Optimization On Androstenedione Production Process Conditions By Microbial Conversion Method

Androst-4-ene-3, 17-dione(AD) is the key intermediates of many synthetical steroid drugs,and be also one of the important raw materials for steroid hormones drugs, such as glucocorticoids, sex hormones, anabolic hormone and so on.It was established the analysis method of substrate and product in the process of microbial transformation of phytosterol to androstenedione in the transformation system. Compared with the measuring results of TLC, UV, HPLC, and GC, a kind of measuring method, the analysis system of AD production was established using TLC and GC, which could be used to qualitatively and quantitatively analyse the concentration AD and phytosterols in the transformation liquids.The effects of flask seed medium's major ingredients on the Mycobacterium sp. growth were studied. Orthogonal methodology was used to optimize the seed medium. The effect of different cosolvents on phytosterols degradation in aqueous and two phase system was studied. It was found that AD production was significantly increased in the presence of an organic-aqueous of two-phase system, the optimal organic phase was vegetable oil. By constructing orthogonal methodology, the organic phase and its proportion were soybean and 16%, and the concentration of the substrate was 1.5%, respectively, the fermentation results was the best. The AD conversion rate was increased from 28.37% to 41.57%.The fermentation medium was optimized by the single factor test and the response surface experiments on the basis of above experiments. The best carbon source, nitrogen source,phosphate source and inorganic salt types were determined. The Response Surface Methodology(RSM) based on a three-factor Box-Behnken design of experiments was used to build an analytical model and finally employed as a constraint in an obvious optimization process. The optimized parameters were soybean oil 1.6%, glucose 0.6%, corn steep liquor 2.18%,(NH4)2HPO4 0.055%, citric acid 0.2%, ferric ammonium citrate 0.005%, and fermentation period of 120 hours, respectively. Under those optimization conditions, it was predicted that the highest concentration of AD gained 5.23 g/L, the yield of AD reached 51.88%, the conversation of substrate was 97.78%. After optimization, the concentration of AD increased 22.43% in compared to the former(4.19g/L). According to the RSM analysis to conduct repeated experiments, the AD concentration reached 5.13 g/L, the yield of AD 50.89%,the conversation of substrate 97.22%and the experimental data basically in line with the predicted values. Moreover, the effect on fermentation result of culture conditions, such as inoculation quantity, temperature, initial pH,ratating speed, liquid medium etc, was studied to obtain the optimal conditions. The condition of AD came to 5.38 g/L, the yield of AD 53.37%, the conversation of substrate 98.42%. Incomparison with the former(4.19g/L) the concentration of AD increased 28.40%.Finally, The AD extraction process in the two-phase system was also studied in the article.After using those optimal conditions including appropriate extraction solvent, temperature, etal,the extraction rate reached 75.78%, and the product purity reached 98.32%.