Abstract
The extracellular cholesterol oxidases produced by an inducible mutant, U-S-A-18, and a constitutive mutant, U-S-3011, of Arthrobacter simplex were purified 18.9- and 15.2-fold as to specific activity, respectively, from the fermentation broth through successive purification steps involving DEAE-cellulose column chromatography, ultrafiltration and gel filtration on Sephadex G-75. The overall yields of the purified enzymes were 28% and 40%, respectively. Both the purified enzymes were confirmed to be homogeneous, and the molecular weights of both enzymes were estimated to be 57, 000 on gradient SDS-polyacrylamide gel electrophoresis. Both the enzymes were stable in the pH range of 6 to 10 at 30°C for 2hr. The optimal pH and temperature for both enzymes were found to be 7.5 and 50°C, respectively. The Km values of the enzymes were 28.4 μM and 30.8 μM, respectively, when cholesterol was the substrate. The activities of both enzymes were remarkably stimulated by Triton X-100 and partially inhibited by sulfhydryl reagents. Due to the similarities of the properties of the two enzymes, it is concluded that the inducible and constitutive cholesterol oxidases produced by the mutants, U-S-A-18 and U-S-3011, are similar in nature.
