydrolysis of cotton fibers. In the hope that could be estimate
quantitatively
the
different
effects
of
three
major
cellulase
components, CBHI, EGI and one -glucosidase, alone and in combination,
in cellulose solubility and glucose formation during the hydrolysis of
cotton fibers.
MATERIALS AND METHODS
Cellulase and substrate
A cellulolytic fungi, Trichoderma pseudokoningii S-38, was isolated
previously in our laboratory(8), the properties of CBHI and EGI from
the crude enzyme of this strain have reported in previous papers(9,10).
The de-waxed cotton fibers was selected as substrate. These fibers were
simply cut and selected passed 100 mesh but was retained by 120 mesh To
obtain substrate containing particles of reasonably uniform size, a 1%
cotton fibers suspensions were subjected to a floatation technique as
suggested by Rautela and King(11). The uniform fraction was obtained by
3
followed them at a given flow rate. The dimensions of which were 100 
25 m long, and
15-20 m broad.
Enzymatic hydrolysis of cotton fibers by crude cellulase.
250 ml flask which consisted of 1 % uniform size particles of cotton
fiber suspended in 50 ml pH 4.8,50 mM acetate buffer, added crude
cellulase solutions(0.005 FPU /per mg cotton fibers, it is about
contained 0.0083 IU CBH,0.104 IU Endo- and 0.002 IU
glucosidase for
per mg cotton fibers). Added 0.001% NaN3 w/v, to preserve contamination.
Hydrolysis were performed at 45oC in shaking bath at 15 rpm. At every
two days, the hydrolysate was separated by centrifuging at 5,000g for
10 min, and the glucose in supernatant was determined by glucose
oxidase(12),using Bio-seor,SBA_50 type. The turbidity of
cellulose
was
determined
using
integrating
sphere
residue
attachment
(Spectrophotometer UV-VIS 240, Shimadzu). The decrease % of turbidity
was defined as cellulose solubility.
Measurement of total cellulase activity
Measurement
strip(Whatman
of
filter
No.1)
as
paper
activity
substrate(13).
(FPU)
using
Measurement
of
filter
paper
-1,4-glucan
endoglucanase(EG)using CMC-Na carboxymethycellulose-Na(middle viscosity,
Sigma) as substrate. The relative activities were calculated as release
of g glucose/ min-1. Measurement of -1,4-glucan cellobiohydrolas(CBH)
using p-nitrophenal –cellobiose p-NPC as substrate. Measurement of 1,4-glucosidase Using p-nitropheal-glucosee, p-NPG as substrate. The
relative activities were calculated as release of 1  mol of pnitropheol/per min( 20).
4
Multivariate regression analysis
A prediction regression equation with interaction of three independent
variables was selected for this purpose which is one of a set
assumption that are imposed in deviating an appropriate regression
models in enzymatic degradation of cotton fibers(7).
The model
considered here is:
Ŷ =b0 + b1x1 + b2 x2+ b3x3 + b4x4 + b5 x5 + b6x6+ b7x7
Where
Ŷ is a predictor of dependent variable(objective function), in
present case
it is the value of reducing sugars formation or the
cellulose solubility, and X1 , X2 ,and X3 represents CBHI, EGI and one glucosidase
which is based on the hypothesis that the degradation of
cellulose by cellulase complex is a simultaneous action of cellulase
components
on
the
crystalline.
And
X4,X5 ,X6,X7 represents
the
synergistic effect between them. and computed as:
X4 =X1 + X2(CBHI+EGI),
X5=X1 + X3(CBHI+-glucosidaseI ),
X6=X2 +X3
(EGI+-glucosidaseI ), and X7=(X1 + X2) X3(CBHI+EGI )-glucosidaseI.
That is based on the assumption that the synergism between CBH and
Endoglucanase by the effect of its sum, and the randomly act exist by
the hydrolysis of -glucosidase on the products produced from the
synergism of CBHI and EGI. That can be represented by the effect of glucosidase times the sum of CBHI and EGI. The b0 is regression constant,
and b1-7 is standard regression coefficient. The value of regression
coefficients bi can be used as an index of effect of cellulase component,
alone and in combination, in cellulose hydrolysis. As Fi of any one Xi
2.0 ,will no new variable entered. A three factor complete combination
design7 (3 x 3 x 3) was applied that considered each of which is
5
corresponding to the three cellulase component, CBHI(three levels: 0,
5.0
and

10.0
g/mg.cellulose)
g/mg.cellulose),EGI(three
-glucosidaseI(three
and
levels:0,1.0,2.0
levels:0,0.5,1.0
g/mg.cellulose).By doing this design, each of the 27 experiments were
conducted for Ŷ.
selected
regression
as
Both cellulose solubility and glucose formation were
objective
analysis
was
function,
performed
respectively.
by
the
The
Statistics
multivariate
Package
of
ANALYST/REGERS command(Fujitsu, Co. Japan), using M-340 S electronic
computer.
