Studies On The Microbial Dehydrogenation Of 17α-hydroxy-16β-methyl-pregn-4,9(11)-diene-3,20-dione By Arthrobacter Simplex

17 α-hydroxy-16 β-methyl-pregn-4,9(11)-diene-3,20-dione was one of the important intermediates used to manufacture beta-methasone (16 ?-methyl -9 a -fluoroprednisolone) which was one of the most effective anti-inflammatory drug. The anti-inflammatory activity of beta-methasone was 35 times than that of hydrocortisone and 2.5 times than dexamethasone.The commercial production of beta-methasone involves several steps from the precursors (Diosgenin and Tigogenin). One of the important step needed A l -dehydrogenation by Arthrobacter simplex. The reaction as follows:Firstly, the current status of steroids industry > development of the steroids resource > progress on the microbial dehydrogenation of steroids and applications of new technology used on the steroid bio-transformation were reviewed and the prospects were described as well in this paper.Strain Q2-2-2-1 was obtained after isolation and rejuvenescention by UV treatment and Cs137- Y irradiation. The production capability was about 86.54%, which was 5% higher than that of the original strain 02-7-6-14 and the strain had hereditary stability.In this article, the effects of the medium composition and the environmental factors on dehydrogenase production were investigated. The optimal medium composition, which was selected by employing single -factor and orthogonal optimization procedures, was as follows (g/L): 4 glucose, 12 corn steep liquor, 3 peptone, 2 KH2PO4. The optical fermentation conditions were: medium's initial pH value 7.0 ~ 8.0, the inoculum amount 20%, loading amount 100 ml in 250 ml shaken flask, amount of inducer 5 mg/100 ml. Dehydrogenase production was promoted by Co2+ ion and Mg2+ ion, but enzyme production was markedly inhibited in the presence of Cu2+ ion and Fe3+ ion, and somewhat inhibited by Zn2+ ion and Fe2+ ion. The growth of Arthrobacter simplex and dehydrogenase production were inhibited strongly by detergent and SDS. However, Tween 80 and pao-di (triatomic alcohol poly ether) benefited to the enzyme production.Studies on bio-transformation conditions of beta-methasone intermediate 17 α -hydroxy-16 β-methyl-pregn-4,9(11)-diene-3,20-dione were investigated in depth and optimized as follows: inoculum size was 20% , loading amount was 100 ml in 250 ml shake vessel , initial pH was 7.0 ~ 7.5 , amount of inducer was 10 mg/L, amount of ethanol used to improve the solubility of the substrate was 7%, substrate concentration was 0.7%. Optimum concentration of cobalt chloride ^ external electron acceptor (NADP> NAD and menadione) and Tween 80 had considerable effect on the conversion. It was the first time to apply diluting cell culture brothes process to 17 α -hydroxy-16 β -methyl-pregn-4,9(11)-diene-3,20-dione dehydrogenation by Arthrobacter simplex. Aseptic water was selected as the diluent. When the ratio of dilution was 1:1, the conversion ratio was found to be comparable with that of the conventional process, so the production costs could be greatly decreased. The effects of ultrasonic irradiation on free cell and substrate were also investigated. Coupled with the process byKdiluting cell culture brothes, substrate which dissolved in ethanol and then precipitated in the sterile water was irradiated by ultrasound. The conversion rate was over 92.54%. It was found that ultrasound irradiation could induce dispersal of cluster of the substrate.Tests in 10 L fermentor were also carried out. It was found that the conversion cycle could be shortened by enhancing the aeration. In the test the conversion rate was about 86.7%,Studies on kinetic behaviors of transformation of 17 a -hydroxy-16 J3 -methyl-pregn-4,9(ll)-diene-3,20-dione by free cell of Arthrobacter simplex were also investigated, including: the effect of initial substrate and the enzyme concentration on initial rate, the stability of the enzyme, the time course of A '-dehydrogenation of the transformation process. Based on the experiment results, a kinetic model for the dehydrogenation of 17 a -hydroxy-16 {3 -methyl-pregn-4,9(ll)-diene...