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Strategy for cold adaptation of enzyme : studies on structure and molecular evolution of cold active alcohol dehydrogenase

Research Project

Project/Area Number 16550150
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Chemistry related to living body
Research InstitutionKansai University

Principal Investigator

OIKAWA Tadao  Kansai University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (80233005)

Project Period (FY) 2004 – 2005
Project Status Completed (Fiscal Year 2005)
Budget Amount *help
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
KeywordsAlcohol dehydrogenase / Flavobacterium / Thermostable enzyme / Psychrophilic enzyme / Psychrotolerant / Alcohol dehydrogenase / Flavobacterium frigidimaris / Geobacillus stearothermophilus / Psychrotorelant / Cold-active / Thermostable
Research Abstract

An NAD^+-dependent alcohol dehydrogenase from the Antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1 was purified to be homogeneity with an overall yield of about 20% and characterized enzymologically. The native enzyme had an apparent molecular mass of 160 kDa and consisted of four identical 40 kDa subunits. The pI of the enzyme was determined to be 6.7 and its optimum pH for oxidation reaction was 7.0. The enzyme contained 2 zinc atoms/subunit. The enzyme exclusively required NAD^+ as a coenzyme and showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NADH. F.frigidimaris KUC-1 alcohol dehydrogenase showed a remarkable thermal stability similar to its thermophilic counterparts and in contrast to other microbial alcohol dehydrogenases. The enzyme was active in the temperature range of 0 to over 85℃ and the most active at 70℃. The half-life time and k_<cat> at 60℃ were calculated to be 50 min and 27,370 (min^<-1>), respectively. The enzyme also showed high catalytic efficiency at low temperatures (0-20℃) (kcat/K_m at 20℃ ; 25,500 mM^<-1> min^<-1>) similar to its psychrophilic counterpart. The alcohol dehydrogenase gene was composed of 1,035 bp and coded 344 amino acid residues with an estimated molecular mass of 36,823 Da. The sequence identities were found with the amino acid sequences of Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%), and Geobacillus stearothermophilus LLD-R (56%) alcohol dehydrogenases. To our knowledge, this is the first example of a cold-active and thermostable alcohol dehydrogenase.

Report

(3 results)
  • 2005 Annual Research Report   Final Research Report Summary
  • 2004 Annual Research Report
  • Research Products

    (9 results)

All 2006 2005 Other

All Journal Article (9 results)

  • [Journal Article] Alanine racemase of Alfalfa seedlings (Medicago sativa L.) : first evidence for the presence of amino acid racemase in plant2006

    • Author(s)
      Kazutoshi Ono
    • Journal Title

      Phytochemistry (印刷中)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Alanine racemase of Alfalfa seedlings (Medicago sativa L.) : first evidence for the presence of amino acid racemase in plant2006

    • Author(s)
      Kazutoshi Ono
    • Journal Title

      Phytochemistry (in print)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Flavobacterium frigidimaris sp. nov., isolated Antarctic Seawater2005

    • Author(s)
      Yuichi Nogi
    • Journal Title

      System. Appl. Microbiol. 28

      Pages: 310-315

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel Antarctic psychrotolerant. Flavobacterium frigidimaris KUC-12005

    • Author(s)
      Tadao Oikawa
    • Journal Title

      Biosci. Biotechnol. Biochem. 69

      Pages: 2143-2154

    • NAID

      130000030489

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Flavobacterium frigidimaris sp. nov., isolated Antarctic Seawater2005

    • Author(s)
      Yuichi Nogi
    • Journal Title

      System.Appl.Microbiol. 28

      Pages: 310-315

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Final Research Report Summary
  • [Journal Article] Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel Antarctic psychrotolerant, Flavobacterium frigidimaris KUC-12005

    • Author(s)
      Tadao Oikawa
    • Journal Title

      Biosci.Biotechnol.Biochem. 69

      Pages: 2143-2154

    • NAID

      130000030489

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2005 Annual Research Report 2005 Final Research Report Summary
  • [Journal Article] Flavobacterium frigidimaris sp.nov., isolated Antarctic Seawater2005

    • Author(s)
      Yuichi Nogi
    • Journal Title

      System.Appl.Microbiol. 28

      Pages: 310-315

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Flavobacterium frigidimaris sp.nov., isolated from Antarctic Seawater2005

    • Author(s)
      Y.Nogi, K, Soda, T.Oikawa
    • Journal Title

      Systematic and Applied Microbiology (印刷中)

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Alanine racemase of Alfalfa seedlings (Medicago sativa L.) : first evidence for the presence of amino acid racemase in plant

    • Author(s)
      Kazutoshi Ono
    • Journal Title

      Phytochemistry (印刷中)

    • Related Report
      2005 Annual Research Report

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Published: 2004-04-01   Modified: 2016-04-21  

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