title: Crystalline NADP-Dependent D-Mannitol Dehydrogenase from Gluconobacter suboxydans.
abstract: D-Mannitol dehydrogenase (EC 1.1.1.138) was purified and crystallized for the first time from the cell-free extract of Gluconobacter suboxydans IFO 12528. The enzyme was purified about 100-fold by a procedure involving ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography, and gel filtration by a Sephadex G-75 column. The enzyme was completely separated from a similar enzyme, NAD-dependent D-mannitol dehydrogenase (EC 1.1.1.67), during enzyme purification. There being sufficient purity of the enzyme at this stage, the enzyme was crystallized, by the addition of ammonium sulfate, to fine needles. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.6 s. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-PAGE and gel filtration chromatography. Oxidation of D-mannitol to D-fructose and reduction of D-fructose to D-mannitol were specifically catalyzed with NADP and NADPH, respectively. NAD and NADH were inert for the enzyme. Since the reaction equilibrium declined to D-fructose reduction over a wide pH range, the enzyme showed several advantages for direct enzymatic measurement of D-fructose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the D-fructose reduced.
authors.affiliation: a Laboratory of Applied Microbiology, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: O
authors.initials: O
authors.lastname: Adachi
authors.affiliation: a Laboratory of Applied Microbiology, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: H
authors.initials: H
authors.lastname: Toyama
authors.affiliation: a Laboratory of Applied Microbiology, Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University.
authors.forename: K
authors.initials: K
authors.lastname: Matsushita
keywords: D-fructose measurement; Gluconobacter suboxydans; NADP-dependent D-mannitol dehydrogenase; polyol dehydrogenase