NAD+-dependent formate dehydrogenase was screened in various bacterial strains. Facultative methanolutilizing bacteria isolated from soil samples, acclimated to a medium containing methanol and formate at pH 9.5, were classified as members of the genus Moraxella. From a crude extract of Moraxella sp. strain C-1, formate dehydrogenase was purified to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.9 and a molecular weight of approximately 98,000. The enzyme is composed of two identical subunits with molecular weights of about 48,000. (9,17,24,30). While NAD+-dependent formate dehydrogenases from methanol-utilizing yeasts have been well characterized (11,12,15,25), the bacterial enzymes have been purified to homogeneity and their properties have been investigated only in Pseudomonas oxalaticus (21) and Achromobacter parvulus (7,8). There has been no report of the utilization of methanol or formate by a Moraxella strain. In this study, we screened for a stable NAD+-dependent formate dehydrogenase from methanolutilizing bacteria, and we purified and characterized the enzyme from Moraxella sp. strain C-1, isolated from soil in an alkaline medium. MATERIALS AND METHODSMaterials. Butyl-Toyopearl and high-pressure liquid chromatography (HPLC) columns G-3000 SW, DEAE-5PW, and Phenyl-5PW were purchased from Tosoh Corp., Tokyo, Japan; Sephadex G-100 and Sephadex G-200 were obtained from Pharmacia, Uppsala, Sweden; alcohol dehydrogenase (Saccharomyces cerevisiae, EC 1.1.1.1) and marker proteins for molecular weight determinations were obtained from Oriental Yeast, Tokyo, Japan; and ampholites were obtained from LKB Produkter AB, Uppsala, Sweden. The membrane filter (Diaflo, PM 30) 4 ,ug of thiamine hydrochloride, 2 jig of pyridoxine hydrochloride, 2 ,ug of p-aminobenzoic acid, 2 ,ug of riboflavin, and 0.1 jLg of folic acid in 1 liter of tap water (pH 7.0). Basal medium supplemented with 0.8% (wt/vol) methanol, 1% sodium formate, and 1% sodium carbonate was used as an enrichment medium. The pH of the enrichment medium was 9.5. Methanol and sodium carbonate were added after the medium was autoclaved. About 100 soil samples from Sagamihara in the Kanagawa Prefecture, Japan, were suspended in 200 ml of the enrichment medium in a 500-ml Erlenmeyer flask and incubated at 30°C on a rotary shaker. Half of the supernatant was removed every other day, and the same volume of the fresh medium was added. Consumption of methanol in the enrichment medium was monitored by a gas-liquid chromatograph (model 802; Ohkura, Tokyo, Japan) with a glass column of Porapack Q (Waters Associates, Inc., Milford, Mass.