Molecular characteristics of the cholesterol oxidase and factors influencing its activity

Abstract The purpose of this study was to characterize Pseudomonas cholesterol oxidase (cholesterol:oxygen oxidoreductase, EC 1.1.3.6) and define factors influencing its activity with a view to performing electrochemical determinations of cholesterol. The enzyme was purified by gel filtration and its molecular characteristics were determined. Its amino acid composition presents a high proportion of glutamic and aspartic acids. Its isoelectric point is between 5.0 and 5.5 unit/pH and its molecular mass is equal to 65.1 kDa, as determined by analytical ultracentrifugation. The influence of reaction products on enzyme activity was studied. The amount of hydrogen peroxide produced is not great enough to inhibit the enzyme. Adding exogenous flavin adenine dinucleotide increases enzyme activity. Bile salts stabilize cholesterol oxidase structure increasing its activity without changing its affinity for cholesterol.