Screening For Strains Capable Of Hydroxylating Steroids (4AD) And Identification Of Products

Androst-4-ene-3,17-dione(4AD)is a key intermediate of steroid drugs.The hydroxylated products of 4AD are commercially valuable steroids for therapeutic use.Microbial hydroxylation of steroid drugs plays crucial role in the production of steroid drugs,but the screening of strains with efficient transformation ability as well as capable of producing novel steroidal drugs with novel pharmaceutical activities are still desirable.This dissertation focused on the screening of strains with specific hydroxylation of 4AD,characterization of the corresponding product,and preliminary exploration of the hydroxylation technique of 4AD.Four strains,including Penicillium decumbens?strain TCCC 41667?Penicillium raistrickii and Aspergillus ochraceus,have been screened to evaluate hydroxylation activity of 4AD.Among these,strain TCCC 41667,with high specificity of 4AD hydroxylation,was identified as Aspergillus sp.by morphological characteristics and the phylogenetic analysis of 18S rDNA.Transformation products were separated and purified by silica gel column chromatography and re-crystallization,and then were identified by MS,NMR and single crystal X-ray diffraction.Results indicated that:P.decumbens biotransformation of 4AD afforded la-hydroxy-4-ene-3,17-dione,1?,6?-dihydroxy-ene-3,17-dione;Aspergillus sp.TCCC 41667 biotransformation of 4AD afforded 15a-hydroxy-4-ene-3,17-dione;P.Raistrickii biotransformation of 4AD afforded the same product 15a-hydroxy-4-ene-3,17-dione(5);A.ochraceus biotransformation gave lla-hydroxy-4-ene-3,17-dione.Among these,la-hydroxylation of 4AD and 1?,6?-dihydroxylation of 4AD were first bio-synthesized.So,beside the a-hydroxylation at steroid nucleus,such as 1?,5? and 7? et al,which was usually reported in literatures,P.decumbens had the ?-hydroxylation of steroid.This will enhance the application of P.decumbens,and lay foundations to discover more and more(3-hydroxylation of steroid.Effect of the biotransformation condition on the accumulation of the product was also investigated.In the hydroxylation process using P.decumbens,sucrose in the medium improved the yield(59.4%)of 1?,6?-dihydroxy-ene-3,17-dione(3),while the yeast extract increased the accumulation of the 1?-hydroxy-4-ene-3,17-dione(2)which is 71%.For Aspergillus sp.TCCC 41776,maltose enhanced the production of the 15a-hydroxy-4-ene-3,17-dione(4)to about 70.4%.These indicated that the composition of the medium plays important role in the hydroxylation of 4AD.