Membrane-bound D-glucose dehydrogenase from Pseudomonas sp.: 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 metho sulfate 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.

Membrane-bound, Electron Transport-linked,d-Glucose Dehydrogenase ofPseudomonas fluorescens. Interaction of the Purified

“…P. jluorescens FM-l isolated in our laboratory was used in this study. The strain was used in the previous work 4 ) as an unidentified Pseudomonas sp. D-Glucose dehydrogenase was prepared from membrane of the organism as described previously.4)…”

“…10 ) The purified enzyme could reduce coenzyme Q-l (Q-l) in spite of being a single polypeptide dehydrogenase. 4 ) No primary dehydrogenase has been known so far to reduce ubiquinone directly. Thus, it is interesting to elucidate whether the glucose dehydrogenase can reduce long-chain Q as well as Q-I.…”

“…Purified o-glucose dehydrogenase was found to be able to reduce long-chain Q homologs, Q-2,Q-4 and Q-6, as well as Q_1 4 ) in a high concentration of ethanol, which is required to solubilize Q (Table III). As described above, the enzyme was stable in ethanol; at least 10% ethanol had no effect on the enzyme activity in the reaction mixture.…”

“…3 ) We have already reported the purification and some properties of the membrane-bound glucose dehydrogenase from Pseudomonas jluorescens. 4 ) The enzyme was solubilized with Triton X-lOO in the presence of KCl and purified to near homogeneity. The purified enzyme was a monomer in the high concentration of Triton X-lOO, but it became an oligomer or aggregated upon removal of the detergent.…”

Purification and Properties of Membrane-bound Sulfite Dehydrogenase fromThiobacillus thiooxidansJCM7814

“…2 ) The mixed suspension was gently stirred at 30°C for 30 min. Method 2: Triton X-IOO and potassium chloride were added to membrane fraction (20 mg/ml) to give a final concentration of 1.0% and I M according to a method described by Matsushita et al 10) The mixed suspension was gently stirred at 4°C for 16h. Method 3: eight kinds of detergents (Detergent-kit, Wako pure chemicals, Japan) were used.…”