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Studies on the cofactors and reaction mechanisms of nitrile hydratase which is useful for the synthesis of amides

Research Project

Project/Area Number 02660114
Research Category

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

Allocation TypeSingle-year Grants
Research Field 応用微生物学・発酵学
Research InstitutionNagoya University (1991)
Kyoto University (1990)

Principal Investigator

NAGASAWA Toru  Nagoya University, Food Science & Technology Associate Professor, 農学部, 助教授 (60115904)

Co-Investigator(Kenkyū-buntansha) SHIMIZU Sakayu  Kyoto University, Agricultural Chemistry, Associate Professor, 農学部, 助教授 (70093250)
Project Period (FY) 1990 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥1,300,000 (Direct Cost: ¥1,300,000)
KeywordsNitrile hydratase / Acylamide / Iron enzyme / Cobalt enzyme / ESR / Iron-active centrer / hydrated PQQ / Pyrroloquinoline quinone / 省エネルギ-
Research Abstract

We have established, the enzymatic production process of acrylamide using the microbial nitrile hydratase and the industrial production of acrylamide was started since several years ago. Compared to the high development of the applied works, the basic studies on nitrile hydratase do not progress fully. Thus, the present works focused on more basic works, the elucidation of the cofactors and reaction mechanisms of nitrile hydratase. The following results are summarized.
(1) There are two kinds of nitrile hydratases, iron or cobalt is involved as a cofactor.
(2) ESR studies showed the following : nitrile hydratase is the first known nonheme iron enzyme with a typical low-spin Fe (III) coordination environment, the axial position of the iron (III) site in the native enzyme may be occupied by thiolate and equo groups, and aliphatic nutrile-substrates directly bind to the iron (III)-active center through water substrate replacement. Cobalt also probably plays the same role, the binding of nitrile and activation of it.
(3) Iron nitrile hydratase involves an active carbonyl cofactor as a second prosthetic group, probably pyrrologuinoline quinone. There may be an interaction, such as a hydrogen bond, between the ferric site and a form of hydrated PQQ.
(4) The iron and cobalt nitrile hydratases were cloned. The sequence of these genes was determined and the extreme high homology of two genes was found in spite of the difference of confactors.

Report

(3 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • Research Products

    (37 results)

All Other

All Publications (37 results)

