Novel Microbial Biotransformation Technique In Hydroxylation And Dehydrogenation Processes Of Epoxyprogesterone

Adrenal cortical hormone-type medicines are a main class of steroids and have become the important category of pharmaceuticals used clinically in recent years. Generally,the drugs are synthesized by the combination of chemical reactions and microbial biotransformation using the natural steroids as the initial substrates.The two steps of biotransformation,namely C₁₁-hydroxylation and C_1,2-dehydrogenation are crucial in the whole synthetic route.Research indicates that C₁₁-hydroxylation is an important group to behave anti-inflammatory activity,and after dehydrogenation and double-bond formation in C_1,2 of steroid ring system,the anti-inflammatory activity of hormone-type drugs would be many times higher than that of the original ones.In this dissertation,the reactions of C₁₁-hydroxylation by Rhizopus nigricans cells,C₁₁β-hydroxylation by Cunninghamlla blakesleeana ATCC 8688a cells and C_1,2-dehydrogenation by free and immobilized Arthrobacter simplex cells were studied in detail.Some novel techniques were explored for process optimization.Firstly,Rhizopus nigricans cells were used to catalyze epoxyprogesterone to 11α-hydroxyepoxyprogesterone.The fermentation conditions were optimized and a novel water-suspending feed mode containing 2.0%sodium dodecylbenzenesulfonate was proposed.Using the optimized medium and feed mode,when the feed concentration was as high as 15g/L,the substrate conversion in the 3.7L fermentor could achieve 49.2%,which shows the potential of the bioconversion in the large-scale production.Secondly,in the process of C₁₁β-hydroxylation by Cunninghamlla blakesleeana ATCC 8688a cells,the conversion product was purified and identified.The characterization results indicated the main product was 11β-hydroxyepoxyprogesterone.However,some byproducts were observed and low activity of hydroxylase was measured.The operation parameters were further optimized to increase the 11β-product yield.Using the optimized medium and feed mode,the yield in the 10L fermentor was stabilized at 35%when feed concentration was 2g/L.Thirdly,Arthrobacter simplex cells were assayed for the ability to convert 11α-hydroxyepoxyprogesterone to 11α-hydroxy-16α,17α-epoxy-pregenene-1,4-diene- 3,20-dione.Tween-80 emulsification feed mode was proposed and Response Surface Methodology(RSM) was used for the dehydrogenation process optimization.Then the suitable aqueous/n-hexane two-liquid-phase system was established.The microcapsule immobilized cells by sodium cellulose sulphate (NaCS)/poly-dimethyl-diallyl-ammonium chloride(PDMDAAC) were used on bioconversion in an air-lift internal loop reactor under the aqueous/n-hexane two-liquid-phase system.Using optimized tween-80 emulsification feed mode,when the time of reaction was 2h,the conversion was as high as 97.54%.In the semi-continuous production,the cultivation with the encapsulated cells was carried out for 5 batches and the conversion in each batch was above 95%,and the activity of enzymes was maintained quite high.Finally,Rhizopus nigricans cells and Arthrobacter simplex cells were used to conduct the mixed fermentation,converting epoxyprogesterone to 11α-hydroxy-16α, 17α-epoxy-pregenene-1,4-diene-3,20-dione by one-pot catalysis.The suitable pH of reaction medium was 6.0.When the substrate concentration was 2g/L,after 5 batches culture,the conversion remained quite high,which showed the stability of the novel biotransformation process.