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Molecular design of lysozyme for the improvement of protein function.

Research Project

Project/Area Number 63571046
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Biological pharmacy
Research InstitutionKyushu University

Principal Investigator

IMOTO Taiji  Kyushu University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (90038282)

Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1988: ¥1,600,000 (Direct Cost: ¥1,600,000)
KeywordsChicken lysozyme / Protein engineering / Mutant lysozyme / Amino acid sequence / Protein function / Protein folding / ニワトリリゾチーム / 遺伝子工学 / 大腸菌 / 酵母 / 変異リゾチーム / 酵素機能
Research Abstract

In order to improve the function of chicken lysozyme by protein engineering, the following experiments were carried out.
1. Various mutant chicken lysozymes which always contain an extra methionine residue at the N-terminal were expressed in E. coli, and folded to the active conformations.
2. A method to process the N-terminal sequence of lysozymes thus obtained was developed.
3. A method for the expression and secretion of lysozyme in yeast was established and various mutant lysozymes were prepared by the method.
4. From the properties of the mutant lysozymes, the following informations were obtained. (1) When catalytic carboxyls, Glu35 and Asp52, were mutated, lysozyme activity was almost completely lost. (2) Lysozyme with the mutation at Asp52 has the weak substrate binding ability. (3) The pK of dissociable residue located at position 35 was abnormal to depress the appearance of charges at this position. (4) Replacement of Trp108 with Gln make the enzyme inactive and unstable. (5) Substitution of Asp for Asn46 which is in the beta-sheet lowered the activity to less than 25%. (6) Substitution of Gly for Asn37 or Asp101 increased the lytic activity. (7) Deletion of Arg14-His15 enhanced the chitinase activity. (8) The folding of the reduced lysozyme where the calcium binding site similar to alpha-lactalbumin is constructed was controlled by the concentration of calcium.
5. In order to learn the way to improve lysozyme function for nature, various chicken type-lysozymes were isolated, and their amino acid sequences and properties were determined.

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (28 results)

All Other

All Publications (28 results)

  • [Publications] K.Fujita: "Enzymatic Synthesis of specifically modified linear oligosaccharide from γ-cyclodextrin derivatives.Study on importance of active sites of Taka amylase A." Bull.Chem.Soc.Japan. 62. 3150-3154 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Fujita: "Regiospecifically bifunctional cyclodextrin having two amino groups on secondary hydroxyl side." J.Org.Chem.53. 1943-1947 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Y.Ito: "Purification,amino acid sequence and some properties of rabbit kidney lysozyme." J.Biochem. 107. 236-241 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] H.Yamada: "Solubilization of reduced and denatured proteins by S-alkylation with charged reagents." J.Biol.Chem.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] H.Yamada: "Reaction of hen egg-white lysozyme with tetranitromethane.Abnormal bond cleavage at glycine 104 and sequentialnitration of three tyrosine residues." J.Biol.Chem.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Ueda: "Effects of artificial intramolecular cross-links on the renaturation rate of reduced lysozyme." Biochemistry.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T.Imoto: "Protein Function:A Practical A pproach(T.E.Creighton,ed.)Chapter 10.Chemical Modification" IRL Press, 31 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 井本泰治: "タンパク質工学への招待" 南江堂, 123 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Fujita: "Enzymatic synthesis of specifically modified linear oligosaccharide from gamma-cyclodextrin derivatives. Study on importance of active sites of Taka amylase A" Bull. Chem. Soc. Japan. 62. 3150-3154 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Fujita: "Regiospecifically bifunctional cyclodextrin having two amino groups on secondary side" J. Org. Chem.53. 1943-1947 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Y. Ito: "Purification, Amino acid sequence and some properties of rabbit kidney lysozyme" J. Biochem.107. 236-241 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] H. Yamada: "Solubilization of reduced and denatured proteins by Salkylation with charged reagents" J. Biol. Chem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] H. Yamada: "Reaction of hen egg-white lysozyme with tetranitromethane. Abnormal bond cleavage at glycine 104 and sequential nitration of three tyrosine residues" J. Biol. Chem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Ueda: "Effects of artificial intramolecular cross-links on the renaturation rate of reduced lysozyme" Biochemistry.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Imoto: Chemical Modification (Chapter 10) in "Protein Function : A Practical Approach" (T.E. Creighton, ed.). IRL Press, 31 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] T. Imoto: Invitation to Protein Engineering. Nankodo, 123 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K.Fujita: "Enzymatic synthesis of specifically modified linear oligosaccharide from γ-cyclodextrin derivatives.Study on importance of active sites of Taka amylase A." Bull.Chem.Soc.Japan. 62. 3150-3154 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Fujita: "Regiospecifically bifunctional cyclodextrin having two amino groups on secondary hydroxyl side." J.Org.Chem.53. 1943-1947 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Y.Ito: "Purification,amino acid sequence and some properties of rabbit kidney lysozyme." J.Biochem.107. 236-241 (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] H.Yamada: "Solubilization of reduced and denatured proteins by S-alkylation with charged reagents." J.Biol.Chem.

    • Related Report
      1989 Annual Research Report
  • [Publications] H.Yamada: "Reaction of hen egg-white lysozyme with tetranitromethane.Abnormal bond cleavage at glycine 104 and sequential nitration of three tyrosine residues." J.Biol.Chem.

    • Related Report
      1989 Annual Research Report
  • [Publications] T.Ueda: "Effects of artificial intramolecular cross-links on the renaturation rate of reduced lysozyme." Biochemistry.

    • Related Report
      1989 Annual Research Report
  • [Publications] T.Imoto: "Protein Function:A Practical Approach(T.E.Creighton,ed.)Chapter 10.Chemical Modification" IRL Press, 31 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 井本泰治: "タンパク質工学への招待" 南江堂, 123 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] K.Fujita.: Chem.Lett.543-546 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] T.Miki.: Protein Engineering.

    • Related Report
      1988 Annual Research Report
  • [Publications] T.Imoto.: J.Biochem.

    • Related Report
      1988 Annual Research Report
  • [Publications] Taiji Imoto.(T.E.Creighton,Editor): "Protein Function: A Practical Approach Chapter 10 Chemical Modification" IRL Press, 31 (1989)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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