Breeding Of 16α, 17α-Epoxy Progesterone Biotransformation Strains And Optimization Of Its Conversion

The hydroxylation of steroids by microorganisms is an important role in biotransformation of steroids.The 11α-hydroxylation is one of the key technologies in the field of steroids medical industry because of the mild conditions,high selectivity and productivity.As a kind of steroid, 16α,17α-epoxy progesterone is an important intermediate for many hormone pharmaceuticals,such as hydrocortisone.In the process of semi-chemical synthesizing steroid drugs,11α-hydroxylation catalyzed by microorganism is the most key steps.Therefore,the aim of this study was focused on the 11α-hydroxylation of 16α,17α-epoxy progesterone by microbial transformation.From 30 different strains of fungi,Cunninghamella elegans 40250 was selected out,according to their ability to hydroxylate 16α,17α-epoxy progesterone.It was found that C.elegans 40250 possessed the highest efficiency of 11α-hydroxylation of 16α,17α-epoxy progesterone among the selected strains.Ion implantation and ⁶⁰Coγradial were used to breeding high conversion rate strain.C.elegans D60-9 was screened out through low energy nitrogen ion beam implantation.At the liquid culture condition,the conversion rate was increased by 10.29%,compared with the original strain.Then ⁶⁰Coγradial was conducted on it,and other mutant was selected.C.elegans DC12-9 genetics nature is stable,It biomass and conversion rate were increased by 3.97g/L and 17.36% respectively.The fermentation conditions of 16α,17α-epoxy progesterone by C. elegans DC12-9 was study.The optimum fermentation conditions have been established.The result show that,glucose 3.3%,corn steep liquor 2.1%,peptone 0.53%,（NH₄）₂SO₄ 0.3%,MnSO₄ 0.02%,BaCl₂ 0.03%, ZnSO₄ 0.01%,LiCl 0.03%,and initial pH 5,inoculation 3%,medium volume 100mL/500mL,reciprocating shake,fermentation time 22h,the extracted had been determined after enzymatic conversion for 48h.Under these condition,the determination showed that the conversion rate of C₁₁α-hydroxylation reaching 77.31%.The kinetic characteristics of C.elegans DC 12-9 which was applied for the 11α-hydroxylation of 16α,17α-epoxy progesterone were investigated.The effects of substrate and product on the conversion process of hydroxylation were studied.The results show that there are inhibitory effects on the hydroxylation process at high substrate concentration and with initial product presence.Based on experimental results,a noncompetitive inhibitory model was proposed to describe the hydroxylation process,and the parameters of the kinetic equation were calculated.