Studies On The Production Of S-Adenosylmethionine By Biosynthesis

There are three ways to produce adenosyl-L-methionine, including chemical synthesis, microbial fermentation and enzymatic synthesis. Chemical synthesis is carried out with S-adenosylhomocysteine and CHsI as the substrates. Adenosyl-L-methionine can be divided into (-)-diasterioisomer and (+)-diasterioisomer according to the configuration. Only (-)-diasterioisomer has the active activity. In the product of chemical synthesis, there is a little (+)-diasterioisomer and it is not easy to be separated. Microbial fermentation was carried out with L-methionine as the substrate in the medium. It had been the main way to produce (-)-diasterioisomer with the fermentation of yeast. Compared to chemical synthesis and microbial fermentation, Enzymatic synthesis has the virtues like high aim product concentration, easily separated, short reacting time and no pollution. Therefore, it has the effective production way hi the industry. Enzymatic synthesis is to use the enzyme, S-adenosylmethionine synthetase (also called ATP: L-methionineS-adenosyltranserase, EC 2.5.1.6), with L-methionine and ATP as the its substrates.In this experiment, 587 strains, which could produce SAM, were screened from the soil picked from all over Zhejiang Province. In these strains, 8 strains were selected with high yield of SAM, especially Qian-95, with 956 y g/ml broth.In the optimization of culture conditions, the initial broth pH, fermentation temperature and the concentration of L-methionine in the broth were examined. The optimized conditions were pH 5.0, temperature 30℃ and substrate concentration 1%. In the optimization of media, the C source, N source, P source and mineral ion were studied. The optimal C source was 8% sucrose, N source 0.5% NRtNOs, P source 0.08% KH2PO4 and 0.016%K2HP04 and mineral ion 0.01%MgSO4 ?7H20, 0.03%CaCl2,0.04%FeSO4 '7H20 and 0.01%LiCl. Also the experiments were carried to investigate the fermentation of SAM in the airlift bioreactior and mechanically stirred fermentor. The results showed the yield of SAM in the airlift bioreactor washigher than that in the mechanically stirred fermentor.The yeast cell disruptions were researched with abrasives, ultrasonic and toluene. The results of disruption was the best with ultrasonic. The operation conditions were 22 KHz and 15 minutes and 0.934 U/ml was obtained as the enzyme activity. It was investigated that the optimal reaction conditions of S-adenosylmethionine synthetase were reacting time 20 min, temperature 28-30℃, pH7. 8-8. 3, L-methionine 5u M and ATP 5 u M. But with the increase of the concentration of ATP, the enzyme activity was inhibited. The penetrating ability of the yeast cell walls could be increased by adding detergents, such as SDS and Tween-80. The results of Tween-80 was better than that of SDS. The optimal concentration of Tween-80 was 0.10g/5g cells and the rate of SAM penetration was 62.3%. The treated cells were immobilized with entrapment of calcium alginate. The optimal enzyme reacting conditions of immobilized cells were temperature 30 ℃ andpH 7.8-8.2. Compared to the free enzyme, the immobilized cells were less sensitive the environment. The half life period of free enzyme was 32 days at the room temperature.The isolation of SAM was established and a experimental sample of SAM salts of sulphuric acid and p-toluenesulphonic acid, which had the purity of 98.2%, was obtained.