Structure analysis of cellulose-cellobiohydrolase complex by in situ three-dimensional magnetic orientation NMR

2023 Fiscal Year Final Research Report

• Project/Area Number: 20H03043
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 40020:Wood science-related
• Research Institution: Forest Research and Management Organization (2022-2023); Kyoto University (2020-2021)
• Principal Investigator: Kusumi Ryosuke 国立研究開発法人森林研究・整備機構, 森林総合研究所, 主任研究員 等 (70546530)
• Co-Investigator(Kenkyū-buntansha): 和田 昌久 京都大学, 農学研究科, 教授 (40270897)
• Project Period (FY): 2020-04-01 – 2024-03-31
• Keywords: 固体NMR / in situ測定 / 単結晶振動パターン / セルロース

Outline of Final Research Achievements

To elucidate the mechanism of cellulose hydrolysis by cellulases, it is essential to analyze the binding sites on an atomic scale in a water environment. To achieve this, we established a novel NMR method based on the three-dimensional magnetic orientation of organic microcrystals. We developed a probe that enables modulation rotation of the sample tube in NMR and tilting of the modulated rotation axis only during NMR measurement. The chemical shift tensor of a model compound was determined precisely by using the probe, even for μm-sized microcrystals in a liquid. The developed probe can be also combined with the multidimensional sequences, which are required for complex compounds such as carbohydrate-binding modules.

Free Research Field

木質科学

Academic Significance and Societal Importance of the Research Achievements

バイオリファイナリーを加速させる上で、セルラーゼ糖結合性モジュールと結晶セルロースの結合状態の原子レベルでの実態解明が欠かせない。しかしながら、基質が水に不溶の結晶セルロースであり、加水分解環境下における結合部位の精密解析は、回折法や電子顕微鏡法、溶液NMR法など従来法では困難な状況にあった。開発したin situ三次元磁場配向NMRは水中での三次元磁気拘束を通じて対象の“単結晶NMR”を可能とし、セルラーゼに加え他の酵素や生体分子・生理活性物質などの液体・反応プロセス中での構造解析にも展開できる。有機・無機材料学・構造生物学・分子工学・創薬・医療工学など幅広い分野への波及が大いに期待される。