The Study Of Technology On11α-Hydroxylation Of Canrenone By Microbial Organisms

Enzyme catalysis reaction is very important in drug synthesis occupies, which iscontemporary the direction of development of organic synthesis. In semi-chemical synthesisof eplerenone from canrenone,11α-hydroxylation of canrenone by microbial transformationis the first step and also one of the most critical steps. So to study the11α-hydroxylation ofcanrenone has important significance in theory and practice.The fermentation conditions of Aspergillus ochraceus SIT34205were investigated.Through the single factors test, the optimal composition of biotransformation medium was asfollows (g/L): glucose25, yeast extract15, cornstarch3, peptone5, NH4H2PO40.5, NaCl20.The optimum fermentation conditions were determined: inoculation4%, the substrateconcentration10g/L, DMF4%(v/v), temperature of transformation28°C. Before thetransformation of canrenone4h, low concentration of canrenone (0.4g/L) was added forinducement of11α-hydroxylase, which could improve the rate of11α-hydroxycanrenoneremarkably. Transformation of canrenone by SIT34205on the scale of5L fermenter wasperformed with optimal medium and optimal fermentation condition. And the rate oftransformation reached94.1%.In order to study the mechanism of microbial conversion of canrenone by SIT34205, thebyproduct was separated and purified by silica gel column chromatography, and its structurewas characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Theresults indicated that the byproduct was1β,11α-dihydroxycanrenone, suggesting a novelcompound based on the search results in the Reaxys and SciFinder databases. Thefermentation conditions was optimized by SAS to inhibit the formation of the byproduct. Andthe K is0.23. In the late stage of the biotransformation, the Zn2+was added in optimalfermentation condition and the K is0.18.The effects of different kinds of surfactants on canrenone dissolution and bioconversionwere also studied in this paper. The results indicated that the surfactants had positive effectson the substrate dissolution, but tw80and tw80-sp60(HLB=10) had positive effects onbioconversion. Then the emulsifying systems were established. The results showed that thesolubility of canrenone were increased obviously in the emulsifying system and shortened thebiotransformation time from60h to48h.