Screening Of Higher Glutathione-producing Strain And Purification Of Its Metabolites

Glutathione （GSH） is a kind of active tripeptide, which is composed of glutamine,cysteine and glycine. Glutathione are extensively used in foods, medicine, health maintenance,anti-aging agents because of its important physiological function.The selection of S. cerevisiae YZM-14, the optimization of cultural condition, and theisolation and purification of glutathione were investigated in this paper.The S. cerevisia was determined as an initial mutant strain among the30different kindsof yeast. The dry cell weight （DCW）, the intracellular GSH content and the GSH productionwere respectively up to4.67g/L,6.51mg/g and30.40g/L. And the optimum condition of thepreparation and regeneration of the protoplast wre studied.S. cerevisiae YZM-14（ZnCL₂^r,Cys^r） was screened with the mutant processing of S.cerevisia protoplast by combinative mutagens of ultraviolet and nitrite. The glutathione （GSH）production （84.72mg/L）,dry cell weight （7.63g/L） and the intracellular GSH content （11.10mg/g） increased by178%﹑63.38%and70.51%respectively, compared with that of the initialstrain, and the productive characteristic of the mutant strain is stable.The metabolic flux of thepentose phosphate pathway and the GSH precursors biosynthetic pathway of S.cerevisiaeYZM-14increased by8.1%, compared with that of the initial strain. Furthermore, themetabolic flux of the succinic acids secretion of the mutant strain decreased. Through thesemechanisms, the utilization efficiency of the carbon sources was enhanced and highproduction of GSH was obtained.The medium composition for production of GSH by shaking flask cultivation wasoptimized by using the response surface methodology. The results showed that the optimumconditions of GSH production were2.544%of glucose,1.033%of yeast extract,0.5%ofammonia sulfate,5.88of initial pH, with loading volume of50mL in a250mL flask,fermentation temperature at28℃、0.1%of MgSO₄·7H₂O,1%of KH₂PO₄, fermentation time36h. The production of glutathione is125.42mg/L, which was increased by75.37%,compared with that of inorganic fermentation medium. Cysteine was determined as a key kind of precursor amino acids in this experimentation, and the addition concentration was0.4%,the intracellular GSH content and GSH production were increased to16.06mg/gand161.23mg/L respectively.The results showed that the optimum conditions of GSH extraction were40%of alcohol,120min of extraction time and20mL（water）/g（DCW） of liquid-material ratio. The optimalwater-solution preserved condition was pH3.0. GSH was isolated and purified from the S.cerevisiae extracts with the stepwise elution strategy by using the strongly acidiccation-exchange resin001×7（732）. The less retention volume and the enhanced GSH puritywere obtained. After isolation and purification, the recovery rate and the purity for the isolatedGSH were up to61.15%and84.71%respectively.