Abstract
Publisher Summary This chapter describes an assay method for the synthesis of D-glucose dehydrogenase from Pseudomonas fluorescens . D-Glucose dehydrogenase occurs on the outer surface of the cytoplasmic membrane of oxidative bacteria, such as Pseudomonas and Gluconobacter species, and initiates a direct oxidation of D-glucose through an electron transport chain. The enzyme is solubilized from the membrane and further purified to a homogeneous state. A spectrophotometric assay at 25° measures the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity can also be measured with PMS, DCIP, Wurster's blue (WB), ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. Many strains of Pseudomonas aeruginosa and P. fluorescens are suitable sources of D-glucose dehydrogenase. The steps involved in the purification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) polyethylene glycol fractionation, (4) ethanol fractionation, (5) hydroxyapatite column chromatography, and (6) phenyl-sepharose column chromatography. All procedures are carried out at 0–5°C. D-Glucose dehydrogenase has a dual pH optimum depending on the electron acceptor used.
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