Abstract
Publisher Summary This chapter describes anassay method for the synthesis of D-glucose dehydrogenase from Gluconobacter suboxydans . The enzyme activity is measured by reading the increase of absorbance at 340 nm in a recording spectrophotometer thermostatted at 25°C in a thermostatted room. Prior to the assay, the buffer solution and distilled water are warmed to 25°. The complete reaction mixture contains 0.1 ml of NADP, 0.1 ml of o -glucose, 0.3 ml of Tris-HCl, and enzyme solution in a total volume of 3.0 ml. The reaction is initiated by the addition of enzyme. The steps involved in the purification procedure are (1) cell-free extract, (2) diethylaminoethyl (DEAE)-sephadex column chromatography (I, II, and III) (3) affinity chromatography on blue-dextran sepharose, and (4) crystallization. All operations are carried out at 0-5°C, unless otherwise stated. Potassium phosphate buffer containing 1 m M 2-mercaptoethanol is used throughout. The procedure reported in the chapter is for 150 g of wet cells of G. suboxydans . Centrifugations are performed at 12,000 g for 20 min. D-Glucose dehydrogenase is specific to nicotinamide adenine dinucleotide phosphate (NADP) and completely inactive with nicotinamide adenine dinucleotide (NAD).
Published Version
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