Cloning, Expression, Characteristics And Application Of Novel Bifunctional Glutathione Synthetase

Glutathione (GSH) is a significant non-protein thiol compound in organisms. It has many important physiological functions, such as antioxidation, detoxification and immunity improvement. In addition, it is benefit for the synthesis of protein, DNA and the transportation of amino acids. Nowadays, it has been widely used in medicine, food, cosmetic and other industries. In this work, some novel bifunctional GSH synthetase from different sources were cloned and expressed. Their enzymatic characteristics were studied. The recombinant strains were also constructed to produce GSH efficiently.The genes codding bifunctional GSH synthetases (GshFAS, GshFAP, GshFST) in Actinobacillus succinogenes, Actinobacillus pleuropneumoniae and Streptococcus thermophilus were cloned in this study. Using the sequences of GshFAS, GshFAP, GshFST and blasting in database,42protein sequences with high similarity were found. The strains containing GshF include G+strains and G-strains, and most of them belong to γ-Proteobacteria and Firmicutes. The phylogenetic analysis of GshF illustrated that there were five small clusters inculding γ-Proteobacteria cluster, Firmicutes cluster, Streptococcus cluster and other complicated clusters. By the alignment between GshFs and γ-glutamate-cysteine synthetase of Escherichia coli, it was found that almost all of the binding sites of substrates were conserved.The recombinant strains of E. coli BL-21/pET-28a-gshFst, BL-21/pET-28a-gshFap and BL-21/pET-28a-gshFas were constructed successfully. All of the three GshFs were over-expressed significantly. After purification, the characteristics of GshFs were studied. GshFAS had the highest specific activity among the three enzymes. GshFAS and GshFAP had similar characteristics with GshFPM(Pasteurella multocida), while GshFST was similar to GshFSA(Streptococcus agalatiae). The optimal pH and temperature were40℃and9.0for the three enzymes.All of the three recombinant strains could be applied to synthesize GSH with high yield and productivity. With higher concentration of substrates, BL-21/pET-28a-gshFas produced36.6mmol/L of GSH in hours. However, the initial concentration of ATP and Cys influenced the activity of recombinant cells and decreased the reaction rate. Glucose could be substitute for ATP and6.5mmol/L of GSH was achieved. The recombinant Pichia pastoris GS115/pGAPZA-gshFst was also constructed. Although the expression level of GshFST in P. pastoris was still low, the glutathione content in the recombinant cells was2.23times that in the host cells at96h with the repeatedly addition of Cys in the process of fermentation.