The Mutantion Creening Of Glutathione Production Strains And Fermentation Optimization

Glutatthione is a y-glutamoyl and thiol bioactive three peptide compounds, are divided into reduced glutathione (GSH) and oxidized glutathione (GSSG).GSH not only as a reagent is widely used in biochemistry, medicine, biology and chemistry study, but also in the pharmaceutical, food, feed and other industries has important use.A raw GSH producing saccharomycetes was isolated and screened on the YEPD screening plate with antibiotics.Twenty-seven GSH producing saccharomycetes(G1-G27) were screened and rescreened from the soil.The strain G4with the highest GSH producing was selected from Yunnan Province3,200meters above sea level,yieldding GSH119mg/L. In this study,we selected the wild type strain G4as the original strain,using ultraviolet rays(UV),1-methyl-3-nitro-1-nitroso-guanidin (NTG), ethylmethane sulfonate(EMS), diethyl sulfate(DES) single factors, double factors and triple factors.The result showed that the NTG was more sensitive to G4.But UV method was better.In this paper, according to the literature, we selected the ethionine, arginine and methionine, sodium azide as resistance screening. After single factor and double factors, three factors of mutagenesis resistance screening, at last,a selected mutant strain G-112-28which produce highest GSH,reaching332.2mg/L, about179%higher than original strain.The genetic stability experiment of mutant strain G-112-28showed that the GSH production of this strain was stable.When the liquid cultivated conditions of the mutant G-112-28optimized,we explored the influence of different carbon source,nitrogen source, inorganic salt and precursor on GSH production. The result showed that the carbon source in sugar,the nitrogen source in peptone.Inorganic salt K2HPO4、KH2PO4、MgSO4、ZnCl2at low concentration has a certain role in promoting GSH synthesis.Choosing the four factors sucrose,peptone,inorganic salt K2HPO4、KH2PO4、MgSO4applies orthogonal analysis.The optimal composition of fermentation medium for the mutant strain G-112-28is as follow(g/L):sucrose20, peptone50, K2HPO41.2, KH2PO41.8, MgSO43, ZnCl23, L-Cysteine6 mmol, L-Cysteine monohydrochloride2.5. At the same time, we researched the temperature,pH value,fermentation time,the volume of vaccination,and found that the container of25ml,pH value of7.0,the vaccination of10%,30℃,fermentation of48h,GSH production was the highest.The productivity of the new process with G-112-28reached402.3mg/L,which was21.1%higher than optimizing before and238%higher than the original process with G4.