Results and discussion
There appears a sequentially occurred and then paralleling progressed processes:
cellulose solubility and glucose formation during enzymatic degradation of cotton
fibers
In figure.1,as several investigators reported(1,2,4,5,15),a typical
progress curves of native cellulose are presented. It shows that either
for glucose formed or for cellulose solubility, the progress occurred
quickly in the early stage of the reaction. During the first four days,
about 50% of the total cotton fibers has been solubilized and then
converted to glucose. After this stage, the reaction rates of both are
all declining rapidly with time. Although under this reaction condition,
cotton fibers can almost 90%
solubilized
and hydrolyzed to glucose,
but this progress is much slower as compared with the enzymatic
hydrolysis other -1,4-linked polysaccharides, such as mannans and
xylans and is also slower than that of the amorphous cellulose(1-3).
6
Conversion % in initial wt.of cellulose
100
90
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
Time course,days
Figure.1 Time course of degradation of cotton fibers at 45 0C by crude cellulase(0.015 FPU
for per mg cellulose). Glucose formed (■) and cellulose solubility (•). All tests were
determined in triplicate, and the SD is about 5%
Another important behavior can be observed from the Figure 1 is that
both progresses of cellulose solubility and glucose formation appear
approximately
belong
to
the
first
order
reaction.
For
cellulose
solubility, a series of its first, second and third half life of
reaction
is
101.2,102.0
and
103.0
hr,
respectively,
for
glucose
formation, these three steps is 148,146 and 147 hr, respectively
(Figures omitted). This result clearly indicated that both cellulose
solubility and glucose formation are sequentially occurred and then
paralleling progressed processes during enzymatic degradation of cotton
fibers, and means conversion rate of both processes all appears
exponential decrease followed by each steps.
In previous reports, the majority of investigators called the above process as “Hydrolysis
process” which means the reaction is only related to the hydrolysis reaction of -1,4glycosidic bonds in cellulose by cellulase. As it appears in present result and
combination of our previous investigation(16,17) which clearly indicated that the process
of the enzymatic degradation of cellulose not only by hydrolysis reaction but also
7
involved the non-hydrolytic disruption reaction of cellulose structure by cellulase. Thus,
termed this entire process as: ”Degradation process” instead of hydrolysis is suitable.
Quantitatively estimating the action of cellulase components in degradation of
cotton fibers by multivariate regression analysis
Since total enzymatic degradation process of cotton cellulose including,
at least, two sequence and heterogeneous reaction, the kinetic analysis
expression obtained from the any one of which cannot be adapted in
predicting the total progress. Therefor, the applicability of these
kinetic models is somewhat limitation to certain hydrolysis conditions.
As presented above, after the six days of hydrolysis, about 70% of
cotton fibers has solubilized and about 60% of glucose formed, so it
can be used as a foundation for evaluated the main effect of cellulase
components in degradation of cotton fibers. A (3 x 3 x 3) factorial
experimental design(18) was performed, i.e. three factors and true
value of each in three levels and in complete combination. The values
of
cellulose
objective
solubility
function,
components(factors),
and
glucose
respectively
alone
and
in
formation
were
and
three
combination,
were
selected
as
cellulase
selected
as
independent variables. In which each of the 27 experiments were
conducted, and then the data were analyzed to estimate the parameters
based on the following model.
Ŷ =b0 + b1x1 + b2 x2+ b3x3 + b4x4 + b5 x5 + b6x6+ b7x7
Where
Ŷ is a predictor of dependent variable(objective function),
in present case
it is the value of glucose formation or the cellulose
solubility, and b0 is regression constant,b1-7 is standard regression
coefficient. X1 , X2 ,and X3 represents the effect of CBHI, EGI and one glucosidase, and X4,X5 ,X6,X7 represents the synergistic effect between
8
them.
(Details
see
Experimental
protocol).
By
doing
this,
two
regression equations were obtained.
When glucose formation as objective function:
Ŷ = b0+0.018EGI+0.176(EGI+-glucosidase)+0.667(CBHI+EGI) +1.106(CBHI+EGI)
 -glucosidase
Similarly, cellulose solubility as objective function:
Ŷ =b0+0.781(CBHI+EGI)+0.814(CBHI+EGI)-glucosidase
Table 1 is the summary of multvariate regression analysis.
