The Breeding Of High Glutathione Production Strains And Study Of Fermentation Condition

Glutathione is a bioactive tripeptide that widely found in nature.Glutathione can be biosynthesize from L-glutamic acid,L-cysteine and glycine.GSH has very important physiological fixnctions,therefore it is widely usedin clinical treatment,food,health,sports and other fields.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 attempted to improve GSH production of the beer yeast Pichia pastoris Y that preserved in the laboratory.The GSH yield of the initial strain Pichia pastoris Y was 81.43mg/L.we selected the inntial strain,using ultraviolet rays?UV?,l-methyl-3-nitro-1-nitroso-guanidin?NTG?and Na NO2 single factors,double factors and triple factors.The experimental results show that the triple mutagenic effect is best.The initial strain Y were mutagenized by continuous ultraviolet irradiation,the GSH production of the mutant stain named Y1 was 51.57% higher than Y.Then Y1 was continually mutagenized by nitrous acid,the GSH production of the mutant stain named Y2 was 4.7% higher than Y1,and the GSH production reached 147.53mg/L.At the end,a mutant stain named Y3 was obtained after Y2 was continually mutagenized by MNNG,GSH production reached 153.08mg/L,which was 3.76% higher than Y3.This paper optimized the fermentation medium and culture conditions of the mutant strains Y3.The optimum combination of fermentation medium components was glucose 20g/L,yeast3g/L,?NH₄?₂SO₄4g/L,KH₂PO₄4.0g/L,Mg SO₄1.5g/L.The optimal conditions was inoculation 9%,fermentation temperature 28?,the initial p H value of 5.5,medium volume 40ml/250 ml.In the end,the highest production of GSH was 169.88g/L,which was 108.6% higher than the starting strains Y.Through the experiment,the effect of adding precursor amino acids on GSH yield was investigated,the results showed that the influence of L-cysteine on the GSH yield was very significant.The GSH yield was 261.83 mg/L,which was 71.04% higher than the mutant strains Y43.After optimization of fermentation conditions,the final production of GSH ascended to 261.83 mg/L,which was 221.54% higher than the GSH yield of the initial strain Pichia pastoris Y.