Enzymatic Synthesis Of Glutathione Catalyzed By Yeast Cells

Glutathione is a tripeptide compound peptide bond simultaneously has y-glutamyl and thiol two bioactive tripeptide condensation by L-glutamic acid, L-cysteine and glycine. These two structures is the structural basis of many important physiological functions in glutathione. For instance it has a y-glutamine bonds involved in the amino acid transporter, glycine and cysteine residues involved in bile acid metabolism, thiol in molecules to participate in free radical detoxification and other important physiological functions which has important applications in the food industry and pharmaceutical. The main contents of this thesis are as follows.1. The experiment have collected and cultured nine yeast strains., The cell biomass and intracellular GSH content have been measured respectely. The cell dry weight of Saccharomyces cerevisiae SP004reached11.39g/L, and the intracellular GSH content of brewer’s yeast TS013reached1.5282mg/g wet cells. The effects of four different extraction methods on the determination of intracellular GSH content in yeast cells has been investigated, the results showed that the temperature crushing to yeast cells was the extracting GSH optimal method. Meanwhile the GSH synthesis catalytic activity of9yeast trains has been analysed, the results showed that the GSH synthetase activity of flocculation yeast SP5was relatively higher and reached2.385mg/g wet cells, the GSH conversion rate of precursor amino acids was7.76%per gram of wet cells.2. By using single-factor experiment investigated the effect s of different ratio of reaction solution volume, different concentrations of MgSO4·7H2O, potassium phosphate buffer, and glucose, different reaction temperature and oscillating reaction rate. Orthogonal test analysed the effects of different precursor amino acids ratios, different concentrations of MgSO4-7H20, potassium phosphate buffer and glucose.The results showed that appropriate conditions for synthesis of GSH is Glu60mmol/L,Cys20mmol/L and Gly20mmol/L, MgSO4·7H2O20mmol/L, glucose0.5mol/L, pH7.0potassium phosphate buffer100mmol/L,30℃,200r/min, oscillation reaction time6h. Under above conditions, the average GSH synthetase activity of flocculation yeast SP5reached2.9498mg/g wet cells, and the GSH conversion rate of precursor amino acids was9.60%per gram of flocculation yeast SP5wet cells, which has been improved23.68%than that of initiate reactive conditions.3. Single factor experiment was carried out to analyze the effects of several factors on the cell biomass of flocculating yeast SP5and the GSH synthesis, the factors were as follow:fermentation temperature, initial pH value, flask fluid volume, inoculum size, carbon sources, organic nitrogen sources and inorganic nitrogen sources. Orthogonal test analysed the interactions of carbon sources, organic nitrogen sources and inorganic nitrogen sources. The fermentation conditions of flocculating yeast SP5was optimized.Under this conditions, incubation temperature30℃, initial pH value7.0, flask fluid volume20%(250mL erlenmeryer flask), inoculum size5%, medium composition:sucrose30g/L, peptone25g/L, ammonium sulfate5g/L, KH2-PO41.5g/L, MgSO4·7H2O0.5g/L, inositol0.1g/L, the GSH conversion rate of precursor amino acids reached10.98%per gram of wet cells which has increased14.38%than that of the initial conditions, and the cell dry weight was10.47g/L.4. In order to improve the cell permeability of flocculating yeast SP5, the permeating effects experiments of surfactants Triton X-100, Tween80, and SDS in different concentration of0.01%,0.1%,1%were investigated. The results showed that the concentration of1%Triton X-100was the best under the30℃treatment for2h. Under optimized conditions0.5%Triton X-100,30℃treatment for2h, the GSH conversion rate of precursor amino acids reached14.65%per gram wet cells which has increased33.42%than that of the initial conditions.