Studies On The Hydroxylation Of Dehydroepiandrosterone By Biological Modification

1α-Hydroxy-Dehydroepiandrosterone is an important drug intermediate which is used for the chemical synthesis of active vitamin D3 and its analogues. The vitamin D3 which has diverse biological mechanisms could act on variant target tissue. So it is wildly used in treating of osteoporosis, parathyroid(ARF), psoriasis, cancer and immune diseases. The moost critical step in semi-synthesis of vitamin D3 by dehydroepiandro- sterone is the 1α-hydroxylation in the first step.This paper reported the 1α-hydroxylation of dehydroepiandrosterone by the micro- bial transformation.Strains which could hydroxyl the dehydroepiandrosterone at 1αsite were directly screened by enriched cultivation with substrate concentration gradient, and one strain was screened out. Morphological characteristics and the phylogenetic analysis of 18S rDNA indicated that it was a typical Penicillium decumbens. Identification of the product in Subsequent biotransformation experiments confirmed that this strain had the bio-transform activity.Three cycles of UV mutagenesis was introduced and one mutant named ph-13 was screened out for its higher transforming ratio and stable genetic characteristics.Variant conditions of fermentation and transformation of ph-13 were tested and result showed that the optimal carbon and nitrogen sources for seed medium were glucose and yeast extract, while the favorite ones in transforming medium were sucrose and sodium nitrate. Best inoculating concentration was 5×10~7个/mL.After optimizing conditions of the 1α-hydroxylation of dehydroepiandrosterone we got the best conversion medium which contained the components below: sucrose 4.75 g/L, sodium nitrate was 3.69 g/L, magnesium sulfate was 0.3g/L, manganese sulfate to 0.0001 g/L, ferrous sulfate 0.0001 g/L. In this optimum medium,the transformation rate of 1α-OH-DHEA was 18.83 %.Subsequently we optimized the separation and purification or 1α-hydroxyled dehy- Droepiandrosterone. The broth was filtrated afer conversion, and then the supernatant was extracted with methylene chloride. The condensate was separated by silica gel chr- matography, LH-20 gel chromatography, PTLC, crystallization-ecrystallization and fin- ally got two compounds. a white needle-like crystal which was 1α-hydroxyled dehydro- epiandrosterone and another white powder-like product which was identified as 7α-hyd- roxy dehydroepiandrosterone.