Breeding Of An Over Producing Glutathione Strain

Glutathione is a bioactive tripeptide that widely found in nature. Glutathione can be biosynthesize from L-glutamic acid, L-cysteine and glycine. Generally, glutathione exists in living organisims with reduction forms, so glutathione commonly refers to reduced glutathione(GSH). GSH has very important physiological functions, therefore it is widely used in clinical treatment, food, health, sports and other fields. Some foreign companies began to research about improve GSH production and industrial production of GSH from the1970s. Although China also began to research and industrial production of GSH, but for the reason that the production and purity of GSH far less than foreign products. Thus the situation of monopoly of foreign products in China market has not changed until now. Against this background, this paper attempts to improve GSH production of the beer yeast (Saccharomyces cerevisiae)that preserved in our laboratory.First, KZY208-3was selected as parent strain which GSH production was32mg/l. The ultraviolet(UV) mutagenesis method was used to the parent strain and screened UV mutants and zinc chloride resistant mutants. Among them, GSH production of the KRZZ-13is higher than others. Its production of GSH reached62.8mg/l and95.6%higher than the parent strain.Then, a mutant KRN-41was obtained by treating MNNG to the stable genetic mutant KRZZ-13. The productivity of KRN-41reached69.7mg/l and improve the rate of117.1%. KRZN-37was obtained by UV and MNNG combined treatment to the KRN-41and triazole, sodium azide drug resistance screening. The productivity of KRZN-37reached162.0mg/l that404.7%higher than parent strain and the wet weight of KRZN-37was58.5g/l. Genetic stability test shows that KRZN-37can be stably inherit.The medium and cultural conditions of KRZN-37have been optimized. Through single factor designs and orthogonal designs show that the optimum medium composition was sugar20g, beef extract15g, L-cysteine2g,(NH4)2SO46g, NaCl2g,MgSO40.1g, K2HPO41.5g, KH2PO41.5g, distilled water1000ml.Optimum fermentation condition was fermentation temperature32℃,inculum size10%, medium volume30ml/250ml, fermentation time30h.The GSH production of KRZN-37reached173.2mg/l which higher than parent strain439.6%and the wet cell weight of KRZN-37reached53.3g/l after using of optimum medium in optimum culture condition.