The Microbial Transformation Of Phytosterols Production Of Testosterone

Testosterone (TS) is the principal hormone released by the testes, and is produced by the interstitial cells. It acts as a powerful androgen and is responsible for the male sex characteristics. The final step in the biosynthesis of TS is reduction of androst -4-ene-3,17 - dione (AD) to TS by 17- ketosteroid reductase (17β-hydroxysteroid: NADP 17-oxidoreductase, EC 1.1.1.64 ). The enzymatic reduction of AD to TS by 17β-hydroxysteroid dehydrogenase (17β- hydroxysteroid: NAD 17- oxidoreductase, EC 1.1.1.63 ) occurs in bacteria, yeast, and fungi. Although the microbial transformation of sterols to AD is now utilized on an industrial scale, commercial production of TS from AD is still carried out using a chemical synthesis.Microbial transformation is to use the specialized catalyze function of one or more enzyme in one or more microorganism, carry out one or more elementary reactions on some special positions of the organic compound which is used as substrate in fermentation, and get a new compound which has a similar structure of the substrate but more valuable than the substrate. Microbial transformation is widely used in medicine, function material and flavor and so on, because of its high selectivity, mild reaction condition, low cost and little pollution. Fermentation not need to use organic solvent, and the bases in waste liquor are amylase, protein and inorganic salt, so Microbial transformation is a green reaction.A testosterone(TS)-producing mutant, YHT-103, was derived from a androst- 4- ene-3,17- dione(AD)- producing bacterium, YHT-1, using a series of UV and nitrosoguanidine(NTG) mutagenesis. Its production yield of testosterone is 8 times more than the original strain.After mutagenesis, The optimization of fermentation conditions for YHT-103 was carried out, and got the optimal conditions were: glucose 1%, ammonium chloride 0.3%, citric acid 0.2%, disodium hydrogen phosphate 0.5%, magnesium sulphate 0.05%, manganese chloride 0.05%, phytosterol 2%, PEG 20000 8%, pH6.8, strain age 30 h. inoculation quantity 10%, fermentation temperature 28°C, aerobic fermentation 5 days, the rotation speed is 200 rpm, then anaerobic fermentation 2 days.50L magnified fermentation was carried out after optimizing the fermentation conditions. Growth status in seeding tank is better than in shaking table condition. Then the influences of pH and ventilatory capacity on fermentation were discussed. The result is that: constant pH is propitious to accumulate production, and when pH is 6.7, the production yield is higher than other conditions. The ventilatory capacity had little influence on fermentation. Bigger ventilatory capacity is good for shorter the fermentation time, but not convenience for control the foam.The separation and purification of the production was researched. Half volume of ethyl acetate to fermentation liquid was used to excerpt the production for two times. Evaporate and recovery ethyl acetate, then the crude extract was received. The crude extract was distilled by methanol for two times, use five times more than the weights of crude extract, the leaved solid is phytosterol. Then methanol was recovered, and the solid was purified by silica gel chromatographic column. At last, the purity of the production is more than 99%.