Study On The Expression Of New Glutathione Synthase By Recombination Escherchia Coli And The Preliminary Separation And Purification Of Glutathione

Glutathione, the tripeptide y-L-Glutamyl-L-Cysteinyl-Glycine, is one of the important antioxidant molecules, which plays a great role in medical, food, sports and other fields. GSH is synthesized via two steps. The dipeptide γ-glutamylcysteine (γ-GC) is firstly formed from L-glutamic acid and L-cysteine and catalyzed by γ-glutamylcsteine synthetase. Glycine is ligated to the C-terminal site of y-GC to form GSH by gluthathione systhase. Recently a new glutathione synthase GshF is isolated from Streptococcus thermophilus. In this study, the recombinant Escherichia coli BL21containing gshF from Streptococcus thermophilus was investigated in flasks and5L fermentor. Different inducers were applied and the effects of incution dosage, time, temperature, and feeding on cell growth, GshF expression and specific enzyme activity were conducted with different media.The results showed that using auto-induction medium the cell growth and expression of GshF at30℃were better than those at37℃. However, using chemically defiend medium affected cell growth and protein expression. The addition of yeast extract into chemically defiend medium would increase the stability of GshF and result better performance at37℃than30℃. When glycerol was used as feeding mudium, the cell grew slowly and there was no GshF expressed. After the process optimization, the cell density reached at96.8and GshF expression was2g/L, where glucose was used as feeding carbon source and0.05mmol/L of IPTG was added when the cell density reached at50.