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Improvements of Antimicrobial Activity of Lysozyme by Protein Engineering

Research Project

Project/Area Number 05660142
Research Category

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

Allocation TypeSingle-year Grants
Research Field 食品科学・栄養科学
Research InstitutionYamaguchi University

Principal Investigator

KATO Akio  Yamaguchi University, Faculty of Agriculture, Professor, 農学部, 教授 (00035114)

Project Period (FY) 1993 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1994: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1993: ¥1,500,000 (Direct Cost: ¥1,500,000)
Keywordslysozyme / hydrophobic peptide fused lysozyme / Antimicrobial Activity / protein engineering / Gentetic engineering / リゾチームcDNA / 蛋白質工学的機能改変
Research Abstract

The effect of the length of hydrophobic peptide (H3 ; Phe-Val-Pro, H5 ; Phe-Phe-Val-Ala-Pro, H7 ; Phe-Phe-Val-Ala-Ile-Pro) at the C-terminus of lysozyme on the bactericidial action against Escherichia coli was investgated by using the yeast harboring the expression vector inserted the cDNA of the hydrophobic fusion lysozyme. Although these hydrophobic fusion lysozyms were little secreted in the culture medium of yeast harboring the expression plasmid, they were highly experssed in the medium when the substitution of Gly with Asn at the position 49 was combined to the fusion lysozyme (H3/G49N-Lz, H5/G49N-Lz, H7/G49N-Lz) to insert the signal sequence (Asn-X-Ser/Thr) of N-linked glycosylation. Interestingly, the amount of secretion of glycosylated fusion lysozyme was much smaller than that of G49N-Lz. These results suggest that the glycosylated fusion lysozyme become unstable and then is subjected to proteolysis due to quality control in endoplasmic reticulum, because the folding dose not work smoothly. On the other hand, the folding of nonglycosylated hydrophobic fusion lysozyme (H3,5,7/G49N-Lz) works so well in ER when their secretion increase considerably. The hydrophobic fusion lysozyme retained 75-80% lytic activity of wild type-Lz. The antimicrobial activity increased in propotion to the length of hydrophobic peptide in fusion lysozyme, although H5/G49N-Lz and H7/G49N-Lz showed almost the same antimicrobial activity. These results suggested that when the hydrophobic residues form the beta-sheet conformation, the length of pentapeptide is enough to penetrate into the inner membrane through the outer LPS membrane. The denaturated fusion lysozyme without lytic activity did not show any killing action against E.coli, suggesting the importance of catalytic domein.

Report

(3 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • Research Products

    (15 results)

All Other

All Publications (15 results)

  • [Publications] H.R.Ibrahim,A.Kato: "Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus." J.Biol.Chem.269. 5059-5063 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Kato,M.Ban: "Polymannosylation to asparagine-19 in hen egg white lysozyme in yeast." FEBS Letters. 355. 76-80 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Kato,S.Tanimoto: "Relationships between conformational stabilities and surface functional of mutant hen egg-white lysozyme constructed by genetic engineering." J.Agric.Food Chem.42. 227-230 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] S.Nakamura,A.kato: "Hyperglycosylation of hen egg white lysozyme in yeast." J.Biol.Chem.268. 12706-12712 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Kato H.R.Ibrahim: "Food Proteins-Structure and Functionality-" K.D.Schwenke and R.Mothes,VCH, 39 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.R.Ibrahim, M.Yamada, K.Matsushita, K.Kobayashi, and A.Kato: "Enhanced bactericidal action of lysozyme to E.coli by inserting a hydrophobic pentapeptide into its C terminus." J.Biol.Chem.269. 5059-5063 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Kato, H.Takasaki and M.Ban: "Polymannosylation to asparagine-19 in hen egg white in yeast." FEBS Letters. 355. 76-80 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Kato, S.Tanimoto, Y.Oda, Y.Inoue and K.Kobayashi: "Relationships between conformational stabilities and surface functional properties of mutant hen egg white lysozymes constructed by genetic engineering." J.Agric.Food Chem.42. 227-230 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] S.Nakamura, H.Takasaki, K.Kobayashi and A.Kato: "Hyperglycosylation of hen egg white lysozyme in yeast." J.Biol.Chem.268. 12706-12712 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.R.Ibrahim,A.Kato: "Enhanced bactericidal action of lysozyme to Escherichia coli by inserting a hydrophobic pentapeptide into its C terminus" J.Biol.Chem.269. 5059-5063 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] A.Kato,M.Ban: "Polymannosylation to asparagine-19 in hen egg white lysozyme in yeast." FEBS Letters. 355. 76-80 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] A.Kato,S.Tanimoto: "Relationships between conformational stabilities and surface functional properties of mutant hen egg-white lysozyme constructed by genetic engineering." J.Agric.Food Chem.42. 227-230 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] S.Nakamura,A.Kato: "Hyperglycosylation of hen egg white lysozyme in yeast." J.Biol.Chem.268. 12706-12712 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] A.Kato,H.R.Ibrahim: "Food Proteins-Structure and Functionality-" K.D.Schwenke and R.Mothes,VCH,

    • Related Report
      1994 Annual Research Report
  • [Publications] ヒッシャムイブラヒム,山田守,松下一信,小林邦彦,加藤昭夫: "Enhanced Bactericidal Action of Lysozyme to E.Coli by Inserting a Hydrophobic Pentapeptide into C-Terminus" J.Biol.Chem.269. 5059-5063 (1994)

    • Related Report
      1993 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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