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Enhancement of Recombinant Cholesterol Oxidase Expression by Fermentation Optimization

Author: LiChuang
Tutor: WangWu
School: Jiangnan University
Course: Fermentation Engineering
Keywords: Cholesterol oxidase Recombinant E. coli Lactose -induced fermentation Two-way training strategy Constant pH-stat Soluble expression
CLC: TQ925
Type: Master's thesis
Year: 2010
Downloads: 35
Quote: 0
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Abstract


Cholesterol oxidase is an important enzyme detection, in addition to widely used in medical testing also has potential applications in the development of food, health care, bio-pesticides. View of the microbial sources of cholesterol oxidase expression in a small amount of need cholesterol-induced problems, our laboratory has successfully built an intracellular expression of genetically engineered bacteria for cholesterol oxidase, cholesterol oxidase exists mainly in the form of inclusion bodies active Low cholesterol oxidase expression. Inducers of this article be taken to adjust and optimize the fermentation process and other means to improve the activity of cholesterol oxidase yield, efficient expression technology to establish a series of cholesterol oxidase, contribute to the achievement of cholesterol oxidase efficient low-cost preparation. Cholesterol oxidase to an intracellular expression engineered bacteria E. coli BL21 (DE3)-PET28a-COD production strains to reduce the formation of inclusion bodies, increase the activity of cholesterol oxidase production rate target, using external factors to optimize internal optimization combined strategy to optimize the fermentation process of cholesterol oxidase. By the lactose induced cholesterol oxidase expression increased proportion of cholesterol oxidase soluble expression, and optimize the conditions of the galactose-inducible expression of cholesterol oxidase. Galactose-inducible expression of cholesterol oxidase enzyme activity were 0.76U/mL, 0.45U/mg; IPTG induction 4.0 and 2.4 times, significantly increase the level of expression of the activity of cholesterol oxidase. Cholesterol oxidase expression induced induced under conditions optimized, activity reached 5.8U/mL, the initial conditions of 7.6 times. 2. Adjust the temperature and pH of the induction phase to improve the activity of cholesterol oxidase yield, to establish two-way training strategy. The optimum temperature and optimum pH for cell growth and induction phase, results showed that the optimum conditions for cell growth stage: temperature 37 ° C, pH 7.5; induction phase optimal conditions: temperature 30 ° C, pH 6.5. Two-way training strategy cultured engineered bacteria, cholesterol oxidase activity eventually reach 16.9U/mL production strength to 1.30U / (mL · h) were increased by 191%, 347%; recombinase expression accounts for the total bacterial 32% of the protein, the soluble fraction accounted for 68% of the amount of cholesterol oxidase expression significantly improve the soluble expression levels of cholesterol oxidase. 3.7L fermenter research the engineered bacteria cultured strategy to further improve the activity of cholesterol oxidase yield. Compared four carbohydrate supplement strategies, determine the use of glucose fed to late logarithmic growth phase, constant speed flow plus the lactose induction medium induced the recombinase expression the maximum enzymatic activity 20.5U/mL, a 21% increase in cultured cells. Formulated pH-stat fed-glycerol lactose mixed induced fluid induced by engineering bacteria to express the strategy of cholesterol oxidase enzyme production reached 25.8U/mL, a 26% increase.

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