Molecular Modification and Whole-cell Catalytic Optimization of Bifunctional Glutathione Synthase

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. 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. GshFst was successfully expressed in E. coli, and the specific enzyme activity of the mutant GshFstL136K/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%. 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.