Study On The Hydrolytic Characterization And Mechanism Of Cellulase On Chitosan

Cellulases with non-specific action on chitosan depolymerization have been well characterized. Now they are widely used in the chitooligosaccharides production due to their low price and availability comparatively to chitinase and chitosanase. But information regarding the exact portion of cellulase with the unusual susceptibility to chitosan, the mechanism of this non-specific catalysis and the action pattern of the non-specific chitosanolysis is greatly limited. It is necessary to study the characterization and mechanism of non-specific cellulase hydrolysis on chitosn. The results explained the enzyme mechanism and gave the instruction in the applications of non-specific hydrolysis for the production of chitooligosaccharides.On the basis of the comparison of the chitosanolytic activities of four kinds of commercial cellulase, one kind of cellulase had good ability to digest chitosan produced by Trichoderma viride was secreted. This enzyme showed maximum activity towards chitosan at pH 5.2 and 60℃. Its maximum activity towards carboxymethyl cellulose(CMC) was at pH 4.2 and 55℃. Under these optimum conditions, 54.5% of the original velocity of chitosan was decreased within the first 10 minutes. Substrate specificity study showed that there was no effect of deacetylation of chitosan on the activity. The cellulase split chitosan in an endo-manner and synergistic action with the exo-manner andβ-glucosidase as indicated by the composition of the products with different deacetylation degree.One portion with CMCase activity and chitosanolytic activity (CCBE) was purified through sequential steps of DEAE-Sepharose CL-6B ion-exchange chromatography, Phenyl Sepharose CL-4B hydrophobic interaction chromatography and Sephadex G-75 gel filtration from commercial cellulase and its homogeneity was demonstrated by SDS-PAGE, RP-HPLC, IEF. The molecular mass of CCBE was 66kDa as estimated by SDS-PAGE and IEF showed that its pI was about 4.45.The kinetic analysis of the CCBE towards chitosan showed that the optimum conditions was pH 5.2 and 60℃. Km and Vmax were 0.746 mg Glucosamine/mL and 0.0268mg/mL min respectively. CCBE was stable at pH 4.0 - 7.8 and 30～50℃. It was gradually inactivated at 60℃and significantly lost activity above 80℃.