Molecular Modification and Whole-cell Catalytic Optimization of Bifunctional Glutathione Synthase
Objective:GshF derived from Streptococcus thermophilus was expressed in Escherichia coli and site-directed mutagenesis was performed on GshFst to improve the enzyme activity and stability of GshF,and to optimize the whole-cell catalytic conditions of this mutant.Methods:Six different plasmids were tested to find the most suitable one,and then a one-factor test was used to optimize the induction conditions;on this basis,GshFst was modelled and molecularly docked using AlphaFold2,and the mutation sites were obtained by prediction.After single-site mutagenesis and combinatorial mutagenesis,the double mutant GshFstL136K/V498C with improved catalytic conditions was obtained.Results:GshFst was successfully expressed in E.coli,and the specific enzyme activity of the mutant GshFst L136K/V498C was 12.03 U/mg,which was 86.80％higher than that of the wild type.The half-life at 37 ℃ was 134.24 minutes,which was 40.95％higher than that of the wild type.Finally,the whole-cell catalysis process of the mutant was optimized to determine the optimal conditions for whole-cell catalysis:biomass of OD600=30,reaction temperature 40 ℃,reaction pH 9.0,buffer Tris-HCl 50 mmol/L,L-glutamate 40 mmol/L,L-cysteine 25 mmol/L,glycine 40 mmol/L,and ATP 25 mmol/L.After catalysisfor 3 h,the concentration of GSH can reach 24.17 mmol/L,and the conversion of substrate L-cysteine was 96.68％.Conclusion:The suitable plasmid for GshFst and the induction expression condition were obtained,the enzyme activity and stability of GshFst were improved by semi-rational modification,and the whole-cell catalysis process was obtained by optimization.
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