D-Glucose Dehydrogenase of Gluconobacter suboxydans: Solubilization, Purification and Characterization

Abstract

D-Glucose dehydrogenase was purified to homogeneity from the membrane of Gluconobacter suboxydans IFO 12528. The enzyme was solubilized from the membrane with Triton X-100 and fractionated with SE-cellulose and hydroxyapatite columns. Other species of membrane-bound dehydrogenases which were cosolubilized with D-glucose dehydrogenase were eliminated by treating the enzyme at fairly acidic pH in the initial step of enzyme purification. The purified enzyme was homogeneous in analytical ultracentrifugation and sucrose density gradient centrifugation. The enzyme had an apparent sedimentation constant of 4.2 S and showed a molecular weight of 87, 000 by urea-sodium dodecylsulfate gel electrophoresis. The spectral studies with the purified enzyme preparation showed the participation of pyrroloquinoline quinone, a new prosthetic group, in the enzyme. Phenazine methosulfate was found to be the best electron acceptor in D-glucose oxidation with the enzyme. The optimum pH of D-glucose oxidation was at pH 3.0 with potassium ferricyanide, and pH 6.0 with phenazine methosulfate, 2, 6-dichlorophenolindophenol and Wurster's blue as electron acceptor. The substrate specificity of the enzyme seemed to be restricted to D-glucose, and other sugars were not oxidized except for maltose which was oxidized at a low rate. The hydrophobicity of the enzyme was discussed from the standpoint of a typical integral protein of the bacterial membrane.