High Throughput Screening Method and Application for L-glutamate Specific Aminotransferase / 中国生物工程杂志
China Biotechnology
;
(12): 59-65, 2017.
Article
in Chinese
| WPRIM
| ID: wpr-607577
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Type of study:
Diagnostic study
/
Screening study
Language:
Chinese
Journal:
China Biotechnology
Year:
2017
Type:
Article
Similar
MEDLINE
...
LILACS
LIS