Purification and properties of L-gulono-1,4-lactone oxidase from Grifola frondosa

Abstract

L-Gulono-1,4-lactone oxidase activity was detected in G. frondosa; therefore its properties were studied after purification. A 766-fold purified preparation of the enzyme from fresh fruit bodies was obtained by means of a seven-step procedure, the overall yield being 14%. The purified enzyme gave a single band on polyacrylamide gel electrophoresis and its absorption spectrum exhibited the characteristic of a flavoenzyme. The enzyme produced L-ascorbic acid and H2O2, with L-gulono-1,4-lactone (GL) as the substrate and oxygen as the electron acceptor, and was optimally active at around pH 7.0 and 45°C. Its molecular mass was determined to be 250 kDa on gel filtration, while the dissociated enzyme exhibited a molecular mass of 69 kDa on SDS-polyacrylamide gel electrophoresis, but the true molecular weight is unknown because of the trypsin treatment in the purification process. The apparent Km value for GL was 24±1 mm. Its substrate specificity was extremely high and, assuming that for GL to be 100, the following results were obtained: D-mannono-, 25: D-glu-cono-, 4: L-idono-, 3: L-galactono-1,4-lactone, 2: and 15 other lactones tested, O. It is presumed that this enzyme is similar to animal GL-oxidase, ascomycetes D-arabinonolactone oxidase, etc.