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Phenylalanine dehydrogenase produced by Bacillus badius IAM 11059 was purified from the crude extract of B. badius to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.5 and a relative molecular mass, Mr, of 310000–360000. The enzyme is composed of identical subunits with an Mr 41000–42000. The substrate specificity of the enzyme in the oxidative deamination reaction was high for l‐phenylalanine, but rather low in the reductive amination reaction, with phenylpyruvate, p‐hydroxyphenylpyruvate, and 2‐oxohexanoate. The gene for the enzyme was cloned into Escherichia coli with plasmid pBR322 as a vector. The enzyme was expressed in high level in E. coli. The enzyme produced by E. coli transformant was purified to homogeneity and shown to be identical to that of B. badius IAM 11059 with respect to the specific activity, Mr, subunit structure and amino acid composition.

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.

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We report differential scanning calorimetry (DSC) experiments between 10 and 120 °C of Dengue 4 envelope protein domain 3 (DEN4 ED3), a small 107-residue monomeric globular protein domain. The thermal unfolding of DEN4 ED3 was fully reversible and exhibited two peculiar endothermic peaks. AUC (analytical ultracentrifugation) experiments at 25 °C indicated that DEN4 ED3 was monomeric. Detailed thermodynamic analysis indicated that the two endothermic peaks separated with an increasing protein concentration, and global fitting of the DSC curves strongly suggested the presence of unfolded tetramers at temperatures around 80-90 °C, which dissociated to unfolded monomers at even higher temperatures. To further characterize this rare thermal unfolding process, we designed and constructed a DEN4 ED3 variant that would unfold according to a two-state model, typical of globular proteins. We thus substituted Val 380, the most buried residue at the dimeric interface in the protein crystal, with less hydrophobic amino acids (Ala, Ser, Thr, Asn, and Lys). All variants showed a single heat absorption peak, typical of small globular proteins. In particular, the DSC thermogram of DEN4 V380K indicated a two-state reversible thermal unfolding independent of protein concentration, indicating that the high-temperature oligomeric state was successfully abolished by a single mutation. These observations confirmed the standard view that small monomeric globular proteins undergo a two-state unfolding. However, the reversible formation of unfolded oligomers at high temperatures is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation.