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New Stereospecific Synthesis of Bioactive Compounds Using Lipase as Catalyst in Organic Solvent

Research Project

Project/Area Number 04556014
Research Category

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

Allocation TypeSingle-year Grants
Research Field 製造化学・食品
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

ODA Junichi  Institute for Chemical Research, Kyoto University, Professor, 化学研究所, 教授 (50027041)

Co-Investigator(Kenkyū-buntansha) HIRATAKE Jun  Inst.Chem.Res., Kyoto Univ., Research Associate, 化学研究所, 助手 (80199075)
KATO Hiroaki  Inst.Chem.Res., Kyoto Univ., Research Associate, 化学研究所, 助手 (90204487)
NISHIOKA Takaaki  Inst.Chem.Res., Kyoto Univ., Associate Professor, 化学研究所, 助教授 (80026559)
田中 琢治  京都大学, 化学研究所, 助手 (40227145)
Project Period (FY) 1992 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥15,900,000 (Direct Cost: ¥15,900,000)
Fiscal Year 1994: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 1992: ¥9,500,000 (Direct Cost: ¥9,500,000)
Keywordslipase / stereospecific synthesis / organic solvent / cyanohydrin / transesterification / lipase activator / 活性化因子 / 大量発現 / 封入体 / リパーゼ活性化因子 / シャペロン / Catalytic triad / 融合タンパク質 / 動的速度論的光学分割 / 光学活性シアノヒドリン類 / 固定化リパーゼ
Research Abstract

Using lipases as catalyst in organic solvent, optically pure cyanohydrins in (s) -configuration were stereoselectively synthesized by transesterification reaction in almost 100% chemical yeild. When disaccharide such as trehalose or sucrose was added at the immobilization of lipases, the carbohydrate added keeps lipase in optimal hydration state and increases the maximal lipase activity. The addition of carbohydrate also increases the durability of the enzyme in organic solvent. Lipase was inhibited by organophosphorus esterase inhibitors in organic solvent, but not inhibited in aqueous solvent. This indicates that the lipase catalyzes the transesterification reaction as a typical serine esterase in organic solvent. Lipase and its activator genes from Pseudomonas aeruginosa TE3285 were cloned and expressed in Escherichia coli. The product lipase was not secreted, but stored in the cells as inactive inclusion bodies. The activator protein was expressed and purified. The lipase activity were recovered by refolding the inclusion bodies under the presence of the activator protein. They are reactivated through a 1 : 1 complex without ATP.

Report

(4 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • 1992 Annual Research Report
  • Research Products

    (36 results)

All Other

All Publications (36 results)

  • [Publications] Inagaki, M.: "One-pot synthesis of optically active cyanohydrin acetates from aldenhydes via lipse-catalyazaed kinetic resolution coupled with in situ formation and racemization of cynohydrins" J. Org. Chem. 57. 5643-5649 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Chihara-Shiomi, M: "Purification, molecular cloning, and expression of lipase from Pseudomonas aeruginosa" Arch. Biochem. Biophys.296. 505-513 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nakatani, T.: "Chemical inactivation of lipase in organic solvent: A lipase from Pseudomonas aeruginosa TE3285 is more like a typial serine enzyme in an organic solvent than aqueous media" Biosci. Biotech. Biochem. 56. 1118-1123 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Inagaki, M.: "One-pot synthesis of optically active cyanohydrin acetates from aldehydes via quinidine-catalyzed transhydrocyanation coupled with lipase-catalyzed kinetic resolution in organic solvent" Bull. chem. soc. Jpn.65. 111-120 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nakai, K.: "Kinetic resolution of racemic α-aminonitriles Via stereo-selective N-acetylation catalyzed by lipase in organic solvent" Bull. Inst Chen Res. Kyoto Unn.70. 333-337 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Ohshima-Hirayama, N.: "Lipase from Pseudomonas aeruginosa-Production in Escherichia coli and activation in vitro with a protein from the downstream gene" Eur. J. Biochem.215. 239-246 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] 西岡孝明: "Pseudomonas aeruginosa TE3285 リパーゼ活性化遺伝子(lipB)産物の機能〓." 生物工学. 72. 194-196 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Shibata, H.: "LipB, private-chaperone for Pseudmonas lipase: Purification and functional studies of the lipase-activator protein" 投稿準備中.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Inagaki, M., et al: "One-pot synthesis of optically active cyanohydrin acetates from aldehydes via lipase-catalyzed kinetic resolution coupled with in situ formation and racemization of cynohydrins." J.Org.Chem.57. 5643-5649 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Chihara-Shiomi, M., et al: "Purification, molecular cloning, and expression of lipase from Pseudomonas aeruginosa." Arch.Biochem.Biophys.296. 505-513 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nakatani, T., et al: "Chemical inactivation of lipase in organic solvent : A lipase from Pseudomonas aeruginosa TE3285 is more like a typical serine enzyme in an organic solvent than aqueous media." Biosci.Biotech.Biochem.56. 1118-1123 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Inagaki, M., et al: "One-pot synthesis of optically active cyanohydrin acetates from aldehydes via quinidine-catalyzed transhydrocyanation coupled with lipase-catalyzed kinetic resolution in organic solvent" Bull.Chem.Soc.Jpn.65. 111-120 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Nakai, K., et al: "Kinetic resolution of racemic alpha-amino-nitriles via stereoselective N-acetylation catalyzed by lipase in organic solvent." Bull.Inst.Chem.Res.Kyoto Univ.70. 333-337 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Oshima-Hirayama, N., et al: "Lipase from Pseudomonas aeruginosa-Production in Escherichia coli and activation in vitro with a protein from the downstream gene." Eur.J.Biochem.215. 239-246 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Inagaki, M., et al: "Conversion of aldehydes to (s) -2-acetoxy nitriles via reversible cyanohydrin formation" Preparative Transformation, 2nd Suppliment, Wiley & Sons. 1 : 10.30-1 : 10.39 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Shibata.H., et al: "LipB,private-chaperone for Pseudomonas lipase : Purificatin and functional studies of the lipase-activator protein" (To be submitted).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Inagaki,M.: "One-pot synthesis of optically active cyanohydrin acetates from aldehydes via lipase-catalyzed kinetic resolution coupled with in situ formation and racemization of cynohydrins" J.Org.Chem. 57. 5643-5649 (1992)

    • Related Report
      1994 Annual Research Report
  • [Publications] Chihara-Shiomi,M.: "Purification,molecular cloning,and expression of lipase from Pseudomonas aeruginosa" Arch.Biochem.Biophys.296. 505-513 (1992)

    • Related Report
      1994 Annual Research Report
  • [Publications] Nakatani,T.: "Chemical inactivation of lipase in organic solvent:A lipase from Pseudomonas aeruginosa TE3285 is more like a typical serine enzyme in an organic solvent than aqueous media" Biosci.Biotech.Biochem. 56. 1118-1123 (1992)

    • Related Report
      1994 Annual Research Report
  • [Publications] Inagaki,M.: "One-pot synthesis of optically active cyanohydrin acetates from aldehydes via quinidine-catalyzed transhydrocyanation coupled with lipase-catalyzed kinetic resolution in organic solvent" Bull.Chem.Soc.,Jpn.65. 111-120 (1992)

    • Related Report
      1994 Annual Research Report
  • [Publications] Nakai,K.: "Kinetic resolution of racemic α-aminonitriles via stereo-selective N-acetylation catalyzed by lipase in orqanic solvent" Bull.Inst.Chem Res.Kyoto Univ.70. 333-337 (1992)

    • Related Report
      1994 Annual Research Report
  • [Publications] Ohshima-Hirayama,N.: "Lipase from Pseudomonas aeruginosa-Production in Escherichia coli and activation in vitro with a protein from the downstream gene" Eur.J.Biochem.215. 239-246 (1993)

    • Related Report
      1994 Annual Research Report
  • [Publications] 西岡孝明: "Pseudomonas aeruginosa TE3285 リバーゼ活性化遺伝子(lipB)産物の機能" 生物工学. 72. 194-196 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Shibata,H.: "LipB,private-chaperone for Pseudomonas lipase:Purification and functional studies of the lipase-activator protein" 投稿準備中.

    • Related Report
      1994 Annual Research Report
  • [Publications] N.Oshima-Hirayama: "Lipase from Pseudomonas aeruginosa:Production in Escherichia coli and activation in vitro with a protein from the downstream gene" Eur.J.Biochem.215. 239-246 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Chihara-Siomi: "Purification,molecular cloning and expression of lipase from Pseudomonas aeruginosa" Arch.Biochem.Biophys.296. 505-513 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Inagaki: "One-pot synthesis of opptically active cyanohydrin acetates from aldehydes via lipasecatalyzed kinetic resolution coupled with in situ racemization" J.Org.Chem.57. 5643-5649 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] T.Nakatani: "Chemical inactivation of lipase in organic solvent:A lipase from Pseudomonas aeruginosa TE3285 is more like a typical serine" Biosci.Biotech.Biochem.56. 1118-1123 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Inagaki: "One-pot synthesis of optically active cyanohydrin acetates from aldehydes via quinidinecatalyzed transhydrocyanation" Bull.Chem.Soc.Jpn.64. 111-120 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Nakai: "Kinetic resolution of racemic a-aminonitriles via stereoselective N-acetylation catalyzed by lipase in organic solvent" Bull.Inst.Chem.Res.,Kyoto Univ.70. 333-337 (1992)

    • Related Report
      1993 Annual Research Report
  • [Publications] M.Inagaki: "One-Pot Synthesis of Optically Active Cyanohydrin Acetates from Aldehydes via Lipase-Catalyzed Kinetic Resolution Coupled with in situ---" J.Org.Chem.57. 5643-5649 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Chihara-Siomi: "Purification,Molecular Cloning,and Expression of Lipase from Pseudomonas aeruginosa" Arch.Biochem.Biophys.296. 505-513 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] T.Nakatani: "Chemical Inactivation of Lipase in Organic Solvent:A Lipase from Pseudomonas aeruginosa TE3285 is More Like a Typical Serine---" Biosci.Biotech.Biochem.56. 1118-1123 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] N.Oshima-Hirayama: "Lipase from Pseudomonas aeruginosa:Production in Escherichia coli and Activation in vitro with a Protein from the Downstream Gene" Eur.J.Biochem.

    • Related Report
      1992 Annual Research Report
  • [Publications] M.Inagaki: "One-Pot Synthesis of Optically Active Cyanohydrin Acetates from Aldehydes via Quinidine-catalyzed Transhydrocyanation---" Bull.Chem.Soc.Jpn.64. 111-120 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] K.Nakai: "Kinetic Resolution of Racemic α-Aminonitriles via Stereoselective N__--Acetylation Catalyzed by Lipase in Organic Solvent" Bull.Inst.Chem.Res.,Kyoto Univ.70. 333-337 (1992)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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