Screeing And Transformation Conditions Of 11α-hydroxylation Of Androst-4-ene-3, 17-dione By Beauveria Bassiana

The microbial hydroxylation of androst-4-ene-3,17-dione(1,4AD) was investigated in this study. The hydroxylated product of 4AD, 11α-OH-AD is valuable intermediates in the synthesis of pharmaceuticals. In the process of semi-chemical synthesizing steroid drugs from 4AD,11α-hydroxylation catalyzed by microorganism is the most key steps. Therefore the aim of this study was focused on the 11α-hydroxylation of 4AD by microbial transformation.Study on transformation of 4AD by microbial was performed in this paper, including screening of strains and identification of structural characterization of the products, optimization of the technological factors of the process of 11α-hydroxylation by fungi .Furthermore, The transformation of 4AD analogues by microbial has been studied too.From 30 different strains of fungi 11 strains of Beauveria bassiana and 1 strain of Aspergillus fumigatu were selected out, according to their ability to hydroxylate androst-4-ene-3, 17-dione by TLC for the initial screening. Then five of transformation products were purified with the chromatographic methods and identified as 11a-hydroxyandrost-4-ene-3,17-dione(2),6B,11a-dihydroxyandrost-4-ene-3,17-dione(3),1 1a,17B-dihydroxyandrost-ene-3-one(4),6B,11a,17B-trihydroxyandrost-ene-3-one(5)byUV, IR,FAB-MS,¹H NMR and ¹³C NMR. It was found that B. bassiana MEL15 possessed the highest efficiency of 11a-hydroxyalation of 4AD among the selected strains.The hydroxylation conditions of 4AD by B. bassiana MEL15 were studied. Optimal culture medium of B. bassiana MEL15 forlla-hydroxylation was as follows (g/L):glucose 20g, corn steep liquor 20g, K₂HPO₄ 1 g, and MgSO₄·7H₂O 0.5g, FeSO₄·2H₂O 0.02, pH 6.0. The cultivation process were performed in 250 ml Erlenmeyer flask with 40 ml of liquid medium, concentration of seed 10%(V/V), the cell age 48h, the transformation time 60h. the optimal conversion rate of 11α-OH-AD was 43%, increased by 20%. While the optimum transformation conditions were obtained: temperature 28℃, 180 r/min, the concentration of solution-MeOH 2.5%(v/v) and the concentration of substrate 2.5g/L. Then the optimal conversion rate of 11α-OH-AD was 65%, increased by 51.2%.Furthermore, the possible transformation routes of 4AD by B. bassiana MEL15 were analyzed by transformational of 4AD and its analogues. B. bassiana MEL15 strain showed the 11α-hydroxylated efficiency of 4AD analogues and the ability of C17β-ketoxide/reduaseo It implicated that B. bassiana MEL15 was a potential whole cell-catalyst in the modification and synthesis of pharmaceuticals.