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| 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 |
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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.
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Received: 08 February 2017
Published: 25 August 2017
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