| After dehydrogenation and double bond formation in C1,2 of steroid ring system, the anti-inflammatory activity of hormone-type medicines will be many times higher than that of the original one. At mild conditions, (such as low temperature, low pressure, etc.), the dehydrogenation reaction of steroid can be catalyzed by certain microorganisms with higher selectivity and productivity comparing with that of the chemical methods, so that the biodehydrogenation has become an important reaction in the production of hormone-type medicines.Beta-methasone, 16p-methyl -9α-fluoroprednisolone, was one of the most effective anti-inflammatory drug. The anti-inflammatory activity of beta-methasone is 35, 10 and 2.5 times higher than that of hydrocortisone, prednisone and dexamethasone, respectively. 17α-hydroxy-16β-methyl-pregn-l, 4, 9(ll)-triene-3, 20-dione (HMPTD) is an important intermediate in the production of beta-methasone, which is one of the key reactions in beta-methasone production and is a dehydrogenation product of 17 α-hydroxy-16 β-methyl-pregn-4, 9(ll)-diene-3, 20-dione (HMPDD) , which was catalyzed by Arthrobacter simplex. The reaction is as follows:The current status of steroids industry, the progress in microbial dehydrogenation of steroids and the applications of new technology used in the steroid biotransformation are reviewed, and the prospects of steroid medicines are discussed as well. Based on the fact that only a few reports on the bio-dehydrogenation of HMPDD, and low conversion ratio of HMPDD currently, the purposes of this work are: the strain screening, better fermentation conditions for the formation of 1-dehydrogenase, the improvement for efficient bio-dehydrogenation of HMPDD, the research and development of new technology for the bioconversion of HMPDD, and the kinetics of biotransformation processes.A strain 2-76 with higher bioconversion ratio of HMPDD was screened out bybioconversion test in shake bottle after strain separation and purification from 15 strains of Arthrobacter simplex. The strain 2-76 was further mutated by UV treatment arid Cs137-γ irradiation, and a better strain Q4-2-51 was obtained by isolating and rejuvenescention. The conversion ratio of HMPDD catalyzed by Q4-2-51 reached 86.63%, which was 8% higher than that of the original strain 2-76. The hereditary stability of this positive mutant was satisfactory.The effects of medium composition and culture conditions on the dehydrogenase activity of strain Q4-2-51 were evaluated experimentally. The optimized medium composition was as follows (g/L): glucose 4, corn steep liquor 12, peptone 3, KH2PO4 2. The optimized fermentation conditions for cell growth and formation of 1-dehydrogenase were as follows: initial pH value, pH7.0; inoculum volume, 20%; amount of inducer, 0.05g/L; and 100 ml medium in 250 ml shaken flask. Dehydrogenase production was enhanced by Co2+ and Mg2+, and slightly inhibited by Zn2+and Fe2+, but was markedly inhibited in the presence of Cu 2+ and Fe3+. The growth of Arthrobacter simplex and the production of dehydrogenase were also inhibited strongly by detergent and SDS. However, the addition of Tween 80 and a kind of defoamer, triatomic alcohol polyether, was beneficial for the enzyme production.The biotransformation conditions for the dehydrogenation of HMPDD were also investigated in depth, and the optimal conditions were obtained as follows: inoculum volume, 15%; loading amount, 100 ml in 250 ml shake vessel; initial pH value of medium, pH7.0~7.5, amount of inducer, 0.05g/L; amount of ethanol used to improve the solubility of steroid substrate, 7% (v/v); and the concentration of HMPDD should be kept at 0.7%. It was found that the addition of appropriate amount of cobalt chloride, external electron acceptor (NADP NAD and menadione), or Tween 80, had a considerable positive effect on the bioconversion ratio. A new strategy was proposed to perform the biotransformation: the fermentation broth was diluted with aseptic water in the ratio of l:l(v/v), and the conversion ratio was m...
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