A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1.
An NAD(+)-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD(+) as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD(+). F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85 degrees C and the most active at 70 degrees C. The half-life time and k (cat) value at 60 degrees C were calculated to be 50 min and 27,400 min(-1), respectively. The enzyme also shows high catalytic efficiency at low temperatures (0-20 degrees C) (k(cat)/K(m) at 10 degrees C; 12,600 mM(-1)min(-1)) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.
Kazuoka
Takayuki
T
Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka, 564-8680, Japan.
Oikawa
Tadao
T
Muraoka
Ikuo
I
Kuroda
Shun'ichi
S
Soda
Kenji
K
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000864:Antarctic Regions; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002384:Catalysis; D003080:Cold Temperature; D004795:Enzyme Stability; D005417:Flavobacterium / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D012623:Seawater / Q000382:microbiology; D014871:Water Microbiology
D001426:Bacterial Proteins; D000426:Alcohol Dehydrogenase