1997 Fiscal Year Final Research Report Summary
Function-structure of protein-polysaccharide cpmplex constructed by protein engineering.
| Project/Area Number |
08660160
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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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 Univeristy |
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Principal Investigator |
KATO Akio Yamaguchi University, Faculty of Agriculture, Professor, 農学部, 教授 (00035114)
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| Project Period (FY) |
1996 – 1997
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| Keywords | lysozyme / polysaccharide attached lysozyme / polymannosyl lysozyme |
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| Research Abstract |
Protein-polysaccharide conjugates showed the excellent functional properties such as heat stability and emulsifying properties. In order to elucidate the molecular mechanism of the improvements of functional properties, we constructed the glycosylated lysozyme using genetic modification. Complementary DNA encoding hen egg white lysozyme (HEWL) was subjected to site-directed mutagenesis to introduce two N-linked glycosylation sites (Asn^<19>-Try^<20>-Tr^<21> and Asn^<49>-Ser^<50>-Thr^<51>) into both positions 19 and 49 by substituting Arg-21 with Thr and Gly-49 with Asn, respectively. The single and double glycosylated lysozymes (G49N,R21T,R21T/G49N) were expressed in Saccharomyces cerevisiae carrying the yeast expression plasmid inserted the single and double mutant HEWL cDNAs. The mutant lysozymes were predominantly expressed a polymannosyl form with a small amout of two oligomannosyl forms. The polymannosyl lysozymes G49N and R21T were glycosylated at positions 49 and 19 with approximately 300 mannose residues, respectively, while the length of the polymannosyl chains attached to R21T/G49N was approximately 272 and 18 mannose residues. The R21T/G49N showed better emulsifying properties than two types of single polymannosyl lysozymes R21T and G49N.In a case of single polymannosyl lysozyme, G49N showed somewhat better emulsifying properties than R21T.The removal of polymannosyl chain from lysoyme with Endo- beta -N-acetylglucosaminidase (Endo-H) resulted in a dramatic decrease in the emulsifying properties of polymannosyl lysozymes. This suggests that the plysaccharide attachment is essential for the functional property.
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Research Products
(14 results)
