Formation and Purification of a New Enzyme, Glycerol Oxidase and Stoichiometry of the Enzyme Reaction

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Glycerol oxidase, a novel glycerol oxidizing enzyme, has been found in several strains belonging to genera Aspergillus, Neurospora, and Penicillium. Aspergillus japonicus AT 008 showed significantly high enzyme activity. Glycerol oxidase was formed in the mycelia of the fungi grown on glycerol as a carbon source. The enzyme was purified from the cell-free extract of Aspergillus japonicus AT 008 by a procedure involving fractionation with ammonium sulfate, column chromatographies on DEAE-Sephadex A-25 and hydroxylapatite, and gel filtration on Sephadex G-200. The overall purification was approximately 190-fold with a yield of 35.5%. The purified enzyme gave a single band on acrylamide gel electrophoresis. The enzyme catalyzed the oxidation of glycerol using molecular oxygen as a primary electron acceptor and producing glyceraldehyde and hydrogen peroxide according to the following scheme: CH₂OHCHOHCH₂OH+0₂→CHOCHOHCH₂OH+H₂O₂.