32] 2-Keto-d-gluconate dehydrogenase from Gluconobacter melanogenus, membrane-bound

Abstract

2-Keto-D-gluconate dehydrogenase catalyzes the oxidation of 2-keto-D-gluconate to 2,5-diketo-D-gluconate and is localized on the outer surface of the cytoplasmic membrane of Gluconobacter species; the enzyme is the primary dehydrogenase of the 2-keto-D-gluconate oxidizing system in vivo . This chapter describes an assay method for the isolation of 2-Keto-D-gluconate dehydrogenase from Gluconobacter melanogenus . 2-Keto-D-gluconate dehydrogenase catalyzes the reaction in vitro in the presence of dyes, such as 2,6-dichlorophenolindophenol, ferricyanide, or ubiquinone derivatives. Ferricyanide is used because of its simplicity for routine assay. The steps involved in the purification procedure discussed are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) ammonium sulfate fractionation, (4) CM-cellulose column chromatography, and (5) hydroxyapatite column chromatography. All purification steps are performed at 0-5 °C and centrifugations are at 10,000 g for 10 min, unless otherwise stated. The purified enzyme is homogeneous as judged by analytical ultracentrifugation and polyacrylamide gel electrophoresis. The purified enzyme has a characteristic deep rose-red color because of the cytochrome component.