title: Purification and characterization of membrane-bound malate dehydrogenase from Acetobacter sp. SKU 14.
abstract: Membrane-bound NAD(P)-independent malate dehydrogenase (EC 1.1.99.16) was purified to homogeneity from the membrane of thermotolerant Acetobacter sp. SKU 14, an isolate from Thailand. The enzyme was solubilized from the membrane fraction of glycerol-grown cells with 1% Triton X-100 in the presence of 0.1 M KCl, and purified to homogeneity through steps of column chromatographies on DEAE-Sephadex A-50 and DEAE-Toyopearl in the presence of 0.1% Triton X-100. The purified enzyme showed a single protein band in both native-PAGE and SDS-PAGE. The enzyme was a homodimer with a molecular mass of 60 kDa subunit and had noncovalently bound FAD as the cofactor. The enzyme was stable over pH 5 and had its maximum activity at pH 11.0 when ferricyanide was used as an electron acceptor. The enzyme activity was elevated by the addition of ammonium ions. The substrate specificity was very strict to only L-malate, of which the apparent Km was 10 mM and over 20 compounds involving D-malate were not oxidized by the enzyme.
authors: Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai, Japan. emiko@ube-k.ac.jp
authors: Emiko
authors: E
authors: Shinagawa
authors: Terumi
authors: T
authors: Fujishima
authors: Duangtip
authors: D
authors: Moonmangmee
authors: Gunjana
authors: G
authors: Theeragool
authors: Hirohide
authors: H
authors: Toyama
authors: Kazunobu
authors: K
authors: Matsushita
authors: Osao
authors: O
authors: Adachi
mesh_terms: D000091:Acetobacter / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008565:Membrane Proteins; D008291:Malate Dehydrogenase