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Purification and properties of NAD + ‐dependent formate dehydrogenase produced by Agrobacterium sp.
Author(s) -
Iida Mitsugi,
Ohtsuki Shin'Ichi,
Mineki Shigeru
Publication year - 1993
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.3620330405
Subject(s) - formate , formate dehydrogenase , chromatography , homotetramer , size exclusion chromatography , sephadex , chemistry , nad+ kinase , isoelectric point , sodium formate , column chromatography , gel electrophoresis , affinity chromatography , polyacrylamide gel electrophoresis , ammonium formate , enzyme , biochemistry , protein subunit , high performance liquid chromatography , organic chemistry , gene , catalysis
Formate: NAD + oxidoreductase (EC 1.2.1.2) with high affinity to formate was purified from a facultative formate‐utilizing Agrobacterium sp. MIL 1052. From a cell extract, the enzyme was purified to homogeneity by ammonium sulfate fractionation, DEAE‐Sephadex A‐50 column chromatography, Sephadex G‐200 column chromatography, and hydroxyapatite column chromatography. The enzyme amounted to 9.1% of intracellular soluble proteins in strain MIL 1052. The subunit M r , estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was 50 kDa. The M r of the native enzyme was estimated to be 112 kDa by gel filtration, suggesting a dimeric structure. The enzyme had an isoelectric point of 7.0. The pH and temperature optima were 5.5–7.0 and 30–45 °C, respectively. The enzyme was stable at pH 6.0–7.5 and at up to 35 C. The apparent K m values for formate and NAD + were calculated to be 0.13 m M and 0.17 m M , respectively.