Purification And Characteristics Of The Strain Screening, Cholesterol Oxidase, Identification And Enzyme Studies

Cholesterol Oxidase [COD, EC 1.1.3.6] is one of key enzymes to assay the content of cholesterol of blood in the method of enzyme linked immunosorbent assay. With its higher sensibility and specificity, it has already applied to in clinical diagnosis.Any kinds of Cholesterol Oxidase from different microorganism differ from others in the principle of action and their characterizations, so as their nutriton and fermentative conditions. In order to know much about its enzymological characterizations, the strain which could producing Cholesterol Oxidase was screened; optimization fermentation conditions of producing Cholesterol Oxidase by Brevibacterium sp. were investigated; Cholesterol Oxidase from Brevibacterium sp. was isolated, purified and its properties were studied. Major results were as follows:1. A strain Brevibacterium sp., which could produce cholesterol oxidase, was screened from 23 clones.2. Brevibacterium sp. can produce relative high Cholesterol Oxidase (COD). The optimization of cultivation conditions was selected through fermentation tests. The highest enzyme activity was obtained 20h at 24℃ and 200 r/min, when 50mL of the culture in 250mL shake flasks was grown in 2g/Lcholesterol, 3g/Lsucrose, 2g/L yeast extract, 3g/L peptone,3g/L beef extract, 1g/LK₂HPO₄, 0.5g/L MgSO₄, 0.2% cholesterol. The initial pH was adjusted to 6.8. The inoculums volume was 5% (V/V). Under the optimized cultivation conditions, the enzyme activity reached 2439U/L.3. Brevibacterium sp. was cultivated with inducement of substrate including cholesterol. Using the techniques of ammonium sulfate precipitation for crude extraction, cholesterol oxidase was purified to electrophoretic homogeneity from liquid cultured cells of Brevibacterium sp.through the following separation procedures which including CM-Sepharose FF chromatography and Sephacryl S-200 chromatography. The specific activity of pure preparation reached 38.904U/mg, and the yield of enzyme activity is 38.2% with a 14.9-fold purification factor. The purified cholesterol oxidase migrated as a single protein band on reduced / non-reduced SDS-PAGE.4. The purified cholesterol oxidase was studied, and its native molecular masses was about 56.0kDa, measured by Sephacryl S-200 chromatography. Molecular weight of Single peptide chain at non-reduced SDS-PAGE conditions demonstrated 59.0kDa. The enzyme was found to have good pH stability from 5～9.5 and thermal stability, with 6.5 as the optimum pH of enzyme activity and 54℃ as the optimum...