| A classically versatile steroid intermediate,9a-hydroxyandrost-4-ene-3,17-dione(9-OH-AD),can be obtained by phytosterols(PS)bioconversion using Mycobacterium.In this study,9-OH-AD was obtained from PS through biotransformation using the cyclodextrin-resting cell system at a high substrate concentration.The results of the optimization of reaction conditions showed that precise control of temperature,oxygen supply,addition of sodium acetate during conversion and supplementation of cofactors(NAD,VB3,VB7)during the incubation of the bacteria could significantly inhibit the degradation of the products,while stabilization of pH and utilization of pharmaceutical grade HP-β-CD could significantly promote the initial reaction rate of 9-OH-AD.Based on the inhibitory effect of metabolic intermediates on Mycobacterium at high substrate concentrations,a series of metabolic intermediates were separated and identified,it was found that 4-ene-3-keto steroids,which are the first metabolites of sterol degradation,accumulated at the beginning of the bioprocess and had a remarkable inhibitory effect on bioconversion.The bioconversion rate was greatly improved when 5 g/L of macroporous adsorbent resin D101 was added to the reaction system in the initial phase.A certain amount of resin acted as a reservoir to remove the inhibitory intermediate in situ and facilitated the bioconversion process,and the 9-OH-AD space-time yield increased to 8.51 g/L/d,which was 23.15%higher than that without resin addition(6.91 g/L/d)after 72 h bioconversion.For the growing cell oil-water emulsion fermentation system,the results showed that the addition of B vitamins and emulsifier E-1360 could shorten the PS conversion cycle(8 d to 7 d)and increase the yield of 9-OH-AD(40.26%to 42.73%)at the fermenter level,and the percentage of the main product 9-OH-AD increased from 79.86 to 83.08%. |
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