Functional Analysis of Arg7 in Endo-glucanase EGⅠ of Scytalidium thermophilium

Yu Weishuai;Li Duochuan;Plant Protection College of Shandong Agricultural University;

Scytalidium thermophilium;;Endoglucanase;;Saturation mutation;;Enzymatic properties;;Thermal stability

Scytalidium thermophilium is a thermophilic fungus whose thermal stability and catalytic activity are the most important properties of industrial enzymes. In this study,the substrate binding site Arg7 in the endoglucanase EGⅠ of GH45 family was saturated mutated,and the changes of properties and kinetic parameters were analyzed. The results showed that only the K_m of the mutant enzyme R7A decreased,and the k_(cat) of the mutant enzyme R7P significantly increased,but the k_(cat)/K_m of the mutant enzyme significantly decreased compared with the wild enzyme. The optimum reaction temperature of WT was 50℃,while that of R7C,R7P and R7V was 40℃,and that of the residual mutant enzymes was 50℃ .The wild enzyme still had 61% activity after treatment at 70℃ for 2 h,and the thermal stability of the mutant enzymes R7F and R7H improved significantly,and more than 70% of the activity was observed after treatment; the activity of the mutant enzyme R7A was only 20% after treated at 70℃ for 2 h,and the other mutant enzymes had about 40% of activity.The optimum pH of WT was 5. 0,while the optimum pH of the mutant R7C was 6. 0,and the optimum p H of the other mutant enzyme was 5. 0. The above results could provide references for further molecular transformation and function study of the GH45 family.