谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用  

High Throughput Screening Method and Application for L-glutamate Specific Aminotransferase

作　　者：贺霖伟 刘璋敏 冯雁[1] 崔莉[1] 

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240

出　　处：《中国生物工程杂志》2017年第8期59-65,共7页China Biotechnology

基　　金：国家"973"计划(2012CB721000);上海市科委基础研究重大重点项目(14JC1403500)资助项目

摘　　要：目的:建立谷氨酸依赖型氨基转移酶-谷氨酸脱氢酶偶联反应的96孔板高通量筛选方法,并用于大肠杆菌氨基转移酶Wec E突变库的筛选。方法:通过优化偶联指示酶-谷氨酸脱氢酶、信号分子NADH浓度及双酶偶联反应时间,建立了光学法测定氨基转移酶活性的氨基转移酶-谷氨酸脱氢酶偶联反应方法;通过定点饱和突变技术构建了大肠杆菌氨基转移酶WecE的突变库;采用96孔板高通量初筛、摇瓶复筛获得了高活性的转氨酶突变体,并对纯化的突变体进行催化活力分析。结果:建立了谷氨酸依赖型氨基转移酶目标反应与0.5 U/ml L-谷氨酸脱氢酶和0.4 mmol/L NADH信号指示反应相偶联的筛选方法;构建了氨基转移酶WecE Tyr 321饱和突变库,通过96孔板高通量筛选,获得了催化活性比野生型提高3.4倍的突变体Y321F。结论:所建立高通量筛选方法背景干扰小,准确性高,为谷氨酸依赖型氨基转移酶分子进化提供了可行性方案。Objective： The aim is to establish L-glutamate specific aminotransferase-L-glutamate dehydrogenase coupling 96-well high throughput screening method,which is applied to molecular evolution of aminotransferase Wec E from E. coli. Methods： An optical assay for aminotransferase catalytic activity based on aminotransferase-glutamate dehydrogenase coupling system is established by optimization of coupling enzyme loading,signal molecule NADH concentration and coupling time. Mutants library of WecE is obtained by sitedirected saturation mutagenesis. Positive mutants can be screened out through 96-well preliminary screening and flask second screening. Results： The target transamination reaction is coupled with L-glutamate dehydrogenase indicative reaction system which consists of 0. 5 U/ml enzyme loading and 0. 4 mmol/L NADH. A positive mutant Y321F whose catalytic activity increases 3. 4 fold compared to that of wild type is screened out in Tyr 321 saturation mutagenesis library of WecE. Conclusion： An accurate high throughput screening method with weak background interference is established. It offers feasible solution for molecular evolution of L-glutamate specific aminotransferase.

关 键 词：谷氨酸依赖型氨基转移酶 L-谷氨酸脱氢酶 高通量筛选 分子改造 

分 类 号：Q819[生物学—生物工程]