Study On Bioconversion Conditions Of 13-ethyl-estr-4-ene-3,17-dione C15α-Hydroxylation By Penicillium Raistrickii

Gestodene is a new generation progestogen, oral contraceptives. Gestodene's strong progestogen activity with very weak androgenic activity will be of benefit to the cardiovascular system, and lowest dosage used as oral contraceptives will have less side-effect. 15a-hydroxyl-13-ethyl-estr-4-ene-3,17-dione, which is produced from 13-ethyl-estr-4-ene-3,17-dione by chemosynthesis, is the crucial intermediate for Gestodene in industry production. In the study, we investigated the bioconversion conditions of 13-ethyl-estr-4-ene-3,17-dione cl5a-hydroxylation by P. raistrickii(ATCC10490). Compared with chemosynthesis, bioconversion has advantage of stereospecificity, process simplicity and cost effectiveness.The dissolution of substrate, the key step in the bioconversion was focused on. 13-ethyl-estr-4-ene-3,17-dione has low solubility in aqueous solution, which would be difficult to hydroxylation. Six kinds of organic solvent, such as MeOH , EtOH . DMSO, were used to increase the solubility of 13-ethyl-estr-4-ene-3,17-dione with different amounts. The results showed that 4%MeOH and 5%DMS0 had the good effects on the conversion rate. Otherwise, a variety of dispersants, including Tween80 , β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin ,were used to help 13-ethyl-estr-4-ene-3,17-dione disperse in the aqueous solution, respectively. The results indicated that β-cyclodextrin was most efficient to improve conversion rate when molar ratio of β -cyclodextrin and substrate was 0.4:1. Although 2-hydroxypropyl- β -cyclodextrin was also a good dispersant, it is far more expensive than β -cyclodextrin which become a bottleneck in industry. Furthermore, dispersants are more effective to improve the solubility of 13-ethyl-estr-4-ene-3,17-dione than organic solvent. Using 4%MeOH together with 0.15% Tween80 to increase the solubility maybe the best choice .Fermentation conditions involved with nitrogen sources, media component, inoculum amount, initial pH, temperature, aeration, time to add substrate and concentration of substrate were studied using orthogonal design method. The results demonstrated that the optimal media for hydroxylation included 3.0% glucose,2.0%corn steep,0.2%KH2PO4, 0.2%K2HPO4, 0.05%KCl, 0.05%MgSO4,and 0.2%NaNO3 .The optimal inoculum amount was 2.5×108 spores in 50mL culture media. The optimal initial pH was 7.0 .The optimal aeration method was 250mL shake flask containing 50mL medium. Likewise, the optimal time to add substrate was at36h and the optimal concentration of substrate was 0.20%.In conclusion, bioconversion conditions of 13-ethyl-estr-4-ene-3,17-dione C15a-Hydroxylation by P. raistrickii were optimized. Under the optimal conditions, the conversion rate can be up to 60% in shake flask, which would overcome the disadvantage of 15a-Hydroxyl-13-ethyl-estr-4-ene-3,17-dione chemosynthesis and provide a good technics to industry.Furthermore, 15a-hydroxylation of 13-ethyl-estr-4-ene-3,17-dione using immobilized P. raistrickii entrapped in calcium alginate gel was studied. The conversion conditions, such as substrate concentration, the time to add substrate, conversion duration, which affected hydroxylation were investigated. As a result, the immobilized P. raistrickii can be utilized repeatedly five times without obvious decline of hydroxylation ability.Moreover, 15a-hydroxylation of 13-ethyl-estr-4-ene-3,17-dione in PEG/salt aqueous two-phase systems was studied.PEG 10000 and PEG20000 were screen out from PEG with different molecular weight, as well as magnesium sulfate from different kinds of salt. The results show that the high conversion rate will be achieved in PEG10000(PEG20000)/ MgSO₄ aqueous two-phase systems when concentration of MgSO₄ is lower than 5%.