Extraction And Purification Of Glutathione From Yeast Cells

Glutathione (GSH) is a tripeptide with special active groups (thiol and γ-glutamoyl), it composed by L-glutamic acid, L-cysteine and glycine. The thiol in GSH can combine with all kinds of free radicals in the body, maintaining appropriate oxidizing environment in organisms. It can also exert detoxification effect by complexing with heavy metallic salt, including lead and mercury. In addition, GSH possess the functions in enhancing immunity, anti-aging and anti-radiation damage. GSH is widely used in food, medicine, cosmetics, which posses the great value and broad application prospects. In this thesis, the separation and purification process of GSH from Saccharomyces cerevisiae SP5were investigated. The main results were described as follows:1. Through the single-factor experiments, the effect of factors such as carbon source, nitrogen source, L-cysteine and Tween80that affected the GSH productivity of Saccharomyces cerevisiae SP5were tested. The optimum medium obtained from orthogonal experiment were:glucose40g/L, yeast extract12g/L,(NH4)2SO45g/L KH2PO42g/L, MgS04·7H2O0.5g/L, inositol0.1g/L,4mmol/L L-cysteine and0.1g/L Tween80were added after6h of fermentation. Under the optimum conditions, intracellular GSH reached1.97mg/g, which increased by21%compared with the initial conditions (1.62mg/g).2. Comparison of the six kinds of extraction methods (ethanol, SDS, hydrochloric acid, boiling water bath, microwave, sodium chloride) of GSH in yeast cells. Orthogonal experiment based on single-factor analysis of ethanol concentration, temperature, time, and the amount of ethanol was studied. Results showed the optimal conditions of ethanol extraction method was that per gram of wet cells was mixed with7ml50%ethanol at40℃for7min. The GSH extraction reached2.21mg/g, increased by82.64%compared to that of boiling water extraction (1.21mg/g).3. Three adsorption resins, including001×7, D001, D301were used to separate glutathione from the extract of yeast cells. The effects of eluant (ammonia, ammonia in ethanol, hydrochloric acid and sodium chloride) concentrations, column height of ion exchange column (02.5cm×30cm), sample flow rate and elution flow rate on the adsorption and elution of glutathione were investigated, respectively. The results showed that D301resin had relatively high absorption capacity of glutathione, the absorption rate of which reached84.63%. Using a combination of60%ethanol and10%ammonia as the eluant achieved an elution effect as high as85.51%. When the column height of D301resin was20cm, sample flow rate1.0ml/min, elution flow rate2.0ml/min, the yield rate of glutathione separation reached62.3%.4. The high performance liquid chromatography was used to analyze the GSH extracts purificated by resin D301, the loss rate and concentration ratio of GSH extracts were detected by vacuum-concentration experiment. The temperature and time of concentration were optimized to determine the best vacuum concentration condition. Results showed that the GSH extracts isolated by resin D301possessed the least miscellaneous peaks, and the GSH purity was as high as68.3%. The extract concentration factor could reach4.23folds under the conditions of0.095MPa,55℃for60min.