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Cholesterol oxidase (COD, EC1.1.3.6) is one of the most important enzyme in the cholesterol metabolism in the process, it can catalyze cholesterol generated cholesteric 4 - en - 3 - one (Cholest-4-en-3-one) and hydrogen peroxide. In recent years, with the deepening of the study, cholesterol oxidase more and more people attention, more applied clinical pharmaceutical, food processing, bio-pesticides. The main content of the research include: cholesterol oxidase producing strain breeding; the SG03 strain enzyme production conditions optimization; the SG03 strain 30L fermentation process; preliminary study of the COD separation, purification and characterization. Initial enzyme activity higher strain is first screened from the different sources strain screening, rescreening series of experiments, and found that higher initial enzyme activity of the Bacillus cereus producing cholesterol oxidase, 0.4108 U / mL. Compound Mutation of Bacillus cereus UV diethyl sulfate (DES), mutagenic strains SG03 enzyme activity reached 1.087 U / mL, 2.64 times the original strain. Genetic stability detection experiment found that the strain passaged production of enzyme activity was relatively stable. SG03 strain as COD obtained composite mutagenesis producing bacteria, and conducted a series of its fermentation medium composition, culture conditions, the culture in the process of optimization. Single factor experiments, glucose is the best carbon source, peptone was the best nitrogen source, cholesterol levels 0.5%. Optimize culture conditions: pH of 7.0, the fermentation broth COD enzyme activity reached 1.153 U / mL; incubation temperature of 30 ℃, COD enzyme activity in the fermentation broth is 1.239 U / mL; the Shaker's better speed 200 r / min. Inoculation amount was 10%, the extracellular COD enzyme activity, of 1.203 U / mL. Strains cultured for 24 h, the extracellular enzyme activity reached a peak of 1.237 U / mL. The response surface optimization the test medium best group was divided into: glucose 5.43 g / L, peptone 7.11 g / L, cholesterol 3.04 g / L. Optimal medium and better training conditions, the enzymatic activity of cholesterol oxidase 1.463U/mL, 34.6% higher than the unoptimized. On this basis, using a 30L fermentor, SG03 strain fermentation the fermentation process for producing cholesterol oxidase. Found by the determination of biomass in the process of cell growth, the growth of bacteria into the stable phase, the maximum biomass reached 14.13 g / L at 36 h when. The SG03 strain extracellular secretion of cholesterol oxidase, fermentation 40 h late in cell growth with stable activity reached the highest value was 1.246 U / mL. pH, stirring rate of enzyme production test: When the pH was 7.0, the bacteria in the fermentation cultured for 44 h the highest activity of 1.271 U / mL, followed by enzyme activity began to decline; When stirring rate of 250 r / min, 42 h of fermentation cholesterol oxidase production reached 1.317 U / mL. Found that the result of the determination of the concentration of residual sugar in the fermentation process, glucose content has been very low, when the fermentation time of 48 h, cell growth also has entered a decline phase. SG03 fermentation supernatant passes through the 70% ammonium sulfate precipitation, CM-Sepharose FF ion exchange chromatography, and Sephadex G-75 gel chromatography purified cholesterol oxidase activity reached 23.467 U / mg, the purification factor was 11.32 times, the recovery rate was 21.6%. Preliminary study the enzymatic properties of the purified cholesterol oxidase enzyme optimum pH was 7.0, the enzyme activity was relatively stable between 5.0 to 8.0; optimum temperature of 30 ° C, the enzyme activity is relatively stable below 30 ℃.
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