Study On The Microbial △～1-Dehydrogenation Of Steroids In Organic Solvent/Water Two-liquid-phase System

In the paper, the organic solvent / water two-liquid-phase system was first applied to the Δ1 dehydrogenation of 17 α -hydroxy-16β -methyl-pregn-4,9(11)-diene-3,17-dione (HMPDD) by free and immobilized Arthrobacter simplex cell.The solubility of 17 a -hydroxy-16 0 -methyl-pregn-1,4,9(11)-triene-3,17-dione (HMPTD) and HMPDD in twelve organic solvents were determined. The results show that benzene, toluene, xylene, ethyl acetate and butyl acetate had higher solubility to HMPTD and HMPDD. The solubility of HMPTD and HMPDD in ethyl acetate is 39.7 g/L and 87.5 g/L respectively, which is the best among the five organic solvents. The solubility of HMPTD and HMPDD in butyl acetate is 32.6 g/L and 53.4 g/L respectively. Then, the effects of the five organic solvents to the viability of Arthrobacter simplex cell were investigated. It was found that the xylenehas the least effect to the viability of Arthrobacter simplex cell. At last, the biotransformation of HMPDD in two-liquid-phase system which is composed of the above five organic solvent and water was conducted. The result show that the conversion ratio of HMPDD is the best in butyl acetate / water two-liquid-phase system. The conversion ratio of HMPDD in this system is 51.8%.The biotransformation of HMPDD by free Arthrobacter simplex cell was investigated in butyl acetate / water two-liquid-phase system. It was found that the following factors, such as substrate concentration, Menadione sodium bisulfite concentration, biotransformation time and agitation speed have great effects on the conversion ratio of HMPDD. Then the conditions for HMPDD biotransformation were optimized by response surface analysis. Under optimum conditions, the conversion ratio of HMPDD was 73.3%, better than the conversion ratio before optimization.The biotransformation of HMPDD by immobilized Arthrobacter simplex cell was also investigated in butyl acetate / water two-liquid- phase system. Compared with other three kinds of cell immobilization methods, calcium alginate entrapment method was adopted for immobilization of Arthrobacter simplex cell. The immobilized conditions, activation time of the immobilized cell, the concentration of K2HPO4 in the activation medium and the biotransformation conditions of immobilized cell in two-liquid-phase system were optimized.In addition, according to the optical property of HMPDD and HMPTD, a new method was developed for the determination of HMPTD content.