Abstract
This chapter describes an assay method for the isolation of D-gluconate dehydrogenase from bacteria. D-gluconate dehydrogenase occurs on the outer surface of cytoplasmic membrane of oxidative bacteria, such as Pseudomonas, Klebsiella, Serratia , and acetic acid bacteria. The enzyme activity is linked to the electron transport chain in the cytoplasmic membrane constituting a D-gluconate oxidase system. The assay is performed spectrophotometrically at 25°C by measuring the decrease of absorbance at 600 nm of 2,6-dichlorophenolindophenol (DCIP) mediated with phenazine methosulfate (PMS). The activity is also measured with PMS, DCIP, ferricyanide, or coenzyme Q (CoQ) as an electron acceptor. The purification procedures of the enzyme from P. fluorescens and K. pneumoniae are described in the chapter. Potassium phosphate buffer, pH 6.0, containing 5 m M MgCl 2 is used throughout the purification of P. fluorescens . The steps involved in the purification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) ammonium sulfate fractionation, and (4) hydroxyapatite column chromatography. Purified D-gluconate dehydrogenase shows a visible absorption spectrum of the cytochrome c type having an asymmetrical α-peak.
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