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2001 Fiscal Year Final Research Report Summary

Mechanisms of protein foldingand molecular breeding in halophilic enzymes

Research Project

Project/Area Number 11660094
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionKagoshima University, Faculty of Agriculture

Principal Investigator

TOKUNAGA Masao  Kagoshima University, Faculty of Agriculture, Professor, 農学部, 教授 (20112782)

Co-Investigator(Kenkyū-buntansha) ISHIBASHI Matsujiro  Kagoshima University, Faculty of Agriculture, Research Associate, 農学部, 助手 (20305163)
Project Period (FY) 1999 – 2001
KeywordsExtremely halophilic archaea / Halophilic / Halobacterium / Nucleoside diphosphate kinase
Research Abstract

We first isolated nucleoside diphsphate kinase (NDK) from Halobacterium cutirubrum which does not require high concentration of salts for its stability and activity. This is first one showing salt-free characteristics among enzymes isolated from extremely halophilic archaea. NDK was highly purified by one step ATP affinity column with 540-fold. It was further purified to homogeneity by hydrophobic chromatography. This enzyme was stable in the presence of 0〜4 M NaCl, and showed maximum activity at 2 M NaCl. It showed about 70 % of maximum activity at 0 and 4 M NaCl.
The ndk gene was cloned and sequenced from H, cutirubrum chromosomal DNA and its nucleotide sequence has been deposited to DDBJ with accession number of AB036344.
We have constructed the expression vector for this gene in E. coli. The expressed Ndkp in E. coli was localized in soluble fraction but did not have enzyme activity. We found that the high salt concentration was required for the activation of this expressed enzyme, and once enzyme was activated in the presence of high salt, it was stable after removal of salt. This fact indicated that Ndkp does not require high salt for stability and activity, but does require it for the protein folding.

  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] Tokunaga, et al.: "Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum"J. Prot. Chem.. 18. 837-844 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Ishibashi et al.: "NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt"FEBS Letters. 493. 134-138 (2001)

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      「研究成果報告書概要(和文)」より
  • [Publications] Yonezawa et al.: "Characterization of nucleoside diphosphate kinase from moderately halopihilic eubacteria"Biosci. Biotecihnol. Biochem.. 65. 1379-1387 (2001)

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      「研究成果報告書概要(和文)」より
  • [Publications] Tokunaga,Hiroko, Kara,Shinichi, Arakawa,Tsutomu, Ishibashi,Matsujiro, Radhey,S.Gupta and Tokunaga,Masao: "Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum."J. Prot. Chem. 18. 837-844 (1999)

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      「研究成果報告書概要(欧文)」より
  • [Publications] Ishibashi,Matsujiro, Tokunaga,Hiroko, Hiratsuka,Kazushi, Yonezawa,Yasushi, Tsurumaru,Hirohito, Arakawa,Tsutomu and Tokunaga,Masao: "NaCl-activated nucleoside dipho sphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt."FEBS Letters. 493. 134-138 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Yonezawa,Yasushi, Tokunaga,Hiroko, Ishibashi,Matsujiro and Tokunaga,Masao: "Characterization of nucleoside diphosphate kinase from moderately halophilic eubacteria."Biosci. Biotechnol. Biochem. 65. 1379-1387 (2001)

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Published: 2003-09-17  

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