| Project/Area Number |
08660165
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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 | Kagoshima University |
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Principal Investigator |
IBRAHIM Hisham Kagoshima University, Faculty of Agriculture Dept. Biochemical Science & Technology (Assistant Professor), 農学部, 講師 (90274836)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1996: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | lysozyme / muramidase / active site / site-directed mutagenesis / antimicrobial / Gram-positive bacteria / membrane-binding / homodimer / リゾチーム / 遺伝子工学 / グラム陰性菌 / 疎水性ペプチド / 卵白リゾチーム / 量体化 / 酵母発現系 / ムラミダーゼ活性 / 抗菌活性 / 構造安定性 |
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| Research Abstract |
The genetic modification of lysozyme was attempted to clarify the role of enzyme activity in its inherent antimicrobial activity against Gram-positive bacteria and to improve bactericidal action to ward Gram-negative ones. To elucidate the role of catalytic function in the antibacterial activity of lysozyme, two mutants (E35A-Lz or D52S-Lz) each of which lacking one of the catalytic residues (Glu35 and Asp52) were generated by sitespecific mutagenesis. Lysozyme attached with different lengths of hydrophobic peptides to its C-terminus were also constructed through genetic fusion, in order to investigate the effective degree of hydrophobicity for killing of Gram-negative bacteria. The oligonucleotides encoding F-V-P (H3), F-F-V-A-P (H5) and F-F-V-A-I-I-P (H7) were fused to the C-terminus Leul29 of lysozyme cDNA.The reconstruted cDNAs were inserted into a yeast expression vector and recombinant proteins were expressed in S.cerevisiae AH22. The enzymatically inactivated lysozymes (D52S or
… More
E35A) exhibited antibacterial activity against Gram-Positive bacterial comparable to the activity of native enzyme, indicating that the antibacterial activity of lysozyme to Gram-positive bacteria is operationally independent of its catalytic function. All of hydrophobic peptide-fused lysozymes retained 75-80 % enzyme activity and their antibacterial action to E.coli were proportionally increased with the increase of the length of hydrophobic peptides. The results suggest that the hydrophobicity of lysozyme play an important role in killing Gram-negative bacteria. However, when the catalytic activity of fusion lysozyme (H5) was diminished by mutating Glu-35 to Ala, its antubacterial activity to E.coli was greatly decreased, suggesting that not only the increased hydrophobicity but also the catalytic function is important for the novel bactericidal activity against Gram-negative bacteria of lysozyme. The most striking data is that the catalytic function of lysozyme play no direct role in its inherent antibacterial activity against Gram-positive bacteria. Less
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