Abstract
D-Gluconate dehydrogenase catalyzing the oxidation ofD-gluconate to 2-keto-D-gluconate was solubilized with Triton X-100 from the membrane of Gluconobacter dioxyacetonicus IFO 3271 and purified to an almost homogeneousstate by chromatographies on DEAE-cellulose and CMToyopearl in the presence of 0.1% Triton X-100. The enzyme had three subunits with molecular weights of 64, 000, 45, 000 and 21, 000, and contained approximately 2mol of heme per mol of the enzyme. The prosthetic group of the dehydrogenase was found to be a flavin covalently bound to the enzyme protein. The substrate specificity of the purified enzyme was very strict for D-gluconate and the apparent Michaelis constant for D-gluconate was 2.2mM.The optimum pH and temperature of the purified enzyme were 6.0 and 40°C, respectively.