  • [Publications] Toru Nagasawa: "A novel nitrilase,arylacetonitrilase,of Alcaligenes faecalis JM3 Purification and characterization" European Journal of Biochemistry. 194. 765-772 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toru Nagasawa: "e-Caprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase" Archieves of Microbilolgy. 155. 13-17 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toru Nagasawa: "Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1" European Journal of Biochemistry. 196. 581-589 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toru Nagasawa: "Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodcoccus rhodochrous J1" Applied Microbiology Biotechnology. 34. 783-788 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Michihiko Kobayashi: "Cloning,nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1" Biochimica et Biophysica Acta. 1129. 23-33 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Makoto Nishiyama: "Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23" Journal of Bacteriology. 173. 2465-2472 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toru Nagasawa: "Large-scale bioconversion of nitriles into useful amides and acids" Van Nostrand Reinhold, 41 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toru Nagasawa: "Industrial use of enzymes" Bernard Wolnak and Associates, 8 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Kobayashi, N. Yanaka, T. Nagasawa, H. Yamada: "Monohydrolysis of an aliphatic dintrile compound by nitrilase from Rhodococcus rhodochrous K22" Tetrahedron. 46. 5587-5590 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Kobayashi, N. Yanaka, T. Nagasawa, H. Yamada: "Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles" J. Bacteriol. 172. 4807-4815 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Kobayashi, N. Yanaka, T. Nagasawa, H. Yamada: "Nitrilase-catalyzed production of pyrazinoic acid an antimycrobacterial agent, from cyanopyrazine by resting cells of Rhodococcus rhodochrous J1" J. Antibiotics. 43. 1316-1320 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] J. Mauger, T. Nakamura, T. Nagasawa, H. Yamada: "Occurrence of a novel nitrilase, arylacetonitrilase, in Alcaligenes faecalis JM3" Arch. Microbiol.155. 1-6 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Nagasawa, T. Nakamura, H. Yamada: "epsilon-Caprolactam, a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase" Arch. Micribiol.155. 13-17 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Nagasawa, T. Nakamura, H. Yamada: "Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase" Appl. Microbiol. Biotechnol.34. 322-324 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Nagasawa, J. Mauger, H. Yamada: "A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3" Eur. J. Biochem.194. 765-772 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Kobayashi, N. Yanaka, T. Nagasawa, V. Nardi-Dei Y. Mihara, H. Yamada: "Hyperinduction of an aliphatic nitrilase by Rhodococcus rhodochrous K22" FEMS Microbiol. Lett. 77. 121-124 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Nagasawa, K. Takeuchi, V. Nardi-Dei, K. Mihara, H. Yamada: "Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1" Appl. Microbiol. Biotechnol.34. 783-788 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Nagasawa, K. Takeuchi, H. Yamada: "Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhochrous J1" Eur. J. Biochem.196. 581-589 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Nishiyama, S. Horinouchi, M. Kobayashi, T. Nagasawa, H. Yamada, T. Beppu: "Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23" J. Bacteriol.173. 2465-2472 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] M. Kobayashi, M. Nishiyama, T. Nagasawa, S. Horinouchi, T. Beppu, H. Yamada: "Cloning, nucleotide sequence, and expression of Escherichia coli of two cobalt-containing J1" Biochim. Biophys. Acta. 1129. 23-33 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] T. Gilligan, T. Nagasawa, H. Yamada: "Production of S-(+)-2-phenylpropionic acid from (R, S)-2-phenylpropionitrile by the combination of nitrile hydratase and streroselective amidase in Rhodococcus eequi TG328" Appl. Microbiol. Biotechnol.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toru Nagasawa: "A novel nitrilase,arylacetonitrilase,of Alcaligenes faecalis JM3 Purification and characterization" European Journal of Biochemistry. 194. 765-772 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toru Nagasawa: "eーCaprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase" Archieves of Microbiology. 155. 13-17 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toru Nagasawa: "Characterization of a new cobaltーcontaining nitrile hydratase purified from ureaーinduced cells of Rhodococcus rhodochrous J1" European Journal of Biochemistry. 196. 581-589 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toru Nagasawa: "Optimum culture conditions for the production of cobaltーcontaining nitrile hydratase by Rhodococcus rhodochrous J1" Applied Microbiology Biotechnology. 34. 783-788 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Michihiko Kobayashi: "Cloning,nucleotide sequence and expression in Escherichia coli of two cobaltーcontaihing nitrile hydratase genes from Rhodococcus rhodochrous J1" Biochimica et Biophysica Acta. 1129. 23-33 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Makoto Nishiyama: "Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23" Journal of Bacteriology. 173. 2465-2472 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toru Nagasawa: "Largeーscale bioconversion of nitriles into useful amides and acids" Van Nostrand Reinhold, 41 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toru Nagasawa: "Industrial use of enzymes" Bernard Wolnak and Associates, 8 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] Jacques Mauger: "Synthesis of various aromatic amide derivatives using nitrile hydratase of Rhodococcus rhodochrous J1" Tetrahedron. 45. 1347-1354 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toru Nagasawa: "Microbial transformations of nitriles" Trends in Biotechnology. 7. 153-158 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toru Nagasawa: "Superiority of Pseudomonas chlororaphis B23 nitrile hydratase as a catalyst for the enzymatic production of acrylamide" Experimentia. 45. 1066-1070 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toru Nagasawa: "Application of nitrile converting enzymes for the production of useful compounds" Pure & Applied Chemistry. 62. 1441-1444 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toru Nagasawa: "Occurrence of a novel nitrilase,arylacetonitrilase,in Alcaligenes faecalis JM 3" Archives of Microbiooogy. 155. 1-6 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toru Nagasawa: "εーCaprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous Ji nitrilase" Archives of Microbiology. 155. 13-17 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toru Nagasawa: "Largeーscale bioconversion of nitriles into useful amides and acids" Van Nostrand Reinhold, 41 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] Toru Nagasawa: "Industrial use of enzymes" Bernard Wolnak and Associates, 8 (1990)

    • Related Report
      1990 Annual Research Report

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Published: 1990-04-01   Modified: 2016-04-21  

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