Abstract
Publisher Summary This chapter describes an assay method for the isolation of 5-keto-D-gluconate reductase from Gluconobacter suboxydans . 5-Keto-D-gluconate reductase catalyzes the reduction of 5-keto-D-gluconate to D-gluconate and occurs only in acetic acid bacteria. It is reduced in the presence of nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) to give D-gluconate and nicotinamide adenine dinucleotide phosphate dehydrogenase (NADP). The reaction rate is measured by the decrcase of absorbancc at 340 nm. NADPH oxidation is followed in a recording spectrophotometer at 25°C. The steps involved in the purification procedure discussed are (1) the preparation of cell-free extract, (2) diethylaminoethyl (DEAE)-sephadex column chromatography, (3) affinity chromatography on blue-dextran sepharose, (4) pH gradient chromatography on diethylaminoethyl (DEAE)-sephadex, and (5) crystallization. All operations are carried out at 0–5°C throughout the purification steps, unless otherwise stated. Centrifugations are at 12,000 g for 20 min. The buffer solution used is potassium phosphate, pH 6.0, which is supplemented with 2-mercaptoethanol, sodium D-gluconate, and glycerol.
Published Version
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