Table 1 Summary of multivariate regression analysis of three cellulase components,
alone and in combination during cotton fibers degradation
0bjective function
Variable entered
Standard
regression coefficient
t-value
EGI
0.118
1.43
CBHI+EGI
0.667
7.30**
EGI+-glucosidase
0.176
2.01
CBHI+EGI)
 -glucosidase
1.106
10.21**
CBHI+EGI
0.781
5.24**
Glucose formation
Cellulose Solubility
(CBHI+EGI)
0.814
7.10**
 -glucosidase
* significance difference tt(30)0.05=2.086, ** extremely significance difference tt(30)0.01=2.843
Because standard regression coefficient is a dimensionless term, so its
absolute
value
is
normally
used
as
a
index
for
quantitatively
evaluating the effect of factor term(variable) for the objective
function (Ŷ). Consequently, here it can be used for the quantitatively
evaluating the positive/negative .i.e. +/component, alone and in combination
effect of each cellulase
on cellulose solubility or glucose
formation. As shown in Table 1,according to the statistic analysis, for
glucose formation, the effect of synergism between CBHI and EGI is the
9
main factor, and adding -glucosidase can significantly increases this
effect. The effect of EGI alone and even in the synergism by glucosidase was little. However, for cellulose solubility, the main
effect only by synergism between CBHI and EGI. This effect by plus glucosidase is also weaker. The results clearly demonstrated that the
effect of these three cellulase components for both sequentially
occurred and then paralleling progressed process is different. Thus,
the effects and act synergistically of three cellulase components in
cellulose degradation can be clearly distinguished by this analysis.
In
previous
studies,
the
value
of
reducing
sugars
(glucose
and
cellobiose) produced during enzymatic hydrolysis of cellulose has been
selected as a standard but a single index for kinetic analysis, as
mentioned
here,
it
only
is
one
of
the
respects
in
the
entire
degradation progress. Supplement of cellulose solubility as an another
index that will be provide more and complete knowledge about the
degradation progress.
Estimating the effect of cellulase components in filter paper
assay(FPA)
Filter paper assay that is a widely used method recommended
by
International Union of Pure and Applied Chemistry for evaluation of
potential saccharifying capacity of a cellulase system(13). But because
of filter paper consisted of both crystalline and amorphous fractions
and the susceptibility of which for cellulase degradation are different.
In usual, several cellulase system have same activities in FP assay but
show
different
saccharifying
capacity
in
practical(1,19).
Thus,
estimating the effect of each cellulase components, alone and in
combination, contributed to the total FPA activity is required for
actually evaluating the saccharifying capacity of a cellulse system.
10
We selected this problem for check the validity of the present
regression equation model. A series experiments were performed by three
factors(CBHI,EGI and one -glucosidase) complete combination design (3
x 3 x 3) and analysis the results using multivariate regression
analysis as before. Experimental conditions and assay procedure are all
based on the report of Ghose .(13). The result was shown in Table 2.
Table 2 Comparison of standard coefficient of three cellulase component in FPA assay
Variable entered
Standard regression
coefficient
t-value
EGI
0.118
1.43
EGI+-glucosidase
0.305
2.75*
CBHI+EGI
0.7368
6.50**
(CBHI+EGI)
 -glucosidase
0.8409
7.42**
0bjective function
Reducing sugars
Formation
* significance difference tt0.05=2.086, ** extremely significance difference tt0.01=2.843
As shown in Table 2, the main effect for reducing sugars produced in FP
assay
cotton
is also by the synergism of CHHI and EGI. But as compared with
fibers(Table
1),
a
new
variable

synergism
of
EGI+-
glucosidase has entered into the equation and appears a significant
effect that clearly indicated the synergism of EGI+-glucosidase has
certain contribution in total FP assay. Which reflected some amorphous
fraction of cellulose in filter paper was hydrolyzed by the synergism
11
of EGI+-glucosidase under FP assay. A previous report in our Lab(20)
suggested that about 72.7% of amorphous fraction of cellulose in filter
paper was hydrolyzed in FP assay. Thus, because of these two equations
have no incompara ability, thus as using the value of a cellulase
system in FP assay for predicted its potential sacchariying capacity
for crystalline cellulose such as cotton fibers, it always got a error
expected value.
For cellulase application, besides total hydrolysis of cellulose into
glucose, several new area are being developed for textile, paper pulp
processing and food, etc(21). These applications are based on the
certain modification of cellulase on cellulose fibers by partial
degradation. The main aspect of the problem in the area is how to
quantitatively estimating the effect of cellulase component, alone and
in combination in the treatment process. This problem seems to be
solved, as mentioned present, by using the factorial experimental
design and combined the method of multivariate regression analysis. The
proposed method can be not only used in establish mechanistic kinetic
model and even more in designing a practical protocol for enzymatic
treatment of cellulosic substrates.
