Screening Saccharomyce Cerevisiae Mutation Strains With High-yield SAM, Fermentation With High Cell Density And Separation & Purification Of SAM

S-adenosyl-L -methionine (SAM, also known as AdoMet, SAMe) is an important intermediate metabolite that participates as a methyl group donor in many key biochemical reactions in all organisms. As medicine and hygienical food, SAM has attracted many interests in clinical research because of its potential to improve many diseases, such as liver disease, depression, and osteoarthritis. Since the research of SAM is still in laboratory interiorly, producing SAM on an industrialized scale can make great sense for our society and create tremendous economic benefits. Screening Saccharomyce cerevisiae mutation strains with high-yield SAM, fermentation with high cell density as well as separation and purification of SAM is studied in this paper.Saccharomyce cerevisiae which accumulates more SAM than other microorganisms is the main strain to produce SAM in fermentable industry. A yeast strain ZJUSC007 is obtained by evaluating SAM throughput of ten kinds of yeast strains. Its SAM concentration and content is 297.82mg·L^-1 and 37.76mggDCW^-1 respectively.One of the main metabolic pathways of SAM in Saccharomyce cerevisiae is thought to be the methylation reaction in the biosynthesis of ergosterol. Mutation strains deficient in ergosterol biosynthesis are acquired by Saccharomyce cerevisiae ZJUSC007 as the original strain with the method of ultraviolation mutation and the marker of nystatin-resistant. The best UV dose is 20s and the proper nystatin concentration is 15mg·L^-1. A mutation strain ZJUSCM186 with higher-yield SAM is got after screening from 364 mutation strains. Its SAM concentration and content achieves 450.07mg·L^-1 and 62.73mg·gDCW^-1, which is 42.24% and 57.02% higher than the original strain respectively. The culture conditions are optimized by single-factor experiments. It is shown that under the optimal condition: initial glucose 30g·L^-1, yeast extract 5g·L^-1 (NH4)₂SO₄ 5g·L^-1 K⁺ 0.15M, L-Methionine 1g·L^-1, transforming time 30h, the final SAM concentration and content can reach 582.59mg·L^-1 and 71.52mg·gDCW^-1Capabilities of accumulating SAM in three kinds of Saccharomyce cerevisiae are investigated by fermenting with high cell density. The result shows: SAM concentration and content as well as the cell density of mutation strain ZJUSCM186is very low, only 0.43g·L^-1, 6.6mg·gDCW^-1 and 55 respectively. ZJUSCM186 is not suitable as the strain to produce SAM by high cell density fermentation. Saccharomyce cerevisiae SC021, the SAM yield, SAM content and cell density of which achieves 3.0g·L^-1, 43mg·gDCW^-1 and 300 respectively, can be used to produce SAM by fermenting with high cell density. The fermentable process of recombinant Saccharomyce cerevisiae ZJUSCG054 which accumulates more SAM is similar to SC021 as the host strain.SAM is separated and purified by weak cation exchange resin HD-2. The optimal adsorption condition is: flow rate 2.0BV·h^-1, SAM concentration 10.11g·L^-1, pH5.0. Using sulphuric acid to elute SAM and the suitable elution condition is confirmed as follows: H₂SO₄ concentration 0.1mol·L^-1, flow rate 2.0BV·h^-1. Under these conditions, the HPLC purity of our product, elution and recovery efficiency can reach 98.16%, 91.78% and 71.29% respectively.