22] d-Sorbitol dehydrogenase from Gluconobacter su☐ydans, membrane-bound

Abstract

Publisher Summary This chapter describes an assay method for the synthesis of D-sorbitol dehydrogenase from Gluconobacter suboxydans . D-Sorbitol dehydrogenase occurs on the outer surface of the cytoplasmic membrane of Gluconobacter species. The enzyme is solubilized from the membrane and further purified to a homogeneous state. D-sorbitol is oxidized by D-sorbitol dehydrogenase and the reaction rate is estimated (a) by spectrophotometry in the presence of 2,6-dichlorophenolindophenol and phenazine methosulfate; (b) by colorimetry in the presence of potassium ferricyanide; (c) by polarography with an oxygen electrode; or (d) by manometry in a conventional Warburg apparatus. The assay method with potassium ferricyanide is employed because of its simplicity for routine assay. The steps involved in the purification procedure are (1) the preparation of membrane fraction, (2) the solubilization of enzyme, (3) fractionation with polyethylene glycol, (4) diethylaminoethyl (DEAE)-cellulose column chromatography, and (5) CM-cellulose column chromatography. D-Sorbitol dehydrogenase is assayed in vitro in the presence of any one of the following dyes as an electron acceptor, namely––potassium ferricyanide, phenazine methosulfate, or nitro blue tetrazolium.