Enhancement Of Recombinant Cholesterol Oxidase Expression By Fermentation Optimization

As an important enzyme, Cholesterol oxidase was widely used in medical testing, on the other side, Cholesterol oxidase also had prospect in food processing, health care, bio-pesticides, etc. With the problems that low cholesterol oxidase production and needing cholesterol as inducer when cholesterol oxidase product by natural microbial, an engineering strain, expressed cholesterol oxidase intracellular, was successfully constructed in the laboratory. However, cholesterol oxidase mainly expressed in the form of inclusion bodies, and cholesterol oxidase activity was low. In this paper, enhanced the soluble cholesterol oxidase expression by adjusting the inducer and optimization of fermentation process, then, established a series of high cholesterol oxidase expression technology, which was conducive to achieving the cost-efficient preparation of cholesterol oxidase.In this study, engineering strain E. coli BL21 (DE3), has the ability to intracellular express cholesterol oxidase, selected for cholesterol oxidase production. Internal and external causes combined optimization strategy was carried out to reduce the inclusion body formation and improve the yield of soluble cholesterol oxidase production.1. Increased the proportion of soluble cholesterol oxidase by lactose induction and optimized the conditions of cholesterol oxidase expression induced by lactose. Induced by lactose, the activity and productivity of cholesterol oxidase were 0.76U/mL and 0.45 U/ (mL·h); 4.0 and 2.4 times the corresponding value by IPTG induction, respectively. Under the optimized condition, activity of cholesterol oxidase reached 5.8 U/mL,7.6 times that under the initial condition.2. Increased soluble cholesterol oxidase production yield by change temperature and pH in induction phase, and established the Two-phase culture strategy. Studied the optimal temperature and pH of cell growth and induction phase, results showed that the optimum condition of cell growth phase were 37℃, pH7.5 and induction phase were 30℃, pH6.5. Strategies for using Two-phase culture works, cholesterol oxidase activity and productivity were 16.9U/mL, and 1.30 U/(mL·h), increased by 191% and 347%. Although the yield of recombinant enzyme decreased to 32%, soluble cholesterol oxidase reached 68% of the total target protein, significantly increased the soluble cholesterol oxidase expression level.3. Studied the culture strategies of engineering bacteria in 7L'fermenter to further improve the yield of cholesterol oxidase activity. Compared four carbohydrate supplement strategies, results showed that induction by lactose after Fed-batch culture, using glucose as the carbon source, maximum activity of cholesterol oxidase reached 20.5 U/mL, which was 21% higher than the corresponding value of batch fermentation. In induction phase, adopted pH-stat to feed the mixed induction solution, enzyme activity reached 25.8 U/mL,26% higher than that of feeding induction solution on constant feeding strategy.