An extracellular cholesterol oxidase producer, Pseudomonas aeruginosa strain PA157, was isolated by a screening method to detect 613-hydroperoxycholest-4-en-3-one-forming cholesterol oxidase. On the basis of a putative cholesterol oxidase gene sequence in the genome sequence data of P. aeruginosa strain PAO1, the cholesterol oxidase gene from strain PA157 was cloned. The mature form of the enzyme was overexpressed in Escherichia con cells. The overexpressed enzyme formed inclusion bodies in recombinant E. coli cells grown at 20 degrees C and 30 degrees C. A soluble and active PA157 enzyme was obtained when the recombinant cells were grown at 10 degrees C. The purified enzyme was stable at pH 5.5 to 10 and was most active at pH 7.5-8.0, showing optimal activity at pH 7.0 and 70 degrees C. The enzyme retained about 90% of its activity after incubation for 30 min at 70 degrees C. The enzyme oxidized 3 beta-hydroxysteroids such as cholesterol, beta-cholestanol, and beta-sitosterol at high rates. The K-m value and V-max value for the cholesterol were 92.6 mu M and 15.9 mu mol/min/mg of protein, respectively. The V-max value of the enzyme was higher than those of commercially available cholesterol oxidases. This is the first report to characterize a cholesterol wddase from P. aeruginosa. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.