Research On The Reduced Products Accumulation In Phytosterol Conversion By Mycobacteria In Cyclodextrin System

The cyclodextrin was usually used to increase the reaction rate in sterols bioconversion because of the extremely low solubility of sterols.In this project,hydroxypropyl-β-cyclodextrin(HP-β-CD),as the cosolvent of sterols,significantly decreased oxygen transfer rate in aqueous phase though it largely increased the bioconversion efficiency of phytosterol in resting cells-cyclodextrin system with Mycobacterium neoaurum NwIB-R10,which caused the oxygen shortage in the biotransformation and further the surplus intracellular NADH,and NAD+ was regenerated by the formation of the reduced product,HBC(22-hydroxy-23,24-bisnorchol-4-ene-3-one),as the final carrier of electron.The following experiments were carried out to solve the problem of surplus NADH because of oxygen shortage.One the one hand,the improved oxygen supply by increased shaking speed slighty decreased the ratio in a low reaction rate per unit cell and concentration of HP-β-CD.On the other hand,ammonium was substituted for nitrate with higher oxidation state,which increased the ratio of AD(androst-4-ene-3,17-dione)/HBC maximally from 2.1 to 5.3 and nitrate utilization led to the decreased NADH/NAD+maximally by 59.5%.The ratio decreased from 66.4%to 52.6%in fed-batch by decreasing the reaction rate.Besides,the ratio decreased from 68.9%to 43.9%by overexpression of 9α-ketosteroid hydroxylase(KshA)in Mycobacterium neoaurum NwIB-R10 through the additional consumption of NADH,and continued to decrease to 22.3%by increasing KshA activity.