嗜热革节孢内切葡聚糖酶EGⅠ中精氨酸Arg7的功能分析  

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

作　　者：于伟帅 李多川[1] Yu Weishuai;Li Duochuan(Plant Protection College of Shandong Agricultural University,Taian 271018,China)

机构地区：[1]山东农业大学植物保护学院

出　　处：《山东农业科学》2020年第2期19-26,共8页Shandong Agricultural Sciences

基　　金：国家科技支撑计划项目(2015BAD15B05);国家高技术研究发展计划(863计划)项目(2012AA10180402);国家自然科学基金项目(31571949)

摘　　要：本研究将嗜热革节孢GH45家族内切葡聚糖酶EGⅠ中的底物结合位点精氨酸Arg7进行饱和突变,测定其性质、动力学参数的变化,以期分析精氨酸Arg7的功能。结果表明,与原酶相比,突变酶R7A的Km有所下降,R7P的kcat显著性提高,但突变酶的kcat/Km均显著性降低。原酶的最适反应温度为50℃,突变酶R7C、R7P和R7V的则为40℃,其余突变酶的均为50℃。原酶于70℃下处理2 h具有61%的活性,相同处理条件下,突变酶R7F、R7H仍具有70%以上的活性,说明其热稳定性明显提高,R7A仅有20%的活性,剩余突变酶均有40%左右的活性。原酶的反应最适pH值为5. 0,而突变酶R7C的为6. 0,剩余突变酶的均为5. 0。本试验探究了精氨酸Arg7饱和突变后嗜热革节孢GH45家族内切葡聚糖酶的性质变化,可为GH45家族进一步的分子改造和功能研究提供参考。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 Km of the mutant enzyme R7A decreased,and the kcat of the mutant enzyme R7P significantly increased,but the kcat/Km 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.

关 键 词：嗜热革节孢 内切葡聚糖酶 饱和突变 酶学性质 热稳定性 

分 类 号：Q783[生物学—分子生物学]