Screening Of Producing High-Yield Strains And Characterization Of Cholesterol Oxidase From Bacillus Cereus

Cholesterol oxidase, (COD, EC1.1.3.6), one of the most important enzymes in cholesterol metabolic process, it can catalyze cholesterol to cholest-4-en-3-one and hydrogen peroxide. In recent years, with the continuous deepening of the study, cholesterol oxidase has been growing emphasis on more applied clinical pharmaceutical, food processing, bio-pesticides and so on.In this paper, the study included:screening of producing high-yield strains, optimization fermentation conditions of producing COD by SG03 were investigated, cultivation conditions of SG03 in a 30L fermentor were studied, COD from the broth of SG03 was purified and the properties were studied.Firstly, the strain producing COD was screened from the different strain, the results showed that a strain Bacillus cereus could produce COD and the enzyme activity reached 0.4108 U/ml. Bacillus cereus was up to ultraviolet and diethyl sulfate (DES) of the compound mutagenesis, the enzyme activity of the strain SG03 reached 1.087U/ml after mutagenesis, improved 2.64 times than that of the original parent strain. The properties of the mutant strain were stable after six generations.The medium components and optimal fermentation conditions of SG03 to produce COD were researched by the single factor experiments and Response Suface Methodology. The suitable culture conditions were as follows:pH 7.0, temperature 30℃, rotation rate 200r/min, inocula amount 10% and cultivation time 24 h. The appropriated fermentation medium was as follow:glucose 5.43 g/L, peptone 7.11 g/L and cholesterol 3.04 g/L. Under the optimized cultivation conditiongs, the enzyme activity reached 1.463 U/mL, improved by 34.6% as compared with that under initial culture conditions.On the basis of the above experiments, the fermentation process of SG03 in a 30 L automatic fermenter was investigated. The growth of bacteria was into the stationary phase and the maximum biomass reached 14.13 g/L after 36 hours. The enzyme activity reached the maximum, 1.246 U/mL, after 40 hours. The initial pH value and the stirring speed experiments results showed that when the pH value was 7.0, the enzyme activity reached the maximum,1.271 U/mL, after 44 hours, when the stirring speed is 250r/min, the cholesterol oxidase production reached 1.317U/ml after 42 hours. From the determination of residual sugar concentration, when the fermentation time reached 48 h, glucose level was very low and the growth of bacteria entered the decline phase.The broth of SG03 was purified with ammonium sulfate precipitation, CM Sepharose FF chromatography and Sephadex G-75 chromatography. The specific activity of pure preparation reached 23.467 U/mg and the yield of enzyme activity was 21.6% with a 11.32 fold purification factor. The enzyme was found to have good pH stability from 5.0～8.0 and thermal stability, with 7.0 as the optimum pH of enzyme activity and 30℃as the optimum temperature. Author:Huang Yin (Microbiology) Supervised by Ge Fei...