Studied On Microbial Conversion Of C21 Steroids

The strains were isolated from soil samples,and have ability to bioconvert C21 steroids after preliminary screening and rescreening with Pregnenolone as the sole carbon source.The strains were identified according to biochemical characteristics,morphology and molecular biology,16 S rRNA gene sequence of bacteria and 18 S rRNA gene sequence of fungi,and homology analysis using the BLAST program.According to the BLAST results were analyzed by the Neighbor-Joining Method using MEGA(5.05)software,determined the phylogenetic position of this strain.It is evaporated,extracted and evaporated to obtain the crude products of transformation C21 steroids.The new compounds were purified by silica gel and Sephadex LH-20 column chromatography.The compounds' chemical structures were elucidated by nuclear magnetic resonance(1H-NMR,13C-NMR(DEPT 135° and DEPT 90°),2D-NMR(HSQC and HMBC))and determined the molecular weight by mass spectrometry(MS).The strain was identified by its DNA sequencing,which was amplified by PCR and electrophoresed to provide a partial 16 S rDNA gene sequence of 1385 base-pairs of DH and 1482 base-pairs of YM3.Two strains were belong to Pseudomonas,were Gram-negative bacteria too.DH and YM3 are Pseudomonas and Pseudomonas aeruginosa,respectively.The 18 S rDNA of the strain T3 had 1314 base-pairs,which is Aspergillus fumigatus and belongs to Aspergillus.Those base sequences were assigned in BankIt and obtained the accession number MF542259.1(DH),MG262387.1(YM3),MF563964.1(T3).The two bioconverted products were phenanthrene-1,2,10-triol and 6H-benzo[c]-chromen-7-ol by NMR and MS elucidated.In addition,phenanthrene-1,2,10-triol showed clear antimicrobial activity.When the concentration of phenanthrene-1,2,10-triol was 0.5 mg/mL,the inhibition zones were 25 mm,18 mm and 17 mm on Candida albicans fungi,Staphylococcus aureus and Escherichia coli bacteria,respectively.The optimal fermentation conditions for the biotransformation of 16,17-alphaepoxypregnenolone to 6H-benzo[c]chromen-7-ol and phenanthrene-1,2,10-triol by DH.The optimum conditions of 6H-benzo[c]chromen-7-ol are summarized as follows: 1.5 g/L of 16,17-alphaepoxypregnenolone SDS emulsion as carbon source,2.0 g/L NH4NO3 as nitrogen source,the initial pH was 7.0,the inoculum concentration was 10%,the liquid medium volume was 100 mL in 250 mL Erlenmeyer flasks,the transformation time was 5 d;The optimum conditions of phenanthrene-1,2,10-triol are summarized as follows: 1.5 g/L of 16,17-alphaepoxypregnenolone SDS emulsion as carbon source,3.0 g/L NH4NO3 as nitrogen source,the initial pH was 6.5,the inoculum concentration was 10%,the liquid medium volume was 80 mL in 250 mL Erlenmeyer flasks,the transformation time was 5 d.The optimal fermentation conditions for the biotransformation of pregnenolone to phenanthrene-1,2,10-triol by T3.The optimum conditions of phenanthrene-1,2,10-triol are summarized as follows: 1.5 g/L of pregnenolone SDS emulsion as substrate concentration,2.0 g/L NH4NO3 as nitrogen source,the initial pH was 7.0,the inoculum concentration was 10%,the liquid medium volume was 70 mL,the transformation time was 6 d.Under optimal conditions,the yields of product was 28.48% and biomass was 4.73 g/L,higher 9.52% and 2.11 g/L,respectively,than the yields obtained prior to optimization.Phenanthrene and its derivatives from various plants and have not been reported to have been obtained from microbial transformation processes.In this paper,microorganisms were used to transform pregnenolone and 16,17-alphaepoxypregnenolone to obtain compound with antibacterial activity.It provides the basis for microbial transformation of C21 steroids.