A mutant producing a pyranose oxidase, which has a higher thermal stability and lower K-m values for D-glucose and 1,5-anhydro-D-glucitol than those of the wild type enzyme, was obtained. A single amino acid substitution Lys for Glu at position 542, had occurred. This altered enzyme, E542K, was not only stable at 55 degrees C, which was 5 degrees C higher than the wild-type enzyme, but was stable in alkaline solution at pH 8.0-11.0. K-m values of E542K for D-glucose and 1,5-anhydro-D-glucitol were 0.7 mM and 14.3 mM, respectively, in contrast with 1.4 mm and 35.3 mM for the wild-type enzyme. A little effect was observed in k(cat) values, and improvement in reactivity was mainly due to the decreases in K-m values. This altered pyranose oxidase is useful for food analysis and diagnosis.