New Technology Research On Steroid Of 11β-hydroxyl Medroxyprogesterone Dehydrogenation By Arthrobacter Simplex

The C_1,2 dehydrogenation of pregna-4-ene-6-methyl-3, 20-dione-11, 17-dihydroxy （11β-hydroxyl medroxyprogesterone） is a key step to synthesis Methyllprednisolone. The dehydrogenation reaction of steroid can be catalyzed by certain microorganisms with higher selectivity and productivity at mild conditions comparing with that of the chemical methods. The substrate was catalyzed by Arthrobacter simplex. The reaction is as follows:Based on the poor solubility of streiods substrate in aqueous media and the inhibiton of products encountered in microorganism dehydrogenation, the new technology of 11β-hydroxyl medroxyprogesterone dehydrogenation by Arthrobacter simplex was studied. With microemulsion and ionic liquids as reaction medium, the composition of systems and process optimization were investigated. The composition of the system and factors in microemulsion were investigated. The experiments indicated that the system was constructed with Arthrobacter simplex UR016 culture broth as aqueous phase; alcohol （7% v/v） was used to dissolve substrate; 4 g/L Tween-80 as surfactant and 10 g/L soybean oil as oil phase adding to aqueous phase. In this Tween-80/ alcohol/ soybean oil/ culture broth microemulsion, the optimal bioconversion temperature was 33℃, with the maximum conversion rate of 88.6% at 46 h in microemulsion system under 4 g/L substrate concentration, an increase of 66.2% compared to that in aqueous system.Two-phase system of Tris-HCl/toluene was constructed, with hydrophilicity ionic liquids as co-solvent adding to aqueous phase. Comparing study on [Bmim]（L）Lac、[Bmim]BF₄、[Emim]BF₄、[Hmim]BF₄、[Bmim]OTF. It was found that [Bmim]（L）Lac as co-solvent could promote cell growth, increase enzyme activity and substrate solubility in aqueous. When the aqueous phase containing 2%（W/V） [Bmim] （L）Lac and 50 g/L wet cells, 10 g/L of steroid in organic phase, the reaction rate reached 80.9% after bioconversion for 16 h.The further experiments on dehydrogenation reaction of 11β-hydroxyl medroxyprogesterone in biphasic system with hydrophilicity ionic liquids as co-solvent were developed. The effects of anion and cation on bioconversion were studied. It was found that 2% [Bmim]BF₄ and 10 g/L calcium lactate adding to aqueous, the conversion ratio was 80.2%, equality with 2% [Bmim]（L）Lac. The immobilized cells with calcium alginate/PEG catalysised dehydrogenation in the optimal syetem: cell concentration and reaction time were 1.00 g/mL and 16h; the conversion ratio could be reach 87.12% under substrate concentration 10g/L.