Purification and Properties of the Glucose Oxidase from Aspergillus niger

Abstract

Glucose oxidase (/3-n-glucose : 02 oxidoreductase, EC 1.1.3.4) has been highly purified from the extracts of three fungi, Penicillium not&urn (1, 2), Penicillium amagasakiense (3, 4), and Aspergillus niger (5, 6).It has also been identified and sometimes partially purified from a number of other sources.Some of these sources are reviewed by Schepartz and Subers (7).Recently Pazur and Kleppe ( 6) have studied the specificity, and Gibson, Swoboda, and Massey (8, 9), the kinetics and mode of action, of the glucose oxidase from A. niger.Pazur, Kleppe, and Ball (10) have made the interesting discovery that this enzyme is a glycoprotein.It has generally been assumed that the glucose oxidases from different sources have the same properties.In this paper some of the physical and chemiral properties of t)he enzyme purified from A. niger are examined and compared with published data for glucose oxidases from other fungal sources.It is felt that the properties of the glucose oxidases isolated from P. not&urn (2, 11) and P. amagusakiense (4, 12) are sufficiently different from those of the il.niger enzyme to warrant more detailed intercomparison in the future.EXPERIMEiW.4LPROCEDURE Xaterials-Dee-O-Concentrate, an extract of .,I. niger mycelia (13), was obtained from the Takamine Laboratories, Miles Chemical Company, New *Jersey.n-Amino acid oxidase was prepared from fresh hog kidneys (14).Catalase with 1260 Keilin units per g was obtained from L. Light and Company, Ltd., Bucks, England.FAD was obtained from Calbiochem and purified by chromatography on DEAE-cellulose, as described previously by Massey and Swoboda (15).DEAEcellulose was manufactured by Whatman and Company, Ltd. (Catalogue No. DE-50).Amberlite CG-50 was obtained from The British Drug Houses, Ltd., Dorset, England.Phenvlmercurie acetate of microanalytical grade was a product of Hopkins and Williams, Ltd., Essex, England.Glass-distilled water was used to make up all solutions.Pretreatment of Zon Exchange agents-To obtain reasonable flow rates with the Amberlite CG-50 resin, fine particles that did not sediment out in water in 40 minutes were siphoned off and discarded.Columns of DIS.-ll+cellulose were washed with 0.2 N NaOH to remove a yellow cont,aminant.The ion exchange agent was then washed successively with 0.2 N HCl, 0.2 ~\i NaOH, and water, and finally it was equilibrated with the buffer to be used in chromatography.Amberlite CG-50 was treated in a like