2003 Fiscal Year Final Research Report Summary
Molecular Designs for Functional Food Proteins by Genetic Modification
| Project/Area Number |
14360077
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
食品科学・栄養科学
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| Research Institution | Yamaguchi University |
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Principal Investigator |
KATO Akio Yamaguchi University, Faculty of Agriculture, Professor, 農学部, 教授 (00035114)
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| 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)
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| Project Period (FY) |
2002 – 2003
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| Keywords | Lysozyme / α-lactalbumin / ovoinhibitor / yeast Pichia pastoris / glycosylated lysozyme / glycosylated α-lactalbumin / inhibition against elastase |
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| 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
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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
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