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Extremophiles

, Volume 11, Issue 2, pp 257–267 | Cite as

A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1

  • Takayuki Kazuoka
  • Tadao OikawaEmail author
  • Ikuo Muraoka
  • Shun’ichi Kuroda
  • Kenji Soda
Original Paper

Abstract

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°C and the most active at 70°C. The half-life time and k cat value at 60°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°C) (k cat/K m at 10°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.

Keywords

Alcohol dehydrogenase Psychrotorelant Thermostable Cold-active Flavobacterium 

Abbreviations

AlcDH

Alcohol dehydrogenase

CHES

N-cyclohexyl-2-aminoethanesulfonic acid

conc.

Concentration

KPB

Potassium phosphate buffer

LMW

Low molecular weight

PVDF

Poly (vinylidene fluoride)

Tris

Tris (hydroxymethyl) aminomethane

UPGMA

Unweighted pair-group method with arithmetic mean

Notes

Acknowledgments

This research was partially supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan, Grant-in-Aid for Scientific Research (C), 2004, No.16550150, and the High-Tech Research Center project for Private Universities, matching fund subsidy from MEXT (2002–2006), and the Kansai University Special Research Fund (2005).

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Copyright information

© Springer 2006

Authors and Affiliations

  • Takayuki Kazuoka
    • 1
  • Tadao Oikawa
    • 1
    • 2
    Email author
  • Ikuo Muraoka
    • 1
    • 2
  • Shun’ichi Kuroda
    • 3
  • Kenji Soda
    • 1
  1. 1.Department of Biotechnology, Faculty of EngineeringKansai UniversitySuita, OsakaJapan
  2. 2.Kansai University High technology Research CenterSuita, OsakaJapan
  3. 3.Institute of Science and Industrial ResearchOsaka UniversityIbaraki, OsakaJapan

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