ISOLATION an‎d IDENTIFICATION OF CHOLESTEROL OXIDASE PRODUCING Pseudomonas Aeruginosa

Cholesterol oxidase (EC:1.1.3.6) is industrially and commercially important for the application in bioconversions for clinical determination of total or free serum cholesterol, and in food industry, for this reason this study was aimed to isolate cholesterol oxidase producing Pseudomonas aeruginosa from different local sources, by taking 55 swap samples from different serological samples includes ears, urine, burns, wounds and sputum, in addition to another 35 environmental samples collected from soil and water from different locations in Baghdad governorate. From these samples 115 bacterial isolate were obtained, and among them only 60 isolate were identified as P. aeruginosa according to the results of biochemical tests and Api 20E system. Ability of these isolates in cholesterol oxidase production was screened in Lauria-Broth medium. Results showed that all of these 60 isolates were able to produce cholesterol oxidase with variable capacities, among them P.aeruginosa H48 was the most efficient isolate in enzyme production. The activity of crude enzyme in culture filtrate of this isolate was 1.71 U/ml. Cholesterol oxidase produced by P.aeruginosa H48 was purified by ammonium sulphate precipitation (70% saturation), dialysis and ion exchange chromatography using CM– cellulose.The enzyme activity of the partially purified enzyme was increased to 3.87 U/ml. Optimum pH and temperature for activity and stability of the partially purified enzyme were estimated. Results showed that the Optimum pH for enzyme activity and stability was pH7.0 and pH 6.5 respectively, while the optimum temperature for enzyme activity and stability was 35 and 37°C respectively.