The Breeding Of High Glutathione-Producing Strains And Optimization Of Culture Condition

The breeding of high glutathione-producing yeast strain and optimization of cultivation conditions were studied.Colorimetric assays for reducing glutathione by Alloxan method, Sodium Nitroprusside method and DTNB method were compared and modified. The result showed that Alloxan method and DTNB method could avoid the disturb of cysteine.DTNB method was more easily and economic。The biomass and GSH content of 295 yeast strains from different species and genera were tested. Diploid strain Saccharomyces cerevisiae Y64 with high biomass and S. uvarum Y247 with high GSH content were chosen as parental strains. Haploids Y64-32 (a) and Y64-14 (a) with high biomass and Y247-6 (() with high cell GSH content were selected through determination of the mating- type, the biomass and the GSH content of the haploids. The three haploid strains were mutagenesised by DES respectively. The mutant Y64-32-76 (a, Lys-) and Y247-6-21 (a, Met-) were chosen as fusion parental strains. The fusant ZJF-71 were selected with higher GSH yield than the parental strains. The GSH content of ZJF-71 was 1.85mg/g wet cell.The mutant Y64-14- 2(α, Ura-Leu-) and Y247-6-7 (a, Arg-) were chosen as hybrid parental strains. The hybrid YHB-12 were selected with higher GSH yield than the parental strains. The GSH content of YHB-12 was 1.49mg/g wet cell. The genetic stability of them were determined and result show they are stable enough in genetic and haven't the separation phenomena of auxotrophic markers. The effect of medium composition and culture condition on the production of glutathione by fusant ZJF-71 and hybrid YHB-12 were studied in shaking-flask culture. The effect of factors such as carbon source, nitrogen source, inorganic salt, that affected the GSH productivity of ZJF-71 and YHB-12 were tested. The optimal fermentation conditions for ZJF-71 which were determined by direct cross experiment was: 2% glucose, 0.7% yeast extract, 0.6% (NH4)2SO4, 0.05% MgSO4?7H2O,0.1% KH2PO4, 0.25% K2HPO4?3H2O, 0.015% CaCl2(2H2O, initial pH 6.0. The effect of amino acids were also tested: cysteine and methionine improved GSH content obviously, glutathine improved biomass. The optimal fermentation conditions for YHB-12 was: 2% glucose, 0.7% yeast extract, 0.6% (NH4)2SO4, 0.05% MgSO4?7H2O, initial pH 6.6. Among the amino acids, cysteine and methionine improved GSH content obviously, glutathine improved biomass.Batch fermentation of glutathione was carried out in an aerated stirred-tank. A typical batch process of glutathione production by ZJF-71 was discussed. Compared with the shaking-flask culture, batch fermentation of glutathione in an aerated stirred-tank had some advantages.