Purification And Enzymatic Properties Of Beta-glucanase. Cellulase Endo Study

An endo-β-glucanase was isolated and purified from the leavening of Trichoderma viride called after MJ1 by means of ammonium sulfate and Sephadex G-100 chromatography, and further fractionated by FPLC (AKTA UPC-900) respectively.The specific activity of the endoglucanase from MJ1 increased 28.6-fold, and coefficient of recovery was 19.7%. The molecular weight was 64.7KD determined by SDS-PAGE. The optimal reaction temperature of the endoglucanase was 53℃, and the optimal pH was 4.2. The values of Km and Vmax calculated from Lineweaver-Burk plots were 1.230×10^-2g/mL and 2.396×10^-2 mg/(mL · min) respectively.The reaction mechanism of carboxymethyl cellulose sodium(CMC-Na) hydrolyzed by cellulase from Trichoderma viride was brought forward on a series of assumption.The kinetic model was established by the influence of pH on endo-β-glucanase activity and the expression of reaction rate was educed, improving upon the "three-state" enzyme catalysis kinetic model. By changing pH of the buffer solution, a series of Km'and Vmax' of CMC-Na hydrolyzed by cellulase of Trichoderma viride were calculated from the Lineweaver-Burk plot. The optimal initial pH value gained by the kinetic model was consistent with data got from experiment. Consequently the kinetic model was validated.