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Creation of stable enzymes for PCR by evolutionary molecular engineering

Research Project

Project/Area Number 09558081
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Structural biochemistry
Research InstitutionTokyo University of Pharmacy & Life Science

Principal Investigator

OSHIMA Tairo  School of Life Science, Tokyo University of Pharmacy & Life Science Professor, 生命科学部, 教授 (60167301)

Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 1999: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1998: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1997: ¥4,900,000 (Direct Cost: ¥4,900,000)
Keywordsisopropylmalate dehydrogenase / tryptophan synthetase / nicking / structural unit / TIM barrel / fragmented enzyme / 高度好熱菌 / PCR / 耐熱酵素 / 実験室内進化 / Thermus thermophilus / 進化的分子工学 / leuB
Research Abstract

The final goal of the present project is to create stable enzymes and polymerases for PCR and the related techniques by laboratory evolution. For this purpose, the investigator devised two laboratory evolution systems ; (1) using an extreme thermophile, Thermus thermophilus as a host, genes coding for mesophile enzymes were integrated into the thermophile chromosome. (2) In the other system, E.coli is used as the host and genes coding for thermophile enzymes were inserted into the chromosomal DNA.When isopropylmalate dehydrogenase is used as a model enzyme, the dehydrogenases from many mesophiles including yeast were successfully stabilized. Likewise, the catalytic activity at room temperature of the Thermus thermophilus enzyme was improved by this method. At present stabilization of E.coli DNA polymerase is under progress.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (30 results)

All Other

All Publications (30 results)

  • [Publications] A.CHIBA et al.: "Coenzyme Activity of NAD Analogs for 3-Isopropylmalate Dehydrogenase from Thermus thermophilus HB8."Biosci. Biotechnol. Biochem.. 63(9). 1647-1649 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] J.Nathaniel et al.: "Structural Conservation of the Isolated Zinc Site in Archaeal Zinc-containing Ferredoxins as Revealed by X-ray Absorption Spectroscopic Analysis and Its Evolutionary Implications."J.Biol. Chem.. 274(33). 23160-23168 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A.CHIBA et al.: "Synthetic and Mechanistic Studies of(2R,3S)-3-Vinylmalic Acid as a Mechanism-Based Inhibitor of 3-Isopropylmalate Dehydrogenase."J.Org. Chem.. 64(17). 6159-6165 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S.AKANUMA et al.: "Further Improvement of the Thermal Stability of a Partially Stabilized Bacillus subtilis 3-Isopropylmalate Dehydrogenase Variant by Random and Site-Directed"Eur. J.Biochem.. 260. 499-504 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] C.MOTONO et al.: "Urea-Induced Unfolding and Conformational Stability of 3N-Isopropylmalate Dehydrogenase from the Thermophile Thermus thermophilus and Its Mesophilic counterpart from escherichia coli."Biochemistry. 38{4}. 1332-1337 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.IWASAKI et al.: "Respiratory Complex II from the Thermophilic Archaeon, Sulfolobus sp.Strain 7 : Genes and Protein."in "Flavins and Flavoproteins 1999" (eds. by S.Ghisla et al.) Rudolf Weber, Berlin. 779-786

