Studies On The Transformation Of DHEA To Produce Steroid Drug Intermediates By Microorganism

Dehydroepiandrosterone (DHEA), a kind of C19adrenal steroid compound,mainly plays important roles in anti-cancer, anti-aging and immune adjustmentfunctions. DHEA can be simply chemical synthesized by some ordinary raw materialsobtained from diosgenin, and we can synthetize many steroid medications whichpossess important physiological activity by intermediate DHEA, for example, theantioxidant activity and antitumor effects of7α-OH-DHEA are significant,1α-OH-DHEA is an important drug intermediate used for the chemical synthesis ofactive vitamin D3and its analogues.This paper reported the structural modification of dehydroepiandrosterone by themicrobial transformation. A fungus which can biotransform DHEA was isolatedthrough the transparent circle method and TLC analysis from preservation strains oflaboratory, and was identified as Streptomyces albus according to its morphologicalcharacteristics and16s rDNA analysis. The expand culture was further performed by20L fermentation tank. The main three transformation products were puried usingsilica gel column chromatography, LH-20gel column chromatography and HPLCpreparation column, and their structures were elucidated as7β-OH-DHEA,7α-OH-DHEA and1α-OH-DHEA respectively by NMR and optical activity analysis.Subsequently, we optimized the fermentation medium and transformtionconditions of Streptomyces albus, and determined that the optimal carbon, nitrogensources and inorganic salt for the medium were glucose20g/L, yeast extrct5g/L,ammonium sulfate8g/L and dipotassium phosphate0.75g/L through the single factorexperiment and the orthogonal experiment. The optimal transformation conditions asfollows: the incubation temperature was30℃, initial pH of culture medium was7.0,rotation speed was160r/min, seed age of the spore was5d, spores concentration was106/mL, substrate concentration was1mg/mL, added the substrate after inoculation48h, the substrate was embebed with β-cyclodextrins, the transformation time afteradding DHEA was48h. Under the optimized conditions above, the transformation rateof unit biomass achieved37.96%，increased by15.2%than before.Finally, We preliminary studied the cytochrome P450of Streptomyces albus, thecontent of P450was determined by CO difference spectroscopy, we analysed thechange of P450in the course of DHEA transformation, studied the inducer(benzoic acid、n-hexane and MEHP) on the effect of P450, confirm that the increase of P450content was most significant while the concentration of inducer n-hexane was10mM,.From the view of the transformation michanism of compound, which also hopes toimprove some conditions of transformation process, get a higher conversion rate ofobjective products and finally applied in industrial production.