ISOLATION AND IDENTIFICATION OF CHOLESTEROLOXIDASE PRODUCING Pseudomonas Aeruginosa

Abstract

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.