Semi-rational Modification Of Glutathione Bifunctional Synthetase And Whole Cell Catalytic Synthesis Of Glutathione

Glutathione（GSH）has many important physiological function and is broadly applied in pharmaceutical,food and cosmetics industries.Glutathione bifunctional synthetase（Gsh F）can simultaneously catalyze the two steps of GSH synthesis,however,Gsh F still has the problem of low catalytic efficiency and poor stability.The purpose of this study was to improve the enzyme activity and stability of Gsh F.Firstly,two different sources of Gsh F were heterologous expressed in Escherichia coli and we demonstrated that the catalytic activity of that derived from Streptococcus thermophilus was higher.On this basis,site-directed mutagenesis was carried out to improve the enzyme activity,and the reason of enzyme activity improvement was analyzed by enzymatic properties,structure analysis and molecular docking analysis.Finally,whole cell catalysis was carried out and the process was optimized.In this study,mutants with improved enzyme activity and thermal stability were obtained by site-directed mutagenesis,which provides reference for engineering Gsh F by using semi-rational modification,and lays a good foundation for the catalyzed synthesis of GSH in industrial scale.The main research contents and results are as follows:（1）In this study,E.coli BL21（DE3）was used as chassis cells,and Gsh F derived from Streptococcus sanguis and Streptococcus thermophilus was heterogeneously expressed,respectively.After the successful construction of strains,induced expression and soluble expression were achieved successfully.Then the synthesis ability of Gsh F from two different sources was compared by whole cell catalysis,the production of BL21（DE3）/p ET28a-gsh Fst and BL21（DE3）/p ET28a-gsh Fss were 5.45 mmol·L^-1 and 5.06 mmol·L^-1,respectively,and it was found that the synthesis ability of Gsh F from S.thermophilus was stronger.On this basis,the expression level of Gsh F was optimized by changing the RBS sequence,the results showed that only the enzyme activity of RH10 and RH20 among all strains increased by 3.88%and5.77%compared with the wild type,so the original RBS was still selected for further work.（2）In order to further improve the activity of Gsh F,the strategy of semi-rational modification was explored.Firstly,the basic physical and chemical properties of the protein were analyzed by bioinformatics,Alpha Fold 2 was used to predict the three-dimensional structure of the protein,and then the mutation site was predicted by Hot Spot Wizard.Semi-rational design and site-directed mutagenesis of Gsh F were carried out,and a mutant S722A with improved enzyme activity was obtained.Compared with wild type,the enzyme activity increased by 48.93%.Enzymatic properties showed that the optimum reaction temperature of S722A was 45℃and the optimum reaction p H was 9.0,which was consistent with that of wild enzyme,but the thermal stability of S722A was further improved than that of wild enzyme.Through molecular docking of S722A,WT and ATP,it was found that the number of hydrogen bonds increased after mutation,which may improve the enzyme activity and thermal stability.In addition,S722 is located near the substrate channel,which may be due to the mutation of this site to alanine with the smallest side chain,which broadens the channel,facilitates substrate entry and product release,and thus improves the enzyme activity.（3）The whole cell catalysis of mutant S722A was performed and the catalytic conditions were optimized.The whole-cell catalytic capacity of the mutant was compared with that of the wild type.After 2 h reaction,the production of GSH achieved by S722A was 20.10 mmol·L^-1,which was 21.30%higher than that achieved by the wild type(16.57 mmol·L^-1).The cysteine molar conversion rate of S722A was 100.5%,which was 17.65%higher than that of wild type（WT was 82.85%）.Furthermore,the catalytic conditions of S722A were optimized,the results showed that the optimal p H and temperature were 8.0 and 37℃,respectively.The biomass had little effect on the whole-cell catalytic synthesis of GSH.The production of GSH achieved by S722A was 28.03 mmol·L^-1 under the condition of high substrate concentration,which increased by 86.12%at lower concentration.The effect of feeding on whole-cell catalytic synthesis of GSH was preliminically explored.Under the condition of low substrate concentration,the production of GSH was 15.03 mmol·L^-1 and unit OD production was 0.50mmol·（L·OD）^-1 at 2 h.After the same substrate concentration was added,the GSH production was 22.22 mmol·L^-1 after 2 h reaction.The unit OD production was 1.48 mmol·（L·OD）^-1,which was increased by 196.00%compared with no feeding.