Establishment And Optimization Of Efficient Biotransformation System For 22-hydroxy-23,24-bisnorchol-4-ene-3-one Production

The preparation of hydroxy-23,24-bisnorchol-4-ene-3-one(HBC)from phytosterols(PS)by mycobacteria is an advanced production method of steroid drug intermediates.In this study,on the basis of the previous cyclodextrin-resting cells transformation system,the growth medium of mycobacteria was optimized,and the biomass and biological activity of the bacteria were increased by 10.4%and 2.4%,respectively.Then the culture conditions of the bacteria were optimized in the fermenter,so that the culture time of the bacteria was shortened from 96 hours to 72 hours,and the biomass of the bacteria was increased by 10.2%.Through the optimization of resting cells transformation conditions and the analysis of product morphology,it was found that when the transformation system of mass ratio of hydroxypropyl-β-cyclodextrin(HP-β-CD)and PS decreased to 1:1,HBC was mainly precipitated by crystal.After the transformation,the product HBC was separated from the transformation system by low speed(900×g)centrifugation,so as to realize the recovery of resting cells and HP-β-CD.Based on this,an efficient and circulating system for the transformation of high concentration PS by mycobacterial resting cells was established.Three continuous cycles of 80 g/L PS transformation can be realized without the addition of resting cells and HP-β-CD.The conversion rate of single batch substrate was over 92%,and the product HBC recovery rate was over 92%.This strategy could significantly reduce the production cost and simplify the downstream purification process of the product,and reduce wastewater discharge.In addition,it was found that there was no production of the by-product 1,4-HBC in the process of PS transformation by mycobacteria growth cells.In order to reduce the by-product production and study the mechanism of by-product production,we preliminarily explored the growth cells transformation system of mycobacteria.Finally,when 50 g/L phytosterol was transformed,there was no 1,4-HBC in the products and the yield of HIBC reached 34.37 g/L.