Purification And Characterization Of Endo-Cellulases From Aspergillus Oryzae

Cellulase is one kind of the enzymes that break down cellulose, includingendo-glucanase（endo-1,4-β-D-glucanase, EC3.2.1.4）, exo-glucanase （exo-1,4-β-D-glucanase,EC3.2.1.91）and β-glucosidase（β-1,4-glucosidase, EC3.2.1.21）.Cellulose is hydrolyzed intoglucose with synergistic effect of these components. Endo-cellulase is also called asendo-beta1,4-glucanase （also known as the Cx enzyme or enzyme CMC）,which belongs tocellulase system.It can degrade macromolecular cellulose into cellobiose and play a key rolein degradation of cellulose. Aspergillus oryzae3.042is commonly used in brewing, itscellulase decomposes cellulose of materials,assisting the proteases to hydrolyze the protein ofmaterials.In this article, Four Cx enzymes were identified by Congo red staining, called as CxenzymeⅠ,Cx enzyme Ⅱ,Cx enzyme Ⅲ and Cx enzyme Ⅳ respectively.DEAE-Cellulose-52ion exchange chromatography was used to primarily separate endocellulases in the crudeenzyme solution,it was found that the active components existed in four eluting peak. Fourpurified Cx enzymes were obtained by preparative electrophoresis after dialysis andconcentration.First three Cx enzymes were monomeric enzyme and their molecular weightswere31.8kDa,34kDa and26kDa respectively determined by SDS-PAGE, the last onecontained four subunits, and their molecular weights were14kDa,23.6kDa,26kDa,33.5kDa respectively.Studies of endo-cellulase of A. oryzae3.042mature Koji have shown that: the optimaltemperature of crude enzyme is50℃,it has better stability at the range from30℃to60℃,andthe optimal pH of enzymatic reactions is4.0,the enzyme was stable between pH3.0and50.Zn²⁺, Fe²⁺and Cu²⁺ions were activators of the cude enzyme,but the Mn²⁺, K⁺, Mg²⁺, Ca²⁺and Na⁺ions could inhibit the activity of enzymes.In this study,four endo-cellulase components were isolated and purified from Aspergillus oryzae huniang3.042brewing, as well as its enzymatic properties was analyzed. The work is part of the basic research work of fungal cellulase system and it has i mportant meanings to deeply study of the fungal source of cellulases and the mutuallyunderstand of synergistic relationships between the components.