Hydroxylations are possibly the most widespread type of steroid bioconversion. Hydroxylations can be used to buildintermediates for further chemical synthesis to provide the steroid molecule with the adequate structure for therapeutic applications. Microorganisms able to hydroxylate steroids in positions C1 to C21 and in position C26 have been reported. 11 α hydroxylation is regarded as essential for anti-inflammatory action. Present study was promoted to extend the investigations to evaluate potentiality of this local oil contaminated soil isolates for transformation of progesterone. Enrichment is carried out in the inorganic medium providing progesterone as an only carbon source. Metabolites are identified by GC/MS and isolates by 16 s rRNA. 11 α hydroxyprogesterone is the metabolite identified having 60 percent of yield. and Bacillus circulans Tex 01-S3c has been found most efficient strain.
Key words: –
Hydroxylation of Progesterone, oil contaminated, Bacillus circulans Tex 01-S3c,
Application of molecular biology techniques and genetic engineering of microorganisms for their improvement as steroid transforming agents are the areas of drug development. Site selection oxygenation, particularly hydroxylation of exogenous steroids is frequent in bacteria genera Bacillus. Several microbial bioconversion of steroids and sterols have been reported, focusing mainly on steroid hydroxylations, ∆1-dehydrogenation and sterol side-chain cleavage.
These biotransformations, mostly associated to chemical synthesis steps, have provided adequate tools for the large scale production of natural or modified steroid analogues. The complex structure of the steroid molecule requires complicated, multi-step schemes for the chemical synthesis of steroid compounds. Microbial steroid conversions are performed in mild temperature and pressure conditions and can provide an efficient alternative to chemical synthesis.
Screening and isolation of active microbial strains for steroid bioconversion is presently an important part of the research and development effort in the steroid drug industry. Oil contaminated soil is a rich source of variety of bacteria capable of biotransformations and degradations of complex chemicals.
MATERIAL AND METHODS
1 gm of oil contaminated soil from different local places was added in the sterile 9 ml saline and 1 ml from this is added in the sterile progesterone liquid enrichment medium (KH2PO4, Na2HPO4. 12 H2O, (NH4)2SO4, MgSO4. 7 H2O, CaCl2. 6H2O, FeSO4. 7 H2O, pH 7) providing progesterone as a only carbon source. As progesterone is not soluble in water, it was dissolved in acetone (50 mg / ml). The flasks are incubated at 300 C for 7 days. Regular microscopic observation and turbidity checking carried out. By serial dilution techniques 0.1 ml was spread on the nutrient agar plates and well isolated colonies are studied for biotransformation potential.
The medium of composition (g/liter): glucose, 40.0; peptone, 1.0; KH2PO4 0.74; MgSO4. 7H2O, 1.0; Yeast extract, 1.0 and asparagine, 0.7, pH was adjusted to 7 used for transformation. The cultivation was performed in 250-ml Erlenmeyer flasks each containing 50 ml medium. The flasks were sterilized and inoculated with 2 ml inoculum of 24 hr cultures of the pure organism. The culture flasks were incubated on incubator shaker at 34° C for 48 hr. Thereafter, 50 mg of progesterone, dissolved in 1 ml acetone, was added to each flask and fermentation was continued for another 72 hr.
At the end of fermentation, metabolites are analyzed by GC/MS. The efficient bacterium identified by 16S rRNA. Standardization of parameters like temperature, pH, incubation period, carried out spectrophotometrically.
RESULTS AND DISCUSSION
11 α hydroxyprogesterone is the metabolite identified having 60 percent of yield. Bacillus circulans Tex 01-S3c is the efficient bacterium identified. Temperature 340 C, pH 6.5, incubation period 60 hours are the optimum parameters for maximum yield.
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