Studies On The Microbial Transformation Of Androst-4-ene-3,17-dione

Steroid drugs are the second medicine, which are widely applied in the field of medicine, a lot of demand to promote the development. Microbial transformation has the advantages of high specificity, safe and efficient, so the method gradually becomes a research focus of steroid drugs.Androst-4-ene-3,17-dione(AD) is an important intermediate for steroid hormones, the lla-hydroxylation catalyzed product of AD is C11α-hydroxy-androst-4-ene-3,17-dione (C11α-OH-AD), it is a key intermediate for Eplerenone, a new drug used in the treatment of hypertension and congestive heart failure.This thesis study on reaction process of the microbial lla-hydroxylation of androst-4-ene-3,17-dione, including the strain screening, transformation and analysis methods, the screening of medium components and conversion process optimization.First, the thesis study on the method of HPLC conditions were as following C18column, mobile phrase of methanol-water (3:2),the flow rate was0.8mL/min,the column temperature was25℃,the detection wave-length was242nm.By experiments determined one-step method and using methanol as extraction agent.Second, high conversion rate of RN-M24strain selected in the four strains, isolated and screened the RN-M24strain, filter out the strain of RN-M246, the optimal medium is(g/L) glucose30, peptone20, cold pressed soybean cake powder12, triammonium citrate1, diammonium phosphate5, sodium chloride1, ferrous sulfate0.5, pH5.0. The conversion rate was40.72%.Finally, optimization the conversion process, including the substrate of adding method, the fermentation time, inoculum size, liquid volume in flask, temperature, rotation speed, and additives were investigated.The results showed that the liquid volume of fermentation medium75mL in500mL flask, add2g/L substrate, with the medium after sterilization ultrasound10min together, inoculum size of4%, at28℃transfer reaction72h,24h before speed is160r/min and24h after adjustment for220r/min, The conversion rate was46.87%.. In addition, in the medium supplemented with100g/L corn oil on the conversion rate has improved significantly. The conversion rate was52.87%.In addition, This thesis also preliminary study on microbial lla-hydroxylation of androst-4-ene-3,17-dione by the MA strain. Activation and screening of MA strain, it was found the strain of MA-18, the optimal conditions (g/L):glucose30, silkworm pupa powder, ammonium sulfate2.5, corn syrup20, dipotassium hydrogen phosphatel, magnesium sulfate0.5,ferrous sulfate0.02, pH6.0. Through the substrate investigated to determine the feeding time is the initiation of the reaction. The product conversion rate can reach to44%.