Side-chain Cleavage Of Phytosterols B Mycobacterium Sp.MB3683

Side-chain cleavage of phytosterols is a main field of steroids transformation, in which products of androst-4-ene-3,17-dione (AD) and androsta-l,4-diene-3,17-dione (ADD) play an important role in steroid pharmaceutical industry and almost all of steroid hormone drugs could be derived from them. Since biotransformation has its advantages in product conversion and purity over chemical synthesis, the production of AD(D) and other intermediates by microbial fermentation has drawn great attention in side-chain cleavege of phytosterols. However, the extremely low solubility of steroids substrate in aqueous solution is a bottleneck that restricts the wide application of bioconversion. This work will focus on the side-chain degradation of phytosterols by Mycobacterium sp. MB3683to increase its solubility so that the bioconversion could be achieved at high substrate feeding concentration.Firstly, a reliable analytical method using HPLC was established, and growth curve of Mycobacterium sp. MB3683in fermentation medium was also determined. By the optimization of culture medium composition and culture condition such as carbon source, nitrogen source and pH, it was found that high carbon source concentration had a negative effect on biotransformation. Although (NH4)2HPO4had a slight influence on reaction, it was important to maintain the constant pH. Favorable fermentation time was ascertained to be168h.Secondly, different organic solvents and cyclodextrins and their derivatives were selected as the solubilizer in an effort to investigate substrates preferences for given solubilizer; other parameters such as feeding time and the amount of solubilizer used in bioconversion were also studied. The results showed that phytosterols (mixture of sterols) was one of the most suitable substrates for producing AD. Different substrates had no their preferences for given solubilizer. When organic solvents were as solubilizer, ethanol and acetone obtained better results. As for cyclodextrins and their derivatives, the methyl-β-cyclodextrin was the best candidate while the concentration of phytosterols was increased to15g/L, the yield of AD could still be over60%at optimized feeding time and the ratio of methyl-β-cyclodextrin to phytosterols (2:1), favorable fermentation time was ascertained to be96h.Finally, eight kinds of vegetable oils, i.e. sunflower, peanut, corn, olive, linseed, walnut, grape seed and rice oil were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols, and the logD of AD in different oil/aqueous systems were determined to be between2.0and3.0. The results indicated that sunflower and corn oils were suitable for phytosterols biotransformation, their AD yield were both over80%at10g/L phytosterols, and the values in other oil/aqueous systmes were also around70%. Based upon that, sunflower oil/aqueous biphasic system was used to explore optimal bioconversion condition such as phase ratio (PR, oil to aqueous), inoculum, feeding time and fermentation time. The result indicated that the highest AD yield was obtained at PR of1:9,8%of inoculum, feeding at48h and120h biotransformation. At even high phytosterols concentration of20g/L, the yield was still over45%. There is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis due to its advantages of low cost and biodegradable.