Shinagawa Emiko, Fujishima Terumi, Moonmangmee Duangtip, Theeragool Gunjana, Toyama Hirohide, Matsushita Kazunobu, Adachi Osao
Department of Chemical and Biological Engineering, Ube National College of Technology, Tokiwadai, Japan.
Biosci Biotechnol Biochem. 2002 Feb;66(2):298-306. doi: 10.1271/bbb.66.298.
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.
膜结合型不依赖NAD(P)的苹果酸脱氢酶（EC 1.1.99.16）从耐热醋酸杆菌属SKU 14（一株来自泰国的分离菌株）的细胞膜中纯化至均一。该酶在0.1 M KCl存在的条件下，用1% Triton X-100从甘油培养细胞的膜组分中溶解出来，并在0.1% Triton X-100存在的条件下，通过DEAE-葡聚糖A-50和DEAE- Toyopearl柱层析步骤纯化至均一。纯化后的酶在天然聚丙烯酰胺凝胶电泳（native-PAGE）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）中均显示出单一蛋白条带。该酶是一种同型二聚体，亚基分子量为60 kDa，以非共价结合的黄素腺嘌呤二核苷酸（FAD）作为辅因子。当以铁氰化物作为电子受体时，该酶在pH 5以上稳定，在pH 11.0时具有最大活性。添加铵离子可提高酶活性。该酶对底物的特异性非常严格，只作用于L-苹果酸，其表观米氏常数（Km）为10 mM，超过20种包括D-苹果酸在内的化合物都不能被该酶氧化。
