25] d-Fructose dehydrogenase from Gluconobacter industrius, membrane-bound

Abstract

This chapter describes an assay method for the synthesis of and the properties of D-fructose dehydrogenase. It occurs on the outer surface of the cytoplasmic membrane of Gluconobacter industrius species. The enzyme is solubilized from the membrane and further purified to a homogeneous state. The reaction rate can be estimated (a) by spectrophotometry in the presence of 2,6-dichlorophenolindophenol and phenazine methosulfate; (b) by colorimetry in the presence of potassium ferricyanide; (c) by polarography with an oxygen electrode; or (d) by manometry in a conventional Warburg apparatus. The ferricyanide method is employed in this purification because of its simplicity for routine use. The steps involved in the prurification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) diethylaminoethyl (DEAE)-cellulose column chromatography, and (4) hydroxyapatite column chromatography. All operations are carried out at 0-5°C, unless otherwise stated. McIlvaine buffer at various pH levels are used. The reaction product of D-fructose oxidation is identified as 5-keto-D-fructose by paper chromatography. The reaction product is specifically reduced to D-fructose in the presence of nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) and 5-keto-D-fructose reductase, which can be crystallized from the cytosol of G. industrius .