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

采用纤维素二糖脱氢酶灵敏测定系统，研究了纯化纤维素酶对结晶纤维素的降解机理。本研究对从里氏木霉中分离得到的内切葡聚糖酶II （EG II）和纤维素生物水解酶I （CBH I）进行了检测。以木醋杆菌生产的结晶纤维素、so2 -胺溶剂体系再生的无定形纤维素和可溶性纤维素糊精为纤维素底物。测定纤维素酶活性在醋酸钠缓冲液（pH4.2）中进行，并估计每种底物的水解速率。CBH I对三种底物的水解速率几乎相同，说明纤维素结构的差异对CBH I的反应速率影响较小。相反，EG II的反应速率受纤维素结构差异的影响较大。可溶性纤维素糊精是EG II的最佳底物，无定形纤维素也容易被EG II水解。然而，结晶纤维素是EG II非常差的底物。CBH 1和EG II对可溶性纤维素糊精的水解没有协同作用，而对固体纤维素，特别是对结晶纤维素有显著的协同作用。此外，CBH I和EG II的组合对可溶性纤维素糊精的反应速率与单独使用EG II的反应速率非常接近。从这些观察结果来看，协同作用的机制可以用CBH I的作用增加EG II的可达点来解释。这些观察结果表明，CBH I是一种具有结晶纤维素组成能力的内型纤维素酶，而不是普通的外型葡聚糖酶。此外，CBH I被认为具有类似氢键酶的纤维素表面溶胀能力。

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.