Production, Purification And Primary Application Of The Cholesterol Oxidase From Rhodococcus Equi 4-2

The significant functions of cholesterol oxidase in food processing, medical assay and biological pest-resistance were recognized gradually and showed more and more potential. In this paper, four topics were investigated: (1) the optimization of the culture media; (2) the comparisons of different extraction conditions and purification methods, and investigations of some physical properties of the extracted endoenzyme; (3) primary applications in food processing and pest-resistance; (4) the acute toxicity test of the endoenzyme and the microbe cells.The periods of cell growth or endoenzyme production were 72 hours. Best nitrogen source was proved to be tryptone. The optimal culture media was 2g/L cholesterol,1ml/L Tween 80 without glycerol, determined by orthogonal array. Further orthogonal regression experiment showed the optimized conditions as 2.192g/L cholesterol and 4.868ml/L Tween 80, and the enzyme activity was predicted to be 610 U/Lwith a high increasing rate of 141%.The potassium phosphate buffer containing 0.5%Triton X-100 could efficiently extract the endoenzyme from within the cells, and the operation time was 10 minutes properly. The extraction effects of sodium chloride solution and phosphate buffer without Triton X-100 were much worse than phosphate buffer containing Triton X-100. The enzyme assay system containing 1% Triton X-100 showed the best color of red with fine stability and the highest enzyme activity.The enzyme protein contained high level of saccharide, the proportion of saccharide and protein was 3.4: 1. The optimal pH for enzyme reaction was 7.5, and the enzyme remained stable within pH5.0 to 10.0. The enzyme had excellent thermal stability lower than 40℃.The microbe cells, endoenzyme and the culture supernatant could all decrease the amounts of cholesterol in egg yolk, the decreasing rate were 49.26%, 40.92% and 37.05% respectively. They also showed high pest-resistant activity, and could strongly inhibit the growth of the cotton boll worms. After 15 days feeding the fatality rate were 100%, 66.7% and 40.0% respectively.The endoenzyme was classified as no toxicity with the median lethal dose (LD50) higher than 21500 mg/kg, moreover, the microbe cells hadn't begotten the acute toxic effects of the biomass lowerthan 1.72×109cfu/ml.