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Molecular basis and functional modulation of carbohydrate epimerases and isomerases useful for carbohydrate conversion

Research Project

Project/Area Number 21K05388
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 38030:Applied biochemistry-related
Research InstitutionHokkaido University

Principal Investigator

Wataru Saburi  北海道大学, 農学研究院, 准教授 (00598089)

Project Period (FY) 2021-04-01 – 2024-03-31
Project Status Completed (Fiscal Year 2023)
Budget Amount *help
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2023: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2022: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2021: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Keywordscellobiose 2-epimerase / mannose 2-epimerase / AGE superfamily / carbohydrate / mannose / cellobiose / mannose isomerase / マンノース2-エピメラーゼ / セロビオース2-エピメラーゼ / オリゴ糖 / 糖質 / 異性化 / キメラ酵素 / エピメラーゼ / イソメラーゼ
Outline of Research at the Start

本研究では,セロビオース2-エピメラーゼ (CE) を用いた反応特異性の分子基盤の解明とマンノース2-エピメラーゼ (ME) の高活性変異酵素の開発を行う.CEに関しては,各種変異酵素の機能・構造解析,基質アナログへの反応解析を通じ反応特異性を支配する構造因子を解明する.MEに関する研究では,大腸菌外膜での組換え酵素生産系とマンノース資化能を失った変異株を用いた効率的高活性変異酵素スクリーニング技術を構築し,高活性変異酵素を開発する.

Outline of Final Research Achievements

Carbohydrate epimerase/isomerases are essential for the diversification and sophistication of enzymatic synthesis of carbohydrates. In this study, two carbohydrate epimerases, cellobiose 2-epimerase (CE) and mannose 2-epimerase (ME) were studied: CE, the molecular basis of the reaction specificity; ME, the structure-function relationship and the development of practical enzymes with high specific activity. In CE, the four α-helices at the N-terminal end of the catalytic domain were shown to contain structures important for isomerase activity. Additionally, the substitution of a small amino acid for Asn, involved in the formation of a hydrogen bond with sugar at reducing end, was shown to drastically enhance the reaction specificity for isomerization. Although a highly active mutant enzyme of ME was not obtained, the crystal structure of ME was successfully determined and structures important for substrate specificity was clarified.

Academic Significance and Societal Importance of the Research Achievements

本研究では糖質の合成の多様化に貢献する糖質異性化酵素が示す反応特異性や基質特異性に関する分子機構の一端を解き明かすことができた.マンノース2-エピメラーゼの構造は本研究により初めて明らかにされたものであり,本酵素の基質特異性の分子基盤が解明されたことの学術的意義は大きい.また本研究で得られた知見は糖質のより高効率な合成酵素の開発や,合成される糖質の大量合成・機能解析へとつながるものと考えられ,機能性糖質の開発など応用に関する波及効果も大きく,社会的意義も大きいと考えられる.

Report

(4 results)
  • 2023 Annual Research Report   Final Research Report ( PDF )
  • 2022 Research-status Report
  • 2021 Research-status Report
  • Research Products

    (8 results)

All 2024 2023 2022 Other

All Journal Article (1 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 1 results) Presentation (5 results) (of which Int'l Joint Research: 2 results,  Invited: 2 results) Remarks (2 results)

  • [Journal Article] Structural insights into the substrate specificity and activity of a novel mannose 2-epimerase from Runella slithyformis.2023

    • Author(s)
      Wang, H., Sun, X., Saburi, W., Hashiguchi, S., Yu, J., Ose, T., Mori, H., and Yao, M.
    • Journal Title

      Acta Crystallographica Section D: Structural Biology

      Volume: 79 Issue: 7 Pages: 585-595

    • DOI

      10.1107/s205979832300390x

    • Related Report
      2023 Annual Research Report
    • Peer Reviewed / Int'l Joint Research
  • [Presentation] Rhodothermus marinus由来セロビオース2-エピメラーゼにおける還元末端糖残基の2-OH基との相互作用残基の反応特異性への寄与の解析2024

    • Author(s)
      佐分利 亘,森 春英
    • Organizer
      日本農芸化学会2024年度大会
    • Related Report
      2023 Annual Research Report
  • [Presentation] Development of functional foodstuffs produced using carbohydrate-metabolizing enzymes2023

    • Author(s)
      Wataru Saburi
    • Organizer
      The 7th Sino-Japan Joint Symposium on Enzyme Technology
    • Related Report
      2023 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] セロビオース2-エピメラーゼが持つイソメラーゼ活性に重要な構造の解析2022

    • Author(s)
      佐分利 亘,森 春英
    • Organizer
      日本応用糖質科学会2022年度大会
    • Related Report
      2022 Research-status Report
  • [Presentation] Runella slithyformis由来マンノース2-エピメラーゼ (RsME) の高活性化を指向したスクリーニング2022

    • Author(s)
      内山 昌典,橋口 早紀,佐分利 亘,森 春英
    • Organizer
      日本応用糖質科学会2022年度大会
    • Related Report
      2022 Research-status Report
  • [Presentation] Expansion of enzymatic synthesis of carbohydrates using novel carbohydrate-active enzymes2022

    • Author(s)
      Wataru Saburi
    • Organizer
      The 17th International Symposium of the Protein Society of Thailand
    • Related Report
      2022 Research-status Report
    • Int'l Joint Research / Invited
  • [Remarks] 北海道大学大学院農学研究院 生物化学研究室ホームページ

    • URL

      http://lab.agr.hokudai.ac.jp/biochem/

    • Related Report
      2023 Annual Research Report
  • [Remarks] 北海道大学大学院農学研究院生物化学研究室ホームページ

    • URL

      http://lab.agr.hokudai.ac.jp/biochem/

    • Related Report
      2022 Research-status Report 2021 Research-status Report

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Published: 2021-04-28   Modified: 2025-01-30  

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