谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用

doi:10.13523/j.cb.20170809

中国生物工程杂志

2017, Vol. 37

Issue (8): 59-65 DOI: 10.13523/j.cb.20170809

技术与方法

谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用

贺霖伟, 刘璋敏, 冯雁, 崔莉

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

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

HE Lin-wei, LIU Zhang-min, FENG Yan, CUI Li

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China

全文: PDF(734 KB) HTML

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WecE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。

关键词： 谷氨酸依赖型氨基转移酶L-谷氨酸脱氢酶; 分子改造; 高通量筛选

Abstract: 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 WecE 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 site-directed 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.

Key words: L-glutamate dehydrogenase Molecular evolution L-glutamate specific aminotransferase High throughput screening

收稿日期: 2017-02-08 出版日期: 2017-08-25

ZTFLH:

Q819

基金资助: 国家"973"计划资助项目（2012CB721000）

通讯作者: 冯雁, 崔莉 E-mail: yfeng2009@sjtu.edu.cn;cuili@sjtu.edu.cn

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引用本文:

贺霖伟, 刘璋敏, 冯雁, 崔莉. 谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用[J]. 中国生物工程杂志, 2017, 37(8): 59-65.

HE Lin-wei, LIU Zhang-min, FENG Yan, CUI Li. High Throughput Screening Method and Application for L-glutamate Specific Aminotransferase. China Biotechnology, 2017, 37(8): 59-65.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170809 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I8/59

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