Acknowledgements: This work was supported by a grant from National
Natural Science Foundation of China, No.394300020 and excellent Ph,D.
Thesis Foundation No
200023,Education Committee in China.
REFERENCES
1. Klyosov, A. A. Trends in biochemistry and enzymology of cellulose degradation.
(1990) Biochemistry, 29,10577-10585
12
2. Wood,T.M.,McCrae,S.I.& Bhat,K.M. The mechanism of fungal cellulase action.
(1989)Biochem.J.260,37-43
3. Tsao,G.T.,Ladisch,M.R.& Bungay,H.R. Biomass refining. In Advanced Biochemical
Engineering. Ed: Sungay, H.R.John & Sons Ina,New York, 1978 p.79
4. Paice,M.G.& Jurasek,L. Structure and mechanistic comparisons of some
-(1-4)
glycoside hydrolases. (1979) In Hydrolysis of cellulose: mechanisms of enzymatic
and acid hydrolysis. Advances in chemistry series. 181 Washington D.C p361-374
5. Fan,L.T,.Lee,Y,H. & David,H. Mechanism of the enzymetic hydrolysis of cellulose:
effects of major structural feature of cellulose on enzymatic hydrolysis. (1980)
Biotech & Bioeng.22,177-1996.
6 Solomon,B.O & Erickson,L.E. Utilization of statistics and experimental design in data
collection and analysis. (1983) Biotech & Bioeng,26,2683- 2705
7. Gao,P.J. et al. The use of multiple regression analysis for estimating the action pattern
of cellulase fraction in hydrolysis of cellulose. (1989) J.Biometrics(China).4,144150
8. Ma,D.B.,Gao,P.J. & Wang,Z.N. Preliminary Studies on the mechanism of cellulose
formation by Trichoderma pserdokoningii S-38(1990). Enzyme Microb.Technol.
12,631-635
9. Yan,B.X. & Sun,Y.Q. Domain structure and confurmation of a
cellobiohydrolase I from Trichodrma Pseudokoningii S-38. (1997)
J.Protein.Chem.16,59-66
10. Yan,B.X.,Sun,Y.Q.& Gao,P.J. Intrinsic fluorescence in endoglucanase
and cellobiohydrolase from Trichoderma Pseudokomingee S-38. (1997)
J. Protein.Chem.18,681-688
11. Rautela, G.S.& King K.W. Significance of the crystal structure of cellulose in the
production and action of cellulase. (1968) Archives Biohem. Biophysc. 123,58960112.
12
Parente,A.H.
et
al.
Glucose
biosensor
using
glucose
oxidase
immobilized in polyaniline(1992) Appl Biochem Biotechnol,37,267-273
13. Ghose,T.K. Measurament of cellulase activities. (1987) Pure & Appl. Chem 59,
257-268
14. Wood,T.M.& Bhat,K.M Methods for cellulase actvities. In: Methods in Enzymology
13
V 160 part A (1988) Eds: Wood, W.A and Kellong, S.T. Academic Press, Inc San
Diego, California 92101
15. Zhang.S.,Wolfgang,D.& Wilson,D. Substrate heterogeneity causes the nonlinear
kinetics of insoluble cellulose hydrolysis. (1999) Biotech & Bioeng.66,35-41
16. Gao,P.J,et al.Non-hydrolysis disruption of crystalline structure of cellulose by
cellulose binding domain and linker sequence of cellobiohydrolase I from
Penicillium janthinellum. (2001) Acta Biochimica et Biophsica Sinica,44,13-18
17. Xiao Z.H,et al. Cellulose-binding domain of endoglucanase III from Trichoderma
reesei disrupting the structure of cellulose. (20001) Biotechnol Letters,23,711-715
18. Gomes,K.A.& Gomes,A.A. (1984) Statistical procedure for agriculture research.
Second edition, John Wiley & Sones Inc,New York
19. Halliwell,G. Microbial -glucanases. (1979) in Progress in
Industrial
Microbiology.Ed: M.J,Bull.V,15,1-58
20. Gao,P.J.,Qu,Y.B 7 Wang,Z.N.,Estimating the pitfals in the assay of
filter paper activities by use of multiple regression analysis.
(1990)J.Biomatrics(China),5,15-2021
21 Béguin,P & Aubert,J.P. The biological degradation of cellulose.
(1994) FEMS Microbiology Reviews,13,25-58
14