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1997 Fiscal Year Final Research Report Summary

Design and Synthesis of Novel Artificial Enzymes by Using Recombinant DNA Technique

Research Project

Project/Area Number 07405060
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Research Field 高分子合成
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

KIMURA Shunsaku  Kyoto University, Graduate School of Engineering, Associate Professor, 工学研究科, 助教授 (80150324)

Co-Investigator(Kenkyū-buntansha) IMANISHI Yukio  Nara Institute of Science and Technology, Department of Material Science, Profes, 物質創成科学研究科, 教授 (00025991)
Project Period (FY) 1995 – 1997
KeywordsMutants / Recombinant DNA Technique / Artifical Enzymes / Esterase / Subtilisin / Enzymatic Activity / Protein Structure
Research Abstract

Hydrophobilized mutant enzymes of esterase from Pseudomonas sp.KWI-56 by connection of 10 or 20 tyrosine residues at the C terminus were synthesized by using the recombinant DNA technique. The mutant protein with a longer poly (tyrosine) chain became more hydrophobic than that with a shorter chain. The structure of the mutant proteins was analyzed by CD spectroscopy, which revealed the decrease of alpha-helicalconformation and the increase of beta-sheet conformation. The enzymatic activity of the mutant esterase was abolished due to the structure change induced by connection of a poly (tyrosine) chain, which should interact with hydrophobic region of the protein. On the other hand, a hydrophobilized mutant enzyme of subtilisin Carsberg by connection of poly (oxyethylene) was synthesized and the structure-activity relationship was investigated in organic solvents. The mutant enzyme showed 150-fold higher activity than the wild type in the ester-exchange reaction in benzen solution. The high activity of the mutant enzyme was also observed in dichloromethane solution, but the activity was diminished in water-miscible organic solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran. In these solvents, the molecular structure of the mutant protein was change significantly probably due to depletion of the essential water from the enzyme.

  • Research Products

    (10 results)

All Other

All Publications (10 results)

  • [Publications] Y.Ito: "Hydrophobilization of Lysozyme by Genetic Combination with Polyproline at the Carboxyl Terminal" Chem.Lett.65-66 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Ito: "Nonradioisotope Assay to Examine Adaptability of Nonnatural Amino Acids to the Active Center of Ribosomal A Site" J.Bioactive And Compatible Polymers. 12. 59-64 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Ito: "Bactericidal activity of human lysozymes carrying various lengths of polyproline chain at the C-termines" FEBS Lett.415. 285-288 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Imanishi: "Glncose-sensitive insuline-relcasing molecular systems" Pure & Appl.Chem.67. 2015-2021 (1995)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] H.Ueda: "Incorporation of hydrophobic helix bundle peptides into lipid bilayer membranes facilitated by a peptide-umbrella structure" J.Chem.Soc.Chem.Commun.363-364 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Y.Ito: "Hydrophobilization of lysozyme by genetic combination with polyproline at the carboxyl terminal" Chem.Lett.65-66 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Ito: "Nonradioisotope assay to examine adaptability of Nonnatural amino acids to the active center of ribosomal A site" J.Bioactive and Compatible Polymers. 12. 59-64 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Ito: "Bactericidal activity of human lysozymes carrying Various lenghts of polyproline chain at the C-terminus" FEBS Lett.415. 285-288 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Y.Imanishi: "Glucose-sensitive insuline-releasing molecular systems" Pure & Appl.Chem.67. 2015-2021 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] H.Ueda: "Incorporation of hydrophobic helix bundle Peptides into lipid bilayr membranes facilitated by a peptide-umbrella structure" J.Chem.Soc.Chemm.Commun.363-364 (1998)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1999-03-16  

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