Purification and characterization of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus.

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Abstract
From membrane fraction of Gluconobacter melanogenus IFO 3293, 2-keto-D-gluconate dehydrogenase was solubilized and purified to an electrophoretically and ultracentrifugally homogeneousstate of about 400-fold in high yields of 41%. Purification was achieved by solubilization with 2% cholate and 0.2 M KCl, subsequent precipitation with ammonium sulfate and polyethylene glycol 6000, and chromatographies on CM-cellulose and hydroxyapatite columns in the presence of Triton X-100. The purified enzyme was tightly bound to a c-type cytochrome existing as a dehydrogenase-cytochrome complex. The molecular weight of the enzyme was determined to be about 133, 000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of three components having molecular weights of 61, 000 (flavoprotein), 47, 000 (cytochrome c) and 25, 000. The dehydrogenase was found to be a flavoprotein having a covalently bound flavin. Only 2-keto-D-gluconate was readily oxidized by the enzyme in the presence of dyes, such as ferricyanide, 2, 6-dichlorophenolindophenol or phenazine methosulfate. NAD, NADP and oxygen did not function as electron acceptors. Optimum pH for enzyme activity was 4.0, and optimum temperature was 39°C. The enzyme activity was not inhibited by sulfhydryl reagents or metal chelators.

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