Purification and properties of NAD+-dependent formate dehydrogenase produced by Agrobacterium sp.

✍ Scribed by Dr. Mitsugi Iida; Shin'Ichi Ohtsuki; Shigeru Mineki

• Publisher: John Wiley and Sons
• Year: 1993
• Tongue: English
• Weight: 550 KB
• Volume: 33
• Category: Article
• ISSN: 0233-111X
• DOI: 10.1002/jobm.3620330405

✦ Synopsis

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 (3-200 column chromatography, and hydroxyapatite column chromatography. The enzyme amounted to 9.1% of intracellular soluble proteins in strain MIL 1052. The subunit Mr, estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was 50 kDa. The M, 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,,, values for formate and NAD+ were calculated to be 0.13 mM and 0.17 mM, respectively.