Quantitative estimating the effect of cellulase
components in degradation of cotton fibers
LuShang Wang, YuZhong Zhang,and Peiji 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 model for enzymatic degradation of cotton fibers has been established that based on a complete factorial experiments and combination by multivariate 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 b 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 depolymerized and solubilized into soluble oligosaccharides, 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 b 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.
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(13).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 general, for deriving a mechanism 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 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 fedbath 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 b 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 S38, 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 dewaxed 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 followed them at a given flow rate. The dimensions of which were 100 ± 25 m m long, and 1520 m 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% NaN_{3 }w/v, to preserve contamination. Hydrolysis were performed at 45^{o}C 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 Bioseor,SBA_50 type. The turbidity of residue cellulose was determined using integrating sphere attachment (Spectrophotometer UVVIS 240, Shimadzu). The decrease % of turbidity was defined as cellulose solubility.
Measurement of total cellulase activity
Measurement of filter paper activity (FPU) using filter paper strip(Whatman No.1) as substrate(13). Measurement of b 1,4glucan endoglucanase(EG)using CMCNa carboxymethycelluloseNa(middle viscosity, Sigma) as substrate. The relative activities were calculated as release of m g glucose/ min^{1}. Measurement of b 1,4glucan cellobiohydrolas(CBH) using pnitrophenal –cellobiose pNPC as substrate. Measurement of b 1,4glucosidase Using pnitrophealglucosee, pNPG as substrate. The relative activities were calculated as release of 1 m mol of pnitropheol/per min( 20).
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:
? =b_{0 }+_{ }b_{1}x_{1 }+_{ }b_{2 }x_{2}+_{ }b_{3}x_{3 }+_{ }b_{4}x_{4 }+_{ }b_{5 }x_{5 }+ b_{6}x_{6}+ b_{7}x_{7}
Where ? is a predictor of dependent variable(objective function), in present case it is the value of reducing sugars formation or the cellulose solubility, and X_{1 },_{ }X_{2} ,and X_{3 }represents CBHI, EGI and one b 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 X_{4},X_{5 },X_{6},X_{7 }represents the synergistic effect between them. and computed as:
X_{4 }=X_{1 }+_{ }X_{2}(CBHI+EGI)_{, }X_{5}=X_{1} + X_{3}(CBHI+b glucosidaseI ),_{ } X_{6}=X_{2} +X_{3} (EGI+b glucosidaseI ), and X_{7}=(X_{1} + X_{2})´ X_{3}(CBHI+EGI )´ b 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 b glucosidase on the products produced from the synergism of CBHI and EGI. That can be represented by the effect of b glucosidase times the sum of CBHI and EGI. The b_{0} is regression constant, and b_{17} is standard regression coefficient. The value of regression coefficients b_{i }can be used as an index of effect of cellulase component, alone and in combination, in cellulose hydrolysis. As F_{i }of any one X_{i< } 2.0 ,will no new variable entered. A three factor complete combination design^{7} (3 x 3 x 3) was applied that considered each of which is corresponding to the three cellulase component, CBHI(three levels: 0, 5.0 and 10.0 m g/mg.cellulose),EGI(three levels:0,1.0,2.0 m g/mg.cellulose) and b glucosidaseI(three levels:0,0.5,1.0 m g/mg.cellulose).By doing this design, each of the 27 experiments were conducted for ?. Both cellulose solubility and glucose formation were selected as objective function, respectively. The multivariate regression analysis was performed by the Statistics Package of ANALYST/REGERS command(Fujitsu, Co. Japan), using M340 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 b 1,4linked polysaccharides, such as mannans and xylans and is also slower than that of the amorphous cellulose(13).
Figure.1 Time course of degradation of cotton fibers at 45^{0}C 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 b 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 involved the nonhydrolytic 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 solubility and glucose formation were selected as objective function, respectively and three cellulase components(factors), alone and in combination, were 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.
? =b_{0 }+_{ }b_{1}x_{1 }+_{ }b_{2 }x_{2}+_{ }b_{3}x_{3 }+_{ }b_{4}x_{4 }+_{ }b_{5 }x_{5 }+ b_{6}x_{6}+ b_{7}x_{7}
Where ? is a predictor of dependent variable(objective function), in present case it is the value of glucose formation or the cellulose solubility, and b_{0} is regression constant,b_{17} is standard regression coefficient. X_{1 },_{ }X_{2} ,and X_{3 }represents the effect of CBHI, EGI and one b glucosidase, and X_{4},X_{5 },X_{6},X_{7 }represents the synergistic effect between them. (Details see Experimental protocol). By doing this, two regression equations were obtained.
When glucose formation as objective function:
? = b_{0}+0.018EGI+0.176(EGI+b glucosidase)+0.667(CBHI+EGI) +1.106(CBHI+EGI)
´ b glucosidase
Similarly, cellulose solubility as objective function:
? =b_{0}+0.781(CBHI+EGI)+0.814(CBHI+EGI)´ b 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 
tvalue 
Glucose formation 
EGI 
0.118 
1.43 
CBHI+EGI 
0.667 
7.30^{**} 

EGI+b glucosidase 
0.176 
2.01 

CBHI+EGI) ´ b glucosidase 
1.106 
10.21^{**} 

Cellulose Solubility 
CBHI+EGI 
0.781 
5.24^{**} 
(CBHI+EGI) ´ b glucosidase 
0.814 
7.10^{**} 
* 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^{ }(?). Consequently, here it can be used for the quantitatively evaluating the positive/negative .i.e. +/ effect of each cellulase component, alone and in combination 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 main factor, and adding b glucosidase can significantly increases this effect. The effect of EGI alone and even in the synergism by b glucosidase was little. However, for cellulose solubility, the main effect only by synergism between CBHI and EGI. This effect by plus b 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.
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 b 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
0bjective function 
Variable entered 
Standard regression coefficient 
tvalue 
Reducing sugars Formation 
EGI 
0.118 
1.43 
EGI+b glucosidase 
0.305 
2.75^{*} 

CBHI+EGI 
0.7368 
6.50^{**} 

(CBHI+EGI) ´ b glucosidase 
0.8409 
7.42^{**} 
* significance difference t> t_{0.05}=2.086, ** extremely significance difference t> t_{0.01}=2.843
As shown in Table 2, the main effect for reducing sugars produced in FP assay is also by the synergism of CHHI and EGI. But as compared with cotton fibers(Table 1), a new variable ¾ synergism of EGI+b glucosidase has entered into the equation and appears a significant effect that clearly indicated the synergism of EGI+b glucosidase has certain contribution in total FP assay. Which reflected some amorphous fraction of cellulose in filter paper was hydrolyzed by the synergism of EGI+b 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.
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