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A.CHIBA et al.: "Coenzyme Activity of NAD Analogs for 3-Isopropylmalate Dehydrogenase from Thermus thermophilus HB8."Biosci.Biotechnol.Biochem.. 63(9). 1647-1649 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] J.Nathaniel et al.: "Structural Conservation of the Isolated Zinc Site in Archaeal Zinc-containing Ferredoxins as Revealed by X-ray Absorption Spectroscopic Analysis and Its Evolutionary Implications."J.Biol.Chem.. 274(33). 23160-23168 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] S.AKANUMA et al.: "Further Improvement of the Thermal Stability of a Partially Stabilized Bacillus subtilis 3-Isopropylmalate Dehydrogenase Variant by Random and Site-Directed Mutagenesis."Eur.J.Biochem.. 260. 499-504 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] C.MOTONO et al.: "Urea-Induced Unfolding and Conformational Stability of 3N-Isopropylmalate Dehydrogenase from the Thermophile Thermus thermophilus and Its Mesophilic counterpart from escherichia coli."Biochemistry. 38{4}. 1332-1337 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A.CHIBA et al.: "Synthetic and Mechanistic Studies of (2R, 3S)-3-Vinylmalic Acid as a Mechanism-Based Inhibitor of 3-Isopropylmalate Dehydrogenase."J.Org.Chem.. 64(17). 6159-6165 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A.CHIBA et al.: "Coenzyme Activity of NAD Analogs for 3-Isopropylmalate Dehydrogenase from Thermus thermophilus HB8"Biosci.Biotechnol. Biochem.. 63(9). 1647-1649 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] J.Nathaniel et al.: "Structural Conservation of the Isolated Zinc Site in Archaeal Zinc-containing Ferredoxins as Revealed by X-ray Absorption Spectroscopic Analysis and Its Evolutionary Im;lications"J.Biol.Chem. 274(33). 23160-23168 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] A.CHIBA et al.: "Synthetic and Mechanistic Studies of (2R,3S)-3-Vinylmalic Acid as a Mechanism-Based Inhibitor of 3-Isopropylmalate Dehydrogenase"J.Org.Chem. 64(17). 6159-6165 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] S.AKANUMA et al.: "Further Improvement of the Thermal Stability of a Partially Stabilized Bacillus subtilis 3-Isopropylmalate Dehydrogenase Variant by Random and Site-Directed"Eur.J.Biochem. 260. 499-504 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] C.MOTONO et al.: "Urea-Induced Unfolding and Conformational Stability of 3N-Isopropylmalate Dehydrogenase from the Thermophile Thermus thermophilus and Its Mesophilic counterpart from escherichaia coli"Biochemistry. 38{4}. 1332-1337 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Akanuma, S.: "Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution." Protein Sci.7. 698-705 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Tamakoshi, M.: "The organization of the leuC, leuD and leuB genes of the extreme thermophile The rmus thermophilus." Gene. 222. 125-132 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] Aosima, M.: "Stabilization of Escherichia coli isopropylmalate dehydrogenase by single amino acid substitution." Protein Eng.10. 1505-1513 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] Qu, C.: "A mutation at the interface between domains causes rearrangement of domains in 3-isopropylmalate dehydrogenase." Protein Eng.10. 45-52 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] Suzuki, T.: "Screening of a mutant plasmid with high expression efficiency of GC-rich leuB ge ne of an extreme thermophile, Thermus thermophilus, in Eschericha coli." J, Biochem. 121. 1031-1034 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] Tamakoshi, M.: "A new Thermus-Escherichia coli shuttle integration vector system." Journal of Bact. 179. 4811-4819 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] M.TAMAKOSHI, et al.: "A New Thermus-Escherichia coli Shuttle Integration Vector System." J.Bacteriol.,. 179〔15〕. 4811-4814 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] M.ISHIDA, et al.: "Highly efficient production of enzymes of an extreme thermophile,Thermus thermophilus:A practical method to overexpress GC-rich genes in Escherichia coli." Extremephiles,. 1〔3〕. 157-162 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] C.Qu, et al.: "A mutation at the interface between domains causes rearrangement of fomains in 3-isopropylmalate dehydrogenase." Protein Eng.10〔1〕. 45-52 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] A.CHIBA, et al.: "Synthesis of Conformationally Restricted Substrate Analogs and Their Interaction with 3-Isopropylmalate Dehydrogenase Derived from Thermus thermophilus." Tetahedron,. 53〔10〕. 3537-3844 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] G.WALLON, et al.: "Purification.catalytic properties and thermostability of 3-isopropylmalate dehydrogenase from Escherichia coli." Biochim.Biophys.Acta,. 1337. 105-112 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] G.WALLON, et al.: "Crystal structure of Escherichia coli and Salmonella typhimurium 3-isopropylmalate dehydrogenase and comparison with their thermophilic counterpart from Thermus thermophilus." J.Mol.Biol.,. 266(5). 1016-1031 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] T.SUZUKI, et al.: "Screening of a mutant plasmid with high expression Efficiency of GC-rich leuB gene of an extreme thermophile,Thermus thermophilus,in Escherichia coli." J.Biochem.,. 121〔6〕. 1031-1034 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] T.SUZUKI, et al.: "Molecular and Phylogenetic Characterization of Isopropylmalate Dehydrogenase of a Thermoacidophilic archaeon,Sulfolobus sp.Strain7." J.Bacteriol.,. 179〔4〕. 1174-1179 (1997)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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