Study On The Mechanism And Process Of The Biosynthesis Of S-adenosyl-L-methionine And Glutathione By The Yeast Cells

The biosynthesis of S-adenosyl-L-methionine ( SAM-e, AdoMet or SAM) and glutathione(GSH) was examined in this work. Also, the separation and purification processes for SAM as well as the preparation of stable SAM salt were studied.Based on the review in literature and analysis of the properties, pharmacology and applications of SAM and GSH, the production and separation processes were evaluated in detail. The major achievements are as followings: 1 . With the addition of L-methionine and glucose, the spent yeast cells from brewers were used for the co-production of SAM and GSH. The most suitable conditions for SAM accumulation are: temperature 27-30癈, pH6.0 and K+ concentration 0.1 2M. When the addition of L-methionine is higher than 0.7g/l, in shake flask cultivation, the SAM accumulation in the yeast cells can reach 25-40 mg/g or 500-800mg/l, and the conversion ratio is up to 30%. Cultivation carried out in a 15-liter fermentor, with the aeration rate of 3.0 vvm and the additions of L-methionine and glucose were 1.0 g/l and 30 g/l, showed the accumulation amounts of SAM and GSH were 45 mg/l and 17.0 mg/l respectively, and the L-methionine convertion ratio was 44%. China is a major beer producer in the world, and there is abundant supply of spent brewer's yeast cells, therefore, the comprehensive utilization of spent yeast cells to produce SAM and GSH will be favorable to increase the economic value. The process is hopeful for commercial production.2. A strain of Saccharomyces cerevisiae ZJUS1 was screened out with high ability to accumulate SAM. A two-step process consisted of cell growth and bioconversion was developed. In which, the yeast cells was cultured for 24 hours, then, L-methionine and glucose were added directly into the cultivation broth for SAM and GSH biosynthesis. The optimal supplements for bioconversion step are: 1 .0 g/1 L-methionine and 30 g/1 glucose. And the optimal initial pH value for SAM and GSH biosynthesis are pH 5.0 and 6.0 respectively. In a 15-liter fermentor, thetwo-step processes were evaluated and the results showed that higher dissolved oxygen level and higher cell density were beneficial for SAM biosynthesis, whereas the GSH biosynthesis was only happened in the late stage of SAM accumulation. When the dissolved oxygen concentration was higher than 40%, the conversion ratio L-methionine was 37.4%, and the productivities of SAM and GSH reached 82.2 mg/g(or 1003.9 mg/1) and 9.2 mg/g(or 140mg/l), respectively. The experimental results indicated that the ability to biosynthesis SAM by freshly cultured yeast cells is much higher than the spent brewer's yeast cells, but less for GSH biosynthesis.3. The mechanism of biosynthesis of SAM and GSH, the importance of energy metabolism on the biosynthesis, and the effects of dissolved oxygen on the aerobic respiration and anaerobic metabolism of yeast cells were discussed and analyzed in detail. Then, an overflow metabolism model was proposed to describe the kinetics of batch culture including cell growth, substrate consumption and products formation. The model simulations were in good agreement with the experimental data. The model analysis can be used to further improve the process technology for SAM and GSH production, also, the information offered from the model will be helpful for the strain improvement by metabolic engineering.4. It was found that picric acid was a good substitute of traditional picrolonic acid for SAM precipitation. The intracellular SAM was extracted by ethyl acetate solution and sulfuric acid solution with a recovery of more than 90%. Then SAM precipitation was carried out by picric acid and SAM precipitated is higher than 90%. After butanone extraction, acetone precipitation and SAM sulfate or SAM double salt of sulfate and p-tosylate formation, the stable SAM salt was obtained. The purity of SAM product was higher than 95%, and the overall recovery of SAM sulfate and SAM double salt of sulfate and p-tosylate were 60% and 50%, respectively.5. A weak acid cationic exchange resin, Zh...