Purification,Characterization And Application Of Cholesterol Oxidase From Recombinant E. Coli

Cholesterol oxidase (EC1.1.3.6, COD) is the key enzyme of degradation cholesterol, which rely on the coenzyme FAD, to catalyze cholesterol into 4 -cholesterol-3-one. The significant functions of cholesterol oxidase (EC1.1.3.6, COD) in food processing, medical assay and biological pest-resistance were recognized gradually and showed potentially use. This research is mainly focused on COD purification from recombinant E.coli, enzymatic properties, and application of COD.The extraction and purification of intracelluLar COD from recombinant E.coli were perfomed. COD was extracted by uLtrasonic treatment, 70% ammonium suLfate precipitation, and purified through DEAE Sepharose F.F, Sephadex G-75. The final specific activity of COD was 47.3 U/mg, and 45-fold concentration was obtained with enzymatic activity recovery of 21%. A single MDH protein band was shown on SDS-PAGE gel finally, the molecuLar weight of the enzyme was 50 KD measured by SDS-PAGE.Enzymatic properties and stabilities of extracted intracelluLar COD from recombinant E.coli ware studied. The enzyme optimum reaction temperature and pH were 37℃and 7.5, the enzyme has good stability when the temperature under 40℃. Meanwhile, the enzyme activity is relatively stable in the pH range from 6 9.The metal cation has a different impact on the enzyme activity, COD was strong inhibited by metal ions such as Ag+and Hg²⁺, while Ca²⁺, Fe3 +, Cu²⁺ had little effect on COD. The enzyme kinetic parameters Km and Vmax values were 8.2×10^-5 mol / L and 0.21 mmol / (L·min).The COD was applied to removal the cholesterol of egg yolk. The best mobile phase conditons of is acetonitrile:methanol:isopropyl alcohol 50:80:10,which is used for determination the cholesterol content of egg yolk by HPLC, and established the standard curve of cholesterol. At the same time, the effects was compared between uLtrasonic treatment and comparison, resuLts showed the separation rate of cholesterol by uLtrasonic treatment is 1.6 times than the comparison. The uLtrasonic treatment conditions were studied, the optimal volume and time of uLtrasonic treatment are 30mL, 15min. Under the optimum operational conditions: treatment time of 9h, COD concentration of 1.07u / g (COD content / egg yolk), the conversion rate of cholesterol was more than 80%. The major nutrients did not change after enzyme treatment, the oxidation products is 4-cholesterol-3-one.