Study On Biotransformation Of Steroid In Two-phase System

Microbial transformation of steroid hormones has important significance of application in the current production of drugs. While the steroid in a very low solubility in aqueous media, traditional biological conversion reaction carried out in the water phase are often restricted on available substrates. Biotransformation of steroids with whole cells in organic/aqueous two-phase system has become one of the research hotspot in recent years.The antitumor drug-exemestane is an irreversible steroidal aromatase inhibitors, whose structure is similar to the natural substrate androstenedione. As an effective drug of the treatment of postmenopausal women with hormone-dependent breast cancer, exemestane is becoming more widespread in its clinical application. Exemestane is traditionally synthesized by chemical method. But some toxic and hazardous chemicals such as DDQ are used in 1-dehydrogenation which is the most important step in the synthesis of exemestane. And it is very difficult to extract exemestane from the by-products. In this article, we studied the biotransformation of steroid in organic medium.The experiment investigated the solubility of steroid substrate and its product in eight different kinds of organic solvents which were n-hexane, n-heptane, dichloromethane, chloroform, toluene, xylene, ethyl acetate and butyl acetate. Five kinds of organic solvents including ethyl acetate, butyl acetate, toluene, xylene and chloroform were selected preliminarily as the organic phase in this two-phase system through the screening tests of steroid solubility. And then the survival of Arthrobacter simplex cells were examined in these solvents respectively. It is found that xylene has a minimal impact on the microbial cells. So we finally determined xylene as the organic phase. Afterwards, it is confirmed that fermentation broth as the water phase and organic solvent in the total volume of 15% was more appropriate.The antitumor drug-exemestane (6-methylene-androsta-1,4-diene-3, 17-dione) was obtained in the xylene/fermentation broth system using 6-methylene-androsta-4-ene-3,17-dione as substrate, which could be 1-dehydrogenated by Arthrobacter simplex. In the meantime, the effects of the conversion time, conversion temperature, shaking speed and bottled fluid volume on the conversion ratio of substrate had been carefully discussed.Results showed clearly that p-xylene had good biocompatibility as organic solvent in the two-phase system, its relative amount was 15%. On the conditions of the medium 20 mL/100 mL flask, temperature 32℃, speed 180 r/min, conversion time 24 h, the conversion rate came to 13.6%.The method of TLC and HPLC was also set up to quantitate the exemestane and related synthetic intermediates.