| Project/Area Number |
05454571
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Nagoya City University |
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Principal Investigator |
IKEZAWA Hiroh Nagoya City Univ., Fac.Pharm.Sci., Professor, 薬学部, 教授 (40080163)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Tomoko Nagoya City Univ., Fac.Pharm.Sci., Assistant, 薬学部, 助手 (70214533)
TSUKAMOTO Kikuo Nagoya City Univ., Fac.Pharm.Sci., Lecturer, 薬学部, 講師 (20183478)
TAGUCHI Ryo Nagoya City Univ., Fac.Pharm.Sci., Assistant Professor, 薬学部, 助教授 (20080210)
田村 悦臣 名古屋市立大学, 薬学部, 講師 (50201629)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1995: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1994: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1993: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Microbial phospholipases / Bacillus cereus sphingomyelinase / Penicillium notatum phospholipase B / Bacillus thuringiensis phosphatidylinositol-specific phospholipase C / Primayr stucture of phospholipases / primary structure-activity relationship / site-directed mutagenesis / GPI-achored proteins / 酸素蛋白質一次構造 / ホスホリパーゼ |
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| Research Abstract |
1. The relationship between the primary structure and the enzyme activity of Bacillus cereus sphingomyelinase was investigated by site-directed mutagenesis, converting Asp and His residues to Gly and Ala residues, respectively, During the study, the structural similarity between bacterial sphingomyelinase and bovine pancreatic DNase I was established by 3D-1D method. Taken together, we concluded that His151, His296 and Asp295 of B.cereus sphingomyelinase are essential for the catalytic and the hemolytic activities and that Asp233 affects the enzyme activity by interacting with His296 whereas Asp126 and Asp156 are involved in substrate recognition by the enzyme.
2. The structure of Penicillium notatum phospholipase B was characterized by us as one of the GPI-anchored proteins. Also, we succeeded in the expression of phospholipase B gene in Escherichia coli as an inert form. We demonstrated that this enzyme was released from the sphaeroplast membrane of P.notatum into the culture medium by the autocatalytic cleavage of GPI anchor.13EA03 : 3. For the study of the structure-activity relationship on Bacillus thuringiensis phosphatidylinositol-specific phospholipase C,we established mass-production system of this phospholipase, using the host-vector system of Bacillus brevis which yielded 500 fold that amount of the enzyme produced by B.thuringiensis.
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