High Efficient Biotransformation Of 11α-Hydroxy-13-Ethyl-Gon-4-Enl-3,17-Dione

Desogestrel, as a new kind of reliable ovulation inhibitor, displayed high affinity for progesterone receptor. At the same time, they showed the slight androgen and lipid metabolism, and no further adverse effect, for androgen receptor. Therefore, compared to the extensive use of contraceptive, like norethisterone, desogestrel attracted much more attention of community.Study on preperation of 11α-hydroxylation by microbial transformation was performed in this dissertation, including screening of strains for the enzymatic reaction, isolation of preperation of 11α-hydroxylation of 13-ethyl-4-gonene-3,17-dione by microbial transformation and structural characterization of the product, optimization of the technological factors of preperation process of 11α-hydroxylation by Aspergillus ochraceus TCCC 41060, and purification of 11α-hydro-13-ethyl-4-gonene-3,17-dione. Furthermore, the byproducts during biotransformation has been studied too.Results of study on the 11 α-hydroxylation showed that rate of transformation by Aspergillus ochraceus TCCC 41060 was better than Aspergillus niger TCCC 41055,Rhizopus oryzae TCCC 40006 and Metarhizium anisopliae TCCC 33676. Structural characterization by HPLC-MS, H-NMR, C-NMR, HMBC and COSY verified the common product as 11α-hydro-13-ethyl-4-gonene-3,17-dione.To improve the efficiency of 11α-hydroxylation of 13-ethyl-gon-4-ene-3,17-dione by A. ochraceus, the bioconversion culture medium of A. ochraceus was studied by one-factor comparative experiments. Results indicated that the most suitable bioconversion culture medium are as follows (g/L):glucose,20, corn steep, 20; yeast extract,20; pH,5.8. Furthermore, the optimal bioconversion conditions of 11α-hydroxylation of 13-ethyl-estr-4-ene-3,17-dione by A. ochraceus were obtained as follows:the liquid volume,50mL/250mL, inoculum spore concentration ,5×106/mL; initial pH,7.5, and the substrates were added to the enthanol medium with 5% as cosolvent, agitated(200r/min) under 28℃ for 120h. While substrate concentration is 2g/L, the higher bioconversion was obtained and it was 57.42% by using HPLC.Isolation and purification of 11α-hydro-13-ethyl-4-gonene-3,17-dione were studied at last. After solid-liqued separation, the mycelium of Aspergillus ochraceus TCCC 41060 was subjected to extraction with ethyl acetate, and the ethyl acetate mixture was boiled with circumfluence for twice for condensation (Dosage of ethyl acetate is 100 mL per 0.2g 13-ethyl-4-gonene-3,17-dione added,50 mL every time). Time of extraction was 60 min each time. Then the raw product was obtained through evaporation, the yield rate of product was 50%.Additionally, there are 4 by-products during the 11α-hydroxylation of 13-ethyl-4-gonene-3,17-dione by using A. ochraceus. Among these, two by-products were separated by preparative liquid chromatograph and were identified by NMR, the results showed that one is 10,11-dihydroxy-13-ethyl-4-gonene-3,17-dione, and the other is 6-hydroxy 13-ethyl-4-gonene-3,17-dione.