Study On Fermentation Conditions Of Recombinant Pichia Pastoris And Preliminary Separation And Purification Of SAM

S-adenosyl-L-methionine is an important metabolic intermediate in organism, which is involved in many biochemical reactions. Its therapeuctic usage could be extensive and varied. Besides its metabolic functions, clinical applications and advance of pharmacological research, process of preparation are emphatic reviewed. In the light of its intrinsic instability, its degradation mechanics, stabilize theory and novel stable salts with polyanions are also introduced.SAM has been used as a prescription drug for many clinical symptom. Although there is supply of SAM as a biochemical regeant in our country, it is too expensive to apply in clinic. The key for generalizing clinical application of SAM is large-scale and economical productive technology of SAM.Fermentation of microorganism and extracting SAM from the cell is the main industrial production process at present. SAM is always produced by fermentation of S.cerevisiae. By changing Pichia pastoris's metabolic path rationally, a constitutive recombinant P. pastoris SIBAS 5071 with riched SAM synthetase was obtained on the basis of research of sulfide's metabolism in yeast in our laboratory. Study on the fermentation conditions of this recombinant P. pastoris in flake level has been performed. Effects of fermentation, such as carbon source, reactant, pH, phosphate buffer, dissolved oxygen on the cell growth and SAM production were considered. The optimum medium is 20 g/L glycerol, 20 g/L peptone, 10 g/L yeast extract, 0.075 mol/LL-methionine, 0.05mol/L phosphate buffer, initial pH 6.0. Medium volume is 40 ml. Under the optimized fermentation conditions, the production of SAM reaches 2.3 g/L (118mg/g dry cell) after three days of fermentation in flask level. P. pastoris possess the advantage of high density fermentation, which hasn't been realized in flake level. The potential productivity of SAM of this recombinant P. pastoris is huge.Our study shows that optimum fermentation condition increases the productivity of SAM of recombinant P. pastoris, therefore the purification technology of SAM could be simplified Our study also provides the necessary information for fermentation in fermentor.SAM is synthesized in the cytosol of every cell. SAM has three ionizable groups besides thesulfonium pole: the carboxyl and the aminic groups of the amino acidic chain with pK 1.8 and 7.8 respectively and the aminic group of the purine with pK 3.4. In an acid environment SAM is positive charged, so it could be adsorbed by weak-acid cation exchange resin. Through reverse adsorption and elution, SAM could be separated from the cell extract.According to the charge of SAM, the processs as follows is adopted . We extractct SAM from the cell by the perchloric acid, then neutralize the extraction with KHCO3. Using the weak-acid caion exchange column chromatography we separate SAM from the mixure. Temterature and pH is controlled well, so SAM has not been decomposed in the experiment. Through the preliminary purification we obtain a pure solution of SAM-HSO4. Recovery ratio is above 90%, content of (S, S)-SAM could reach 95%.The steps of separation and purification of SAM are always complex. When the content of SAM in the microbial cells is lower than a certain level, purification of SAM is very hard to be reached. We prepare SAM from the productive recombinant P. pastoris, the technology could be simplified..