Studies On Biotransformation From Phytosterol To Androstenedione By Mycobacterium Neoaurum ZJUVN-08

Steroid hormone is an important category in our pharmaceutical industry, and androstenedione is an indispensable key intermediate of steroid hormones. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis, and can alleviate the current situation caused by the raw material scarcity of diosgenin, is of great significance to full use of plant steroid resources, as well as prompting the pharmacy development. In the present study, strains with the ability to selectively degrade phtosterol were screened and their AD producing ability was tested. The strain with high AD yield was identified and mutation using low energy N+implantation was followed. As a result, a genetically stable mutant strain with AD as the sole product, designated as ZJUVN-08, was obtained. The effects of medium composition and culture conditions on phytosterol biotransformation were evaluated experimentally. Different cell wall permeabilization factors were used to enhance cell wall permeability and AD production. The effect of different cosolvents in aqueous system on the degradation of phytosterol into AD was studied. AD production was significantly increased in the presence of HP-P-CD. Then the process optimization was investigated using growing mycobacteria cells in depth. Biotransformation of phytosterol to AD using resting cell was optimized. The main results of this study were as follows:Soil samples which were seriously polluted by vegetable oil were collected from Taian surburb, Shandong, China. After incubation in encrichment media, the enrichment broth was spread on the plates with phytosterol as the sole carbon source.76strains were isolated, and then these strains were inoculated to bioconversion media to test the ability to transform the substrate. Transformation products were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). A strain named ZJUVN was found to have the maximum production and was able to transform phytosterol to AD and androsta-1,4-diene-3,17-dione (ADD) with the ratio of10:1. The strain was identified as Mycobacterium neoaurum by morphology, biochemical identification and16S rDNA sequence analysis.The strain was bred using low energy N+implantation method for accumulation of AD as the only product. The most suitable mutation conditions were:energy of10keV, implantation dose of100×2.6×1013ions/cm2, with the death rate of70%. Colonies appearing on the selected plates were chosen and tested for their AD production. Then23strains were selected with AD as the sole product. One of the mutated strains, designated as ZJUVN-08, produced the maximum level of AD (2.29g/L) which increased by71.05%. The strain was serially passaged for10passages and AD production was determined. The results demonstrated that M. neoaurum. ZJUVN-08has good genetic stability within10passages.The effects of medium composition and culture conditions on AD production by strain ZJUVN-08were evaluated experimentally. The optimized medium composition was as follows:glucose1%, peptone0.6%, KH2PO40.15%, MgSO40.05%, FeSO4. The optimized fermentation conditions were as follows:initial pH value7.0; temperature30℃; inoculums volume,10%; and50mL medium in250mL shaken flask; rotate speed200rpm. The product was purified by preparation HPLC and was a white powder, then further identified as AD using NMR, MS and IR analysis.By the study of ultrasound, bacitriacin, protamine and glycine on the strain growth and AD production, it was found that under the condition that with ultrasound power500W, treating time180s, AD yield was2.95g/L and increased by5.8%. After the treatment of different concentration of bacitriacin, the growth of strain was inhibited and AD yield was decreased under all the tested bacitriacin concentration. After the treatment of10g/L protamine, AD yield was3.02g/L and increased by8.6%. It was found that5g/L glycine was in favor of strain growth and AD production, with the AD yield of3.16g/L. Drug sensitivity assays were carried out to establish whether glycine increased AD production by increasing the cell wall permeabilization.The effect of different cosolvents on phytosterol degradation in aqueous system was studied. It was found that AD production was significantly increased in the presence of HP-β-CD. The parameters of biotransformation process by growing mycobacteria cells were optimized using fractional factorial design and response surface methodology. The optimial process conditions were observed at0.1g/L inducer, pH7.0, and molar ratio of hydroxypropyl-β-cyclodextrin to phytosterol1.92:1,8.98g/L phytosterol and at120h of incubation time. Under these conditions, the maximum AD yield was5.96g/L and the maximum phytosterol conversion rate was94.69%, which is a cost-efficient process compared to the non-optimized condition.The effect of phytosterol bioconversion using resting cells of strain Mneoaurum ZJUVN-08was investigated. The optimized conditons were as follows:cell age of72h; cell concentration of45g/L; Tris-HCl buffer solution pH value and concentration were7.0and0.05mol/L, respectively. After repeatedly utilizing four times, phytosterol bioconversion rate was decreased to56.04%.