Creation of enzymes producing primary alcohols with high selectivity from alkanes

• Project/Area Number: 17K06913
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Catalyst/Resource chemical process
• Research Institution: Tokyo Institute of Technology
• Principal Investigator: Miyaji Akimitsu 東京工業大学, 物質理工学院, 助教 (40452023)
• Project Period (FY): 2017-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2019: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2018: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2017: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: 資源有効利用 / 酵素 / 反応場設計 / 直鎖アルカン変換反応 / 第一級アルコール合成 / アルカン資源有効利用

Outline of Final Research Achievements

We aimed to create an enzyme that produces primary alcohols from n-alkanes by substituting amino acid residues near the catalytic site of the enzyme cytochrome P450BM-3. Various enzymes introducing bulky functional groups and controlling the space size near the catalytic site of cytochrome BM-3 were synthesized and their activity for n-alkane oxidation was investigated.　The selectivity for primary alcohols in the oxidation reactions depends on the molecular size of the amino acid residues introducing near the catalytic site. According to the dependency, we synthesized the enzyme introducing two tryptophan residues near the catalytic site of cytochrome BM-3. The enzyme showed primary alcohol selectivity up to 51% in n-alkane oxidation.

Academic Significance and Societal Importance of the Research Achievements

本研究成果は、直鎖アルカンから選択的に第一級アルコールを合成するバイオ触媒開発における〈触媒部位近傍の官能基導入〉と〈反応場の空間サイズの制御〉を指針とした酵素反応場設計の有効性を示したことに学術的意義がある。この酵素設計指針とノウハウは、天然ガス成分である炭素数４以下の直鎖アルカンを室温条件で第一級アルコール（1-ブタノール、1-プロパノール、エタノール、メタノール）へ変換するバイオ触媒開発にも応用可能であり、２１世紀の物質社会に不可欠な省エネルギー・低環境負荷の天然ガス有効利用技術への展開が期待できる。

Research Products

• [Journal Article] Methanol Biosynthesis from Methane Using <i>Methylosinus trichosporium</i> OB3b Grown in Medium Containing High Copper Concentration (2021)
  • Author(s): MIYAJI Akimitsu
  • Journal Title: Journal of the Japan Petroleum Institute Volume: 64 Issue: 1 Pages: 29-35
  • DOI: 10.1627/jpi.64.29
  • NAID: 130007965368
  • ISSN: 1346-8804, 1349-273X
  • Year and Date: 2021-01-01
  • Peer Reviewed / Open Access
• [Journal Article] Growth-promoting effect of cyanocobalamin on Methylosinus trichosporium OB3b culture under high copper concentration for methanol synthesis from methane (2020)
  • Author(s): Miyaji Akimitsu、Furuya Daiki、Orita Izumi、Baba Toshihide
  • Journal Title: Bioresource Technology Reports Volume: 11 Pages: 100473-100473
  • DOI: 10.1016/j.biteb.2020.100473
  • Peer Reviewed
• [Journal Article] Influence of tryptic hydrolysis on the enzymatic function of the membrane-bound form of particulate methane monooxygenase from Methylosinus trichosporium OB3b (2020)
  • Author(s): Miyaji Akimitsu、Satou Keita、Baba Toshihide
  • Journal Title: Journal of Biotechnology Volume: 323 Pages: 98-106
  • DOI: 10.1016/j.jbiotec.2020.08.006
  • Peer Reviewed
• [Journal Article] Influence of copper ions removal from membrane-bound form of particulate methane monooxygenase from Methylosinus trichosporium OB3b on its activity for methane hydroxylation (2019)
  • Author(s): Miyaji Akimitsu、Nitta Muneyuki、Baba Toshihide
  • Journal Title: Journal of Biotechnology: X Volume: 1 Pages: 100001-100001
  • DOI: 10.1016/j.btecx.2018.100001
  • Peer Reviewed / Open Access
• [Journal Article] Influence of amino acid residues near the active site of cytochrome P450 from Bacillus megaterium on the selectivity of n-octane oxidation to octanol regioisomers (2017)
  • Author(s): Miyaji Akimitsu、Baba Toshihide
  • Journal Title: AIP Conference Proceedings Volume: 1878 Pages: 020001-020001
  • DOI: 10.1063/1.5000169
  • Peer Reviewed
• [Presentation] 高濃度銅イオン共存下でのメタン変換生体触媒の調製 (2020)
  • Author(s): 宮地輝光・古谷大稀・馬場俊秀
  • Organizer: 第126回 触媒討論会
• [Presentation] ビタミン B12 によるメタン資化性菌Methylosinus trichosporium OB3b の増殖促進 (2020)
  • Author(s): 宮地輝光・古谷大稀・折田和泉・馬場俊秀
  • Organizer: 第50回 石油・石油化学討論会
• [Presentation] メタンあるいは低級アルカン有効利用への生体触媒利用 (2019)
  • Author(s): 宮地 輝光
  • Organizer: 第124回 触媒討論会 「水素＋天然ガス＋燃料電池＋コンピュータ」研究会横断若手シンポジウム
  • Invited
• [Presentation] シトクロムP450BM-3変異体の直鎖アルカンからアルコール異性体への選択性にパーフルオロアルカン酸が及ぼす影響 (2018)
  • Author(s): 宮地 輝光・テキ ブンカ・ガンスフ ゲレル・馬場 俊秀
  • Organizer: 第122回 触媒討論会
• [Presentation] アミノ酸側鎖を置換したシトクロムP450 BM-3による直鎖オクタンから1-オクタノールへの酸化反応 (2017)
  • Author(s): 宮地 輝光・テキ ブンカ・馬場 俊秀
  • Organizer: 第120回 触媒討論会
• [Presentation] Influence of amino acid residues near the active site of cytochrome P450 from Bacillus megaterium on the selectivity of n-octane oxidation to octanol regioisomers (2017)
  • Author(s): Akimitsu Miyaji, Toshihide Baba
  • Organizer: The 3rd International Conference on Chemical Engineering, Food and Biotechnology (ICCFB2017)
  • Int'l Joint Research
• [Presentation] Lower alkane oxidation in bacterial cells (2017)
  • Author(s): Akimitsu Miyaji
  • Organizer: 47th Petroleum-Petrochemