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Functional and structural analysis of heat-resistant dye-dependent D-lactate dehydrogenase and its application to biosensor devices

Research Project

Project/Area Number 18K14394
Research Category

Grant-in-Aid for Early-Career Scientists

Allocation TypeMulti-year Fund
Review Section Basic Section 38030:Applied biochemistry-related
Research InstitutionThe University of Tokushima (2019-2021)
Ritsumeikan University (2018)

Principal Investigator

HAYSHI Junji  徳島大学, 大学院社会産業理工学研究部(生物資源産業学域), 講師 (20802101)

Project Period (FY) 2018-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2021: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2020: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000)
Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2018: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Keywords酵素工学 / 酵素 / 超好熱菌 / 構造生物学 / FAD / 補酵素特異性 / 応用生物化学
Outline of Final Research Achievements

Dye-dependent dehydrogenase (Dye-DH) has a potential as a biosensor devices.
However, Dye-DH is an unstable enzyme and there are few studies. In this study, we found a large number of dye-dependent D-lactate dehydrogenase (DLDH) in hyperthermophilic bacteria. All DLDHs from hyperthermophilic bacteria showed high thermostable. We succeeded in developing an enzyme electrode on which this high thermostable DLDH is immobilized. Using this electrode, we succeeded in detecting D-lactic acid in food samples. This study is considered to play an important role in the development of new measurement methods for D-lactic acid. We also succeeded in analyzing the X-ray crystal structure of DLDH.

Academic Significance and Societal Importance of the Research Achievements

Dye-DHは不安定な酵素であり、有用性に反して研究例が少ない。そのため応用例は限定的であり、また酵素の機能改良に必須の構造情報も少ない。本研究により開発に成功したD-乳酸測定用バイオセンサーは、D-乳酸に対して特異的で、且つ長期間利用できることが明らかとなった。D-乳酸は診断用バイオマーカーや食品の品質管理の指標として有用であるが、これまでに簡便に測定する方法は無かった。本研究の成果はD-乳酸の新規測定法開発において重要な役割を果たすと考えられる。また、Dye-DHの構造情報の取得によりDye-DHの機能改良も可能となり、高性能センサーや電極の開発にも繋がる成果を得たといえる。

Report

(5 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Research-status Report
  • 2019 Research-status Report
  • 2018 Research-status Report
  • Research Products

    (9 results)

All 2022 2021 2020 2019

All Journal Article (7 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 7 results,  Open Access: 3 results) Presentation (2 results)

  • [Journal Article] Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii2022

    • Author(s)
      Kawakami Ryushi、Ohshida Tatsuya、Hayashi Junji、Yoneda Kazunari、Furumoto Toshio、Ohshima Toshihisa、Sakuraba Haruhiko
    • Journal Title

      International Journal of Biological Macromolecules

      Volume: 208 Pages: 731-740

    • DOI

      10.1016/j.ijbiomac.2022.03.114

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Characterization of a novel moderate-substrate specificity amino acid racemase from the hyperthermophilic archaeon Thermococcus litoralis2021

    • Author(s)
      Kawakami Ryushi、Kinoshita Chinatsu、Kawase Tomoki、Sato Mikio、Hayashi Junji、Sakuraba Haruhiko、Ohshima Toshihisa
    • Journal Title

      Bioscience, Biotechnology, and Biochemistry

      Volume: 85 Issue: 7 Pages: 1650-1657

    • DOI

      10.1093/bbb/zbab078

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Identification, characterization, and cloning of a novel aminoacylase, L-pipecolic acid acylase from <i>Pseudomonas</i> species2021

    • Author(s)
      Hayashi Junji、Ichiki Yoshiaki、Kanda Akiko、Takagi Kazuyoshi、Wakayama Mamoru
    • Journal Title

      The Journal of General and Applied Microbiology

      Volume: 67 Issue: 5 Pages: 186-194

    • DOI

      10.2323/jgam.2020.12.001

    • NAID

      130008120232

    • ISSN
      0022-1260, 1349-8037
    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Mutagenesis and structure-based analysis of the role of Tryptophan525 of γ-glutamyltranspeptidase from Pseudomonas nitroreducens2021

    • Author(s)
      Sano Chiharu、Itoh Takafumi、Phumsombat Putthapong、Hayashi Junji、Wakayama Mamoru、Hibi Takao
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 534 Pages: 286-291

    • DOI

      10.1016/j.bbrc.2020.11.093

    • Related Report
      2020 Research-status Report
    • Peer Reviewed
  • [Journal Article] Crystal structure of the catalytic unit of thermostable GH87 α-1,3-glucanase from Streptomyces thermodiastaticus strain HF3-32020

    • Author(s)
      Itoh Takafumi、Panti Niphawan、Hayashi Junji、Toyotake Yosuke、Matsui Daisuke、Yano Shigekazu、Wakayama Mamoru、Hibi Takao
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 533 Issue: 4 Pages: 1170-1176

    • DOI

      10.1016/j.bbrc.2020.09.133

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Unique active site formation in a novel galactose 1‐phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum2020

    • Author(s)
      Ohshida Tatsuya、Hayashi Junji、Yoneda Kazunari、Ohshima Toshihisa、Sakuraba Haruhiko
    • Journal Title

      Proteins: Structure, Function, and Bioinformatics

      Volume: 88 Issue: 5 Pages: 669-678

    • DOI

      10.1002/prot.25848

    • Related Report
      2020 Research-status Report
    • Peer Reviewed
  • [Journal Article] Structural insights into substrate recognition and catalysis by glycoside hydrolase family 87 α-1,3-glucanase from Paenibacillus glycanilnticus FH112019

    • Author(s)
      T. Itoh, R. Intuy, W. Suyotha, J. Hayashi, S. Yano, K. Makable, M. Wakayama, T. Hibi
    • Journal Title

      FEBS Journal

      Volume: - Issue: 12 Pages: 1-20

    • DOI

      10.1111/febs.15161

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] 超好熱菌由来色素依存性D-乳酸脱水素酵素のX 線結晶構造解析2021

    • Author(s)
      林 順司,大志田達也,川上竜巳,里村武範,若山 守,大島敏久, 櫻庭春彦
    • Organizer
      日本農芸化学会中四国支部第58回講演会
    • Related Report
      2020 Research-status Report
  • [Presentation] D-乳酸分析用酵素の探索と機能解析2019

    • Author(s)
      長濱伸哉、林順司、里村武範、若山守、大島敏久、櫻庭春彦
    • Organizer
      日本農芸化学会関西支部例会(第507回講演会)
    • Related Report
      2018 Research-status Report

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Published: 2018-04-23   Modified: 2023-01-30  

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