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Analytical study of the primary structures of microbial phospholipases on the basis of molecular biology

Research Project

Project/Area Number 05454571
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Biological pharmacy
Research InstitutionNagoya City University

Principal Investigator

IKEZAWA Hiroh  Nagoya City Univ., Fac.Pharm.Sci., Professor, 薬学部, 教授 (40080163)

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)
Project Period (FY) 1993 – 1995
Project Status Completed (Fiscal Year 1995)
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)
KeywordsMicrobial 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
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.

Report

(4 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • 1993 Annual Research Report
  • Research Products

    (28 results)

All Other

All Publications (28 results)

  • [Publications] 田村悦臣: "Bacillus cereus由来スフィンゴミエリナーゼのアスパラギン酸変異体の構築とその性質" 日本細菌学雑誌. 49. 237-237 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 小林とも子: "B. brevisを用いたB. thuringiensis由来PIPLC大量発現系の構築" 生化学. 66. 1018-1018 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 山田敦子: "Site-directed mutagenesisによるB. cereus由来sphingomyelimaseの活性部位に関する研究" 生化学. 67. 949-949 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Masahiro Tomita: "The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase" Biochimica et Biophysica Acta. 1203. 85-92 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 為石香織: "Bacillus cereus由来sphingomyelinaseのアスパラギン酸変異体" 生化学. 65. 1031-1031 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 山田敦子: "B. cereus由来sphingomyelinaseの部位特異的変異体の構築とその解析" 生化学. 66. 818-818 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 小林とも子: "B. brevisを用いたB. thuringiensis由来PIPLC大量発現系の構築" 生化学. 66. 1018-1018 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Hiro-omi Tamura: "Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomylinase" Biochemical Journal. 309. 757-764 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Hiroh Ikezawa: "Studies on the active sites of Bacillus cereus sphingomyelinase Substitution of some amino acids by site-directed mutagenesis" Amino Acids. 9. 293-298 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 田村悦臣: "脂質生化学研究35巻" 日本脂質生化学研究会, 4 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 小林とも子: "脂質生化学研究36巻" 日本脂質生化学研究会, 4 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 田村悦臣: "脂質生化学研究37巻" 日本脂質生化学研究会, 255-257 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] M.Tomita, Y.Ueda, H.Tamura, R.Taguchi and H.Ikezaga: "The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase" Biochimica et Biophysica Acta. 1203. 85-92 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] H.Tamura, K.Tameishi, A.Yamada, M.Tomita, Y.Matsuo, K.Nishikawa and H.Ikezawa: "Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomeylinase" Biochemical Journal. 309. 757-764 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] H.Ikezawa, K.Tameishi, A.Yamada, H.Tamura, K.Tsukamoto, Y.Matsuo and K.Nishikawa: "Studies on the active sites of Bacillus cereus sphingomyelinase-Substitution of some amino acids by site-directed mutagenesis" Amino Acids. 9. 293-298 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Hiro-omi Tamura: "Mutation in aspartic acid residues modifies catalytic and haemolytic activities of Bacillus cereus sphingomyelinase" Biochemical Journal. 309. 757-764 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Hiroh Ikezawa: "Studies on the active sites of Bacillus cereus sphingomyelinase-Substitution of some amino acids by site-directed mutagenesis" Amino Acids. 9. 293-298 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 山田 敦子: "Site-directed mutagenesisによるB.cereus由来sphingomyelinaseの活性部位に関する研究" 生化学. 67. 949-949 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 田村 悦臣: "脂質生化学研究37巻" 日本脂質生化学研究会, 3 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Hiro-omi Tamura: "Mutation in aspartic acids modifies catalytic and hemolytic activities of Bacillus cereus sphingomyelinase" Biochemical Journal. (in press). (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] 山田敦子: "B.cereus由来sphingomyelinaseの部位特異的変異体の構築とその解析" 生化学. 66. 818-818 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 田村悦臣: "Bacillus cereus由来スフィンゴミエリナーゼのアスパラギン酸変異体の構築とその性質" 日本細菌学雑誌. 49. 237-237 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 小林とも子: "B.brevisを用いたB.thuringiensis由来PIPLC大量発現系の構築" 生化学. 66. 1018-1018 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] 小林とも子: "脂質生化学研究36巻" 日本脂質生化学研究会, 4 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Masahiro Tomita: "The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase" Biochimica et Biophysica Acta. 1203. 85-92 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 為石香織: "Bacillus cereus由来sphingomyelinaseのアスパラギン酸変異体の解析" 生化学. 65. 1031-1031 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 田村悦臣: "Bacillus cereus由来スフィンゴミエリナーゼのアスパラギン酸変異体の構築とその性質" 日本細菌学雑誌. 49. 237-237 (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] 田村悦臣: "脂質生化学研究35巻" 日本脂質生化学研究会, 4 (1993)

    • Related Report
      1993 Annual Research Report

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Published: 1993-04-01   Modified: 2016-04-21  

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