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Reaction Mechanisms and Application of New Bacterial Enzymes

Research Project

Project/Area Number 08044218
Research Category

Grant-in-Aid for international Scientific Research

Allocation TypeSingle-year Grants
SectionJoint Research
Research Field 応用微生物学・応用生物化学
Research InstitutionToyama Prefectural University

Principal Investigator

ASANO Yasuhisa  Toyama Prefectural University, Biotechnology Research Center, Professor, 工学部, 教授 (00222589)

Co-Investigator(Kenkyū-buntansha) ヘイデン ブロナ  ダブリン大学, 生化学科, 博士研究員
アーテミュイク ピーター  シェフィールド大学, 分子生物, 博士研究員
ライス デイビット・W  シェフィールド大学, 分子生物, 教授
KOMEDA Hidenobu  Toyama Prefectural University, Biotechnology Research Center, Associate Professo, 工学部, 助手 (50285160)
KATO Yasuo  Toyama Prefectural University, Biotechnology Research Center, Associate Professo, 工学部, 助教授 (20254237)
DAIRI Tohru  Toyama Prefectural University, Biotechnology Research Center, Associate Professo (70264679)
ライス デイビッド・W  シェフィールド大学, 分子生物および生物工学科, 教授
ウォーラー デイビッド・  シェフィールド大学, 分子生物及び生物工学科, 博士研究員
ブリットン K・リンダ  シェフィールド大学, 分子生物及び生物工学科, 博士研究員
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥9,800,000 (Direct Cost: ¥9,800,000)
Fiscal Year 1997: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 1996: ¥4,900,000 (Direct Cost: ¥4,900,000)
KeywordsAmino Acid Dehydrogenase / Opine Dehydrogenase / X-ray Crystallographic Studies / Site Directed Mutagenesis / Molecular Cloning / Leucine Dehydrogenase / Phenylalanine Dehydrogenase / Methylaspartate Ammonia-Lyase / X線構造解析 / ロイシン脱水素酵素
Research Abstract

(1) Phenylalanine dehydrogenase (PheDH) : PheDHs from Bacillus sphaericus, B.badius, Sporosarcina ureae were purified from Escherichia coli transformants in large scales and sent to Prof.Engel (University College Dublin, Ireland) and Prof.Rice (University of Sheffield, U.K.) for X-ray crystallography. Based on informations of X-ray crystallography of glutamate dehydrogenase, chimeric enzymes of PheDH with altered substrate specificities were constructed.
(2) Methylaspartate ammonia-lyase (MAL) : MALs from Enterobacteria such as Enterobacter, Citrobacter, Proteus were purified to homogeneities and their enxymological properties were analyzed in detail. MAL from Citrobacter amalonaticus strain YG-1002 was digested with endo peptidases and N-terminal amino acid sequences were determined. Based on the information, DNA probes were synthesized. The gene for the enzyme was cloned form the genomic library of c.amalonaticus strain YG-1002 by PCR and Southern hybridization. The sequence of the gene was compared with that of a strict anaerobe Clostridium tetanomorphum. MAL was crystallized and sent to Prof.Rice for studies of X-ray crystallography.
(3) Opine dehydrogenase (ODH) : The gene for ODH from Arthrobacter sp.IC was cloned and expressed in E.coli. The enzyme was purified in a large scale and sent to Prof.Rice, and on its X-ray studies were started. The first structure of a (D,L) superfamily member, N-(1-D-Carboxylethyl)-L-norvaline dehydrogenase from Arthrobacter sp.strain 1C,has been solved to 1.8* resolution and the location of the bound coenzyme determined.
(4) Results were presented in some international academic meetings.

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (25 results)

All Other

All Publications (25 results)

  • [Publications] K.L. Britton: "Crystallisation of Arthrobacter sp.strain 1C opine dehydrogenase and its complex with NAD^+." Acta Crystallography D. 54. 124-126 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Y.Asano: "Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp.strain DM-86-1" Archives of Microbiology. 169. 220-224 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Y.Kato: "3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for(S)-glutamate fermentation in Enterobacteriaceae" Archives of Microbiology. 168. 457-463 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] K.Nakamura: "Quantitation of L-amino acids by cycling between an aminotransferase and a dehydrogenase:application to the determination of L-phenylalanine in human serum" Analytical Biochemistry. 234. 19-22 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Y.Asano: "Screening for novel amino acid dehydrogenases-soil of Toyama prefecture as a source of microorganisms-(in Japanese)" Bulletin of Toyama Prefctural University. 6. 101-106 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] S.Y.K. Seah: "Alternation in substrate specificity of phenylalanine dehydrogenase by site directed mutagenesis" FEBS Letters. 370. 93-96 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] A.Yamada, T.Dairi, Y.Ohno, X.-L.Huang and Y.Asano: "Nucleotide Sequencing of Phenylalanine Dehydrogenase Gene from Bacillus badius IAM 11059" Biosci.Biotech.Biochem.59. 1994-1995 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] T.Dairi and Y.Asano: "Cloning, nucleotide Sequencing, and Expressing of an Opine Dehydrogenase Gene from Arthrobacter sp.strain 1C." Apple.Environ.Microbiol.61. 3169-3171 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] S.Y.K.Seah, K.L.Britton, P.J.Baker, D.W.Rice, Y.Asano and P.C.Engel: "Alternation in Substrate Specificity of Phenylalanin Dehydrogenase by Site Directed Mutagenesis" FEBS Letters. 370. 93-96 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Y.Asano, M.Tanetani, and N.Horita: "Screening for Novel Amino Acid Dehydrogenases-Solid of Toyama Prefecture as a Source of Microorganisms- (in Japanese)." Bull.Toyama Pref.Univ.6. 101-106 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Y.Kato, H.Yamada and Y.Asano: "Stereoselective Synthesis of Opine-type Secondary Amine Dicarboxylic Acids by a New Enzyme Opine Dehydrogenase-Use of Recombinant Enzymes-" J.Molec.Catal.B : Enzymatic. 1. 151-160 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] K.Nakamura, T.Fujii, Y.Kato, Y.Asano and A.J.L.Cooper: "Quantitaion of L-Amino Acids by Cycling between an Aminotransferase and a Dehydrogenase : Application to the Determination of L-Phenylalanine in Human Serum" Anal.Biochem.234. 19-22 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Y.Kato, and Y.Asano: "3-Methylaspartate Ammonia-Lyase as a Maker Enzyme of the Mesaconate Pathway for (S) Glutamate Fermentation in Enterobacteriaceae" Arch.Microbiol.168. 457-463 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Y.Asano, and M.Tanetani: "Thermostable Phenylalanine Dehydrogenase from a Mesophilic Microbacterium sp.strain DM-86-1" Arch Microbiol.169. 220-224 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] K.L.Britton, H.F.Rogers, Y.Asano, T.Dairi, Y.Kato, T.J.Stillman, and D.W.Rice: "Crystallisation of Arthrobacter sp.strain 1C Opine Dehydrogenase and Its Complex with NAD+." Acta Cryst.D.54. 124-126 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] K.L.Britton, Y.Asano, and D.W.Rice: "The Crystal Structure and Active Site Location of the N-(1-D-Carboxyethyl)-L-norvaline dehydrogenase from Arthrobacter sp.strain 1C." Nature Structural Biol.(in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] K.L.Britton: "Crystallisation of Arthrobacter sp.strain 1C opine dehydrogenase and its complex with NAD+." Acta Crystallography D. (in press).

    • Related Report
      1997 Annual Research Report
  • [Publications] Y.Asano: "Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp.strain DM-86-1" Archives of Microbiology. (in press).

    • Related Report
      1997 Annual Research Report
  • [Publications] Y.Kato: "3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae" Archives of Microbiology. 168. 457-463 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] K.Nakamura: "Quantitation of L-amino acids by cycling between an aminotransferase and a dehydrogenase : application to the determination of L-phenylalanine in human serum" Analytical Biochemistry. 234. 19-22 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Y.Asano: "Screening for novel amino acid dehydrogenases-soil of Toyama Prefecture as a source of microorganisms-(in Japanese)" Bulletin of Toyama Prefctural University. 6. 101-106 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] S.Y.K.Seah: "Alternation in substrate specificity of phenylalanine dehydrogenase by site directed mutagenesis" FEBS Letters. 370. 93-96 (1995)

    • Related Report
      1997 Annual Research Report
  • [Publications] Stephen Y.K.Seah: "Alteration in relative activities of phenylalanine dehydrogenase towards different substrates by site-directed mutagenesis." FEBS Letters. 370. 93-96 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] T.Dairi: "Cloning,nucleotide sequencing, and expression of an opine dehydrogenase gene from Arthrobacter sp. strain 1C." Applied and Environmental Microbiology. 61. 3169-3171 (1995)

    • Related Report
      1996 Annual Research Report
  • [Publications] Y.Kato: "Stereoselective synthesis of opine-type secondary amine carboxylic acids by a new enzyme opine dehydrogenase. -Use of recombinant enzymes-" Journal of Molecular Catalysis B: Enzymatic. 1. 151-160 (1996)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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