High Throughput Screening Method and Application for L-glutamate Specific Aminotransferase
10.13523/j.cb.20170809
- VernacularTitle:谷氨酸依赖型氨基转移酶的高通量筛选方法及其应用
- Author:
Linwei HE
;
Zhangmin LIU
;
Yan FENG
;
Li CUI
- Keywords:
L-glutamate specific aminotransferase;
L-glutamate dehydrogenase;
High throughput screening;
Molecular evolution
- From:
China Biotechnology
2017;37(8):59-65
- CountryChina
- Language:Chinese
-
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 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.
