Purification and properties of L-gulono-1,4-lactone oxidase from Grifola frondosa.
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 degrees 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-glucono-, 4; L-idono-, 3; L-galactono-1,4-lactone, 2; and 15 other lactones tested, 0. It is presumed that this enzyme is similar to animal GL-oxidase, ascomycetes D-arabinonolactone oxidase, etc.
Okamura
M
M
Laboratory of Biochemistry and Nutrition, Kanagawa Prefectural College of Nursing and Medical Technology, Yokohama, Japan.
D000363:Agaricales / Q000201:enzymology*; D001205:Ascorbic Acid / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050544:L-Gulonolactone Oxidase; D007783:Lactones / Q000378:metabolism; D008970:Molecular Weight; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
D007783:Lactones; D006861:Hydrogen Peroxide; D013401:Sugar Alcohol Dehydrogenases; D050544:L-Gulonolactone Oxidase; D001205:Ascorbic Acid