Endo-beta-glucanase. Cellulase Purification And Kinetic Study

Endo-β-glucanases(endoglucanases) were isolated and purified from a commercial preparation of Aspergillus niger and the leavening of Trichoderma reesei called after MJ1 by means of ammonium sulfate and Sephadex G100 chromatography,and further fractionated by FPLC respectively.The specific activity of the endoglucanase from Aspergillus niger increased 8.1-fold, and recovery coefficient reached 7.5%.The molecular weight was estimated as 26.4KD by SDS-PAGE.The optimal reaction temperature and pH of the endoglucanase were 55℃and 4.8 respectively.The values of Km and Vmax calculated from Lineweaver-Burk plots were 6.838×10^-3 g·mL^-1 and 2.906×10^-2 mg·(mL·min)^-1 respectively.The specific activity of the endoglucanase from MJ1 increased 28.6-fold,and recovery coefficient reached 19.7%.The molecular weight was estimated as 64.7KD by SDS-PAGE.The optimal reaction temperature and pH of the endoglucanase was 53℃and 4.2 respectively.The values of Km and Vmax were 1.230×10^-2 g·mL^-1 and 2.396×10^-2 mg·(mL·min)^-1 respectively.Glucose was added into the electrophoretically pure endoglucanase isolated from Aspergillus niger.Glucose protected effectively the activity during the preservation of solution of endoglucanase.A kinetic model was brought forward based on some speculation and validated.The decreasing of both entropy and enthalpy of deactivation owing to the adding of glucose indicated that glucose made endoglucanase thermodynamically more stable.The reaction mechanism of carboxymethyl cellulose sodium(CMC-Na) hydrolyzed by electrophoretically pure endoglucanase from MJ1 was brought forward on a series of assumption.A kinetic model of influence of pH on endoglucanase activity was established and the expression of reaction rate was educed.The optimal initial pH value gained by the kinetic model was consistent with data got from experiment,consequently the kinetic model was validated.Glucose,galactose,xylose,fructose and sucrose were added into the endoglucanase produced from MJ1 respectively.Both glucose and galactose brought competitive inhibition.Xylose and Surose brought noncompetive and uncompetive inhibition respectively.Fructose didn't affect endoglucanase.The active mechanism was discussed.The endoglucanase from Trichoderma viride was modified by methanol.The results indicated that the enzyme activity decreased resulting from the modification.Substrate and glucose weakened the inactivation degree of endoglucanase.According to FTIR,the carboxyl in the side chain redical of endoglucanase was modified by methanol.The kinetic analysis demonstrated that one carboxyl was essential group for endoglucanase activity. The second derivative and curve fitting results showed that the percentages ofβ-sheet andα-helix increased and those of turn and random decreased.