Investigation on the degradation mechanism of crystalline cellulose by cellulases using a new sensitive detection method.

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Degradation mechanism of crystalline cellulose by purified cellulases was investigated with a new sensitive assay system of cellulase activity using cellobiose dehydrogenase.Endoglucanase II (EG II) and cellobiohydrolase I (CBH I), which had been purified from Trichoderma reesei, were tested in this study. Crystalline cellulose produced by Acetobacter xylinum, regenerated amorphous cellulose from SO2-amine solvent system, and soluble cellodextrin were utilized as cellulose substrates. The measurement of cellulase activity was done in sodium-acetate buffer (pH4.2) and the rate of hydrolysis for each substrate was estimated.CBH I can hydrolyze all three substrates with almost same reaction rate, suggesting that the difference of cellulose structure is less effective to the reaction rate of CBH I.In contrast, the reaction rate of EG II is significantly depending on the difference of cellulose structure. Soluble cellodextrin is the best substrate for EG II and amorphous cellulose is also hydrolyze easily by EG II.However, crystalline cellulose is very poor substrate for EG II.There is no syncrgistic effects of CBH 1 and EG II on the hydrolysis of soluble cellodextrin, whereas the significant synergy is observed for solid celluloses, especially for crystalline cellulose. Furthermore, the reaction rate of CBH I and EG II combination is very comparable to that of EG II alone for soluble cellodextrin. From these observations, the mechanism of synergy can be explained by the increasing of accessible points for EG II by the action of CBH I.These observation suggests us that CBH I is a endo-type cellulase with the composition ability of crystalline cellulose rather than the ordinal exo-type glucanase. Moreover, CBH I is considered to possess hydrogen-bondase like ability for swelling of cellulose surface.