麦芽糖酶-葡萄糖淀粉酶D246A异源表达及性质表征  

Heterologous Expression and Property Characterization of Maltase-Glucoamylase D246A

作　　者：高雨晴 董钢印 张洪瑞 马占山 方丽[1,2] 詹冬玲[1,2] GAO Yuqing;DONG Gangyin;ZHANG Hongrui;MA Zhanshan;FANG Li;ZHAN Dongling(College of Food Science and Engineering,Jilin Agricultural University,Changchun 130118,China;Laboratory of Biomass Active Ingredient Research and High Value of Agricultural Products,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林农业大学食品科学与工程学院,长春130118 [2]吉林农业大学生物质活性成分研究与高附加值农产品实验室,长春130118

出　　处：《吉林大学学报（理学版）》2024年第3期742-749,共8页Journal of Jilin University:Science Edition

基　　金：吉林省自然科学基金(批准号:20230101262JC).

摘　　要：以大型真菌灵芝中的麦芽糖酶-葡萄糖淀粉酶(MGAM)为研究对象,采用同源序列比对、同源模建、底物对接和定点突变等方法成功构建酶活力显著降低的突变体D246A.酶学性质表征结果表明:最适反应温度由野生型(wild type,WT)的65℃减少至60℃,突变体耐热能力下降;最适pH值由WT的6.0升高至7.0,有利于工程菌生长;半衰期由WT的2.0 h下降至1.5 h,酶稳定性降低.酶动力学结果表明:突变体D246A酶动力学曲线符合Michaelis方程,与WT相比,K_(m)值变大,表明酶与底物亲和力下降;V_(max)约降低至原来的1/4.The maltase-glucoamylase(MGAM)from the large fungus Ganoderma lucidum as the research object,and a mutant D246A with significantly reduced enzyme activity was successfully constructed by using methods such as homologous sequence alignment,homologous modeling,substrate docking,and site-specific mutation.The characterization results of enzymatic properties show that the optimal reaction temperature decreases from 65℃for wild type(WT)to 60℃,and the heat tolerance of the mutant decreases.The optimal pH value increases from 6.0 for WT to 7.0,which is beneficial for the growth of engineering bacteria.The half-life decreases from 2.0 h for WT to 1.5 h,the stability of enzyme decreases.The enzyme kinetics results show that the enzyme kinetics curve of mutant D246A conforms to the Michaelis equation,and compared with the WT,the K_(m)value increases,indicating a decrease in affinity of enzyme and substrate.V_(max)decreases to 1/4 of its original value.

关 键 词：定点突变 异源表达 性质表征 麦芽糖酶-葡萄糖淀粉酶 

分 类 号：Q556[生物学—生物化学]