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Molecular Designs for Functional Food Proteins by Genetic Modification

Research Project

Project/Area Number 14360077
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
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)

Co-Investigator(Kenkyū-buntansha) UTSUMI Shigeru  Kyoto University, Faculty of Agriculture, Professor, 農学部, 教授 (40111976)
AZAKAMI Hiroyuki  Yamaguchi University, Faculty of Agriculture, Assistant Professor, 農学部, 助手 (40263850)
Project Period (FY) 2002 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥13,000,000 (Direct Cost: ¥13,000,000)
Fiscal Year 2003: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2002: ¥8,900,000 (Direct Cost: ¥8,900,000)
KeywordsLysozyme / α-lactalbumin / ovoinhibitor / yeast Pichia pastoris / glycosylated lysozyme / glycosylated α-lactalbumin / inhibition against elastase / グリコシル化 / マクロファージ / プロテアーゼ阻害 / タンパク質の分誌設計 / フォスビチン
Research Abstract

(1) The functional properties of lysozyme, α-lactalbumin and ovoinhibitor were improved by genetic modification. The conformational stability of lysozyme was greatly enhanced by substitution of serine with threonine at position 91 to strengthen the intramolecular hydrophobic packing. The bactericidal action of lysozyme was dramatically strengthened by the attachment of serine-rich peptides to the C-terminaus
(2) Glycosylated a-lactalbumins (α-LAs) were found to activate macrophage cell (J774.1). To elucidate the function. we produced the glycosylated and non-glycosylated mutant α-LAs in yeast Pichia pastoris. The glycosylated α-LA secreted in Pichia pastoris remarkably could stimulate the activation of macrophage cell, wheroas the non-glycosylated a-LA could not. To further confirm the role of carbohydrate chain, various mutant α-LAs were constructed. The mutant N45D deleted the N-linked glycosylation site at position 45 produced both non-glycosylated type and glycosylated type, at posi … More tion 76, α-LA. The former greatly decreased the activation for Mφ, the later revealed strong activation for Mφ. The mutant D46N added the glyrcosylation signal at position 46 produced only glycosylated α-LA. The D46N strongly activated Mφ. These results indicate that glycosylated a-LA activates Mφ, while non-glycosylated a-LA does not. Furthermore, the anti-tumor activity using FMA cell of glycosylated α-LA through Mφ was confirmed
(3) The ovoinhibitor cDNA was integrated into yeast Pichia pastoris genome. The secreted recombinant ovoinhibitor was purified by ion-exchange chromatography on a DEAF sepharose column with 10 mM Tris-Hcl buffer (pH-8.0). Recombinant ovoinhibitor has a molecular weight 46 kDa, calculating from SDS-PAGE. Recombinant ovoinhibitor showed the inhibitory activity against trypsin, chymotrypsin and elastase as native ovoinhibitor. Another attempt was carried out to express the small fragment of elastase inhibitory domain in ovoinhibitor in Pichia pastoris. The elastase inhibitory fragment consisting of sixth and seventh domain in ovoinhibitor was successfully integrated into Pichia genome and secreted in cultivation medium. The molecular weight was 15 kDa, calculating from SDS-PAGE. Recombinant fragmented elastase inhibitory domain showed both inhibitory activity against elastase and chymotrypsin. Ovoinhibitor predominantly secreted in the glycosylated form. The effect of glycosylation on the inhibitory activity against proteases was also investigated Less

Report

(3 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] Y.Song, J.Sakai, A.Saito, M.Usui, H.Azakami, A.Kato: "Relatoinship between the stability of lysozymes mutated at the inside hydrophobic core and secretion in Saccharomyces cerevisiae"Nahrung. 46. 209-213 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] A.Saito, M.Usui, Y.Song, H.Azakami, A.Kato: "Secretion of glycosylated α-Lactalbumin in yeast Pichia pastoris."J.Biochem.. 132. 77-82 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] S.T.Liu, A.Saito, H.Azakami, A.Kato: "Expression purification, and characterization of an unstable lysozyme mutant in Pichia pastoris."Protein Expression and Purification. 27. 304-312 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] S.Begum, A.Saito, A.Kato, J.He, H.Azakami: "Expression and characterization of chicken ovoinhibitor in Pichia pastoris"Nahrung. 47. 359-363 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] S.Begum, A.Saito, X.H.Xu, A.Kato: "Improvement of functional properties of ovoinhibitor by conjugation with galactomannan"Biosci.Biotech.Biochem.. 67. 1897-1902 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] A.Saito, Y.Sako, M.Usui, H.Azakami, A.Kato: "Functional properties of glycosylated lysozyme secreted in Pichia Pastoris."Biosci.Biotech.Biochem.. 67. 2334-2343 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Akio Kato: "Handbook of Food Enzymology"J.R.Whitaker, A.G.J.Voragen, D.W.S.Wong (Marcel Dekker). 971-978 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Song, J.Sakai, A.Saito, M.Usui, H.Azakami, A.Kato: "Relatoinship between the stability of lysozymes mutated at the inside hydrophobic core and secretion in Saccharomyces cerevisiae"Nahrung. 46. 209-213 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] A.Saito, M.Usui, Y.Song, H.Azakami, A.Kato: "Secretion of glycosylated α-Lactalbumin in yeast Pichin pastoris"J.Biohem.. 132. 77-82 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] S.T.Liu.A.Saito, H.Azakami, A.Kato: "Expression, purification, and characterization of an unstable lysozyme mutant in Pichia pastoris"Protein Expression and Purification. 27. 304-312 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] S.Begum, A.Saito, X.H.Xu, A.Kato: "Improvement of functional properties of ovoinhibitor by conjugation with galactomannan"Biosci.Biotech.Biochem.. 67. 1897-1902 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] S.Begum, A.Saito, A.Kato, J.He, H.Azakami: "Expression and characterization of chicken ovoinhibitor in Pichia pastoris"Nahrung. 47. 359-363 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] A.Saito.Y.Sako, M.Usui ; H.Azakami, A.Kato: "Functional properties of glycosylated lysozyme secreted in Pichia Pastoris"Biosci.Biotech.Biochem.. 67. 2334-2343 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] X.H.Xu, O.Kashima, A.Saito, H.Azakami, A.Kato: "Structural and functional properties of chicken lysozyme fused serine-rich heptapeptides at the C-terminus"Biosci.Biotech.Biochem.. 68 (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] S.Begum, A.Saito, A.Kato, J.He, H.Azakami: "Expression and characterization of chicken ovoinhibitor in Pichia nastoris"Nahrung. 47. 359-363 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] A.Saito, Y.Sako, M.Usui, H.Azakami, A.Kato: "Functional properties of glycosylated lysozyme secreted in Pichia Pastoris"Biosci.Biotech.Biochem. 67. 2334-2343 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] S.T.Liu, A.Saito, H.Azakami, A.Kato: "Expression, purification, and characterization of an unstable lysozyme mutant in Pichia nastoris"Protein Expression and Purification. 27. 304-312 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 加藤昭夫, 川岸明彦, 河田康志, 内海 成, 吉川正明, 山縣ゆり子: "タンパク質工学-生命科学系分野のための-"医学出版. 305 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Y.Song, H.Azakami, S.Begum, J.He, A.Kato: "Different effect of calnexin deletion in Saccharomyces cerevisiae on the secretion of two glycosylated amyloidogenic lysozymes"FEBS Letters. 512. 213-217 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] A.Saito, M.Usui, Y.Song, H.Azakami, A.Kato: "Secretion of glycosylated α-lacalbumin in yeast Pichia pastoris"J.Biochem.. 132. 77-82 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] S.T.Liu, A.Saito, H.Azakami, A.Kato: "Expression, purification and characterization of an unstable lysozyme mutant in Pichia pastpris"Protein Expression & Purification. 27. 304-312 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Y.Song, A.Saito, M.Usui, H.Azakami: "Relation between the stability of lysozyme mutated at the inside hydrophobic core and secretion in Saccharomyces cerevisiae"Nahrung. 46, No3. 209-213 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Akio Kato: "Handbook of Food Enzymology"Marcel Dekker Inc.. 971-978 (2003)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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