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 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 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.

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.

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 followed them at a given flow rate. The dimensions of which were 100 ± 25 m m long, and 15-20 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% NaN₃ w/v, to preserve contamination. Hydrolysis were performed at 45^oC 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 residue cellulose was determined using integrating sphere attachment (Spectrophotometer UV-VIS 240, Shimadzu). The decrease % of turbidity was defined as cellulose solubility.

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:

X₄ =X₁ + X₂(CBHI+EGI)_, X₅=X₁ + X₃(CBHI+b -glucosidaseI ), X₆=X₂ +X₃ (EGI+b -glucosidaseI ), and X₇=(X₁ + X₂)´ X₃(CBHI+EGI )´ b -glucosidaseI.

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 -1,4-linked polysaccharides, such as mannans and xylans and is also slower than that of the amorphous cellulose(1-3).

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.

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.

When glucose formation as objective function:

Similarly, cellulose solubility as objective function:

Table 1 is the summary of multvariate regression analysis.

* significance difference t> t_(30)0.05=2.086, ** extremely significance difference t> t_(30)0.01=2.843

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.

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.

* 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.

J.Protein.Chem.16,59-66

13. Ghose,T.K. Measurament of cellulase activities. (1987) Pure & Appl. Chem 59,

257-268