Membrane-bound D-Glucose Dehydrogenase fromPseudomonassp.: Solubilization, Purification and Characterization

Abstract

A membrane-bound d-glucose dehydrogenase [E.C. 1.1.99.a] was solubilized from the membrane of Pseudomonas sp. and purified to a nearly homogeneous state. The solubilized enzyme was monomeric in the presence of 1% Triton X–100 but aggregated after removing the detergent. The enzyme was a single peptide having a molecular weight of about 90,000. The enzyme reacted with various artificial electron acceptors such as phenazine methosulfate, 2,6-dichlorophenolindophenol, Wurster’s blue, coenzyme Q₁ and ferricyanide. The enzyme had a dual optimum pH depending on the electron acceptor. Reductase activities of the enzyme for 2,6-dichlorophenolindophenol, ferricyanide and coenzyme Q₁ were found in more acidic pH region, whereas its activities for phenazine methosulfate and Wurster’s blue were observed in more alkaline region. p-Benzoquinone inhibited phenazine methosulfate reductase activity non-competitively but it inhibited 2,6-dichlorophenolindophenol reductase activity competitively against the acceptor. The enzyme possessed fairly broad substrate specificity, and the reaction product was a gluconolactone.