Preparation Of 9?-hydroxy-androst-4-ene-3,17-dione By Biotransformation

Steroidal drugs are widely applied in the field of medicine,which has become the second largest after the antibiotic drugs.Great need of steroidal drugs promotes its development.Because microbial transformation is efficient,environmentally friendly,safe and energy-efficient,it becomes a research hotspot about steroidals drugs.Diosgenin is in shortage,which is semi-synthetic raw materials of sterdoid drugs,phytosterol as a new resource for the steroid industry has injected new vitality.9a-OH-AD is a key intermediate in the industrial production of steroid drugs,it also plays an important role in the industrial production of steroidals drugs.Microbial transformation of phytosterol to produce 9a-OH-AD with side chain of phytosterol degradation,semi-synthetic materials achieve diversified in production of steroid drugs,but domestic research in the area is still in its initial stage.In the paper,microbial transformation of phytosterol to prepare 9a-OH-AD by DG strains was studied.Including,purification and mutagenesis of DG strains,analytical method,screening and optimization of seed medium and fermentation medium and optimization of fermentation condtions.Firstly,the conditions of HPLC was determined,C18 alkyl silane bonded phase column;mobile phase of methanol:water=7:3(v/v);flow rate of 0.8mL/min;column temperature of 25?;wavelength of UV detector 242nm.After several times of purification and mutagenesis of DG strains,a better strain DG-3-14 was got,and also the suitable seed medium and optimal fermentation medium were got.The seed medium(g/L):glucose 3,ammonium nitrate 3,magnesium sulphate heptahydrate 0.5,dipotassium hydrogen phosphate 1,yeast extract 15,pH value about 7.0.Fermentation medium(g/L):Molasses 4,dry yeast powder 20,sodium 1.5,Magnesium sulfate heptahydrate 0.75,Potassium chloride 2.75,Dipotassium hydrogen phosphate 1.3,ferrous sulfate heptahydrate 0.5,zinc sulfate heptahydrate 0.05,copper sulfate pentahydrate 0.01.The fermentation parameters for maximum conversion of phytosterol were optimized.Including,the optimum growing time of strains,temperature,pH value,the amount of inoculum,liquid volume,inoculum,fermentation time,and concentration of substrate fermentation and the additive.Optimization results are:optimum growing time of the strains 36h,temperature 32?,pH value of 6.0,inoculum 15%,liquid volume 70ml,speed 220r/min,concentration of substrate 1%,fermentation 5d,soybean oil added,which increase yield of the product significantly.The yield of the product was improved from 18.78%to 75.12%by screening DG bacteria,optimizing the media and fermentation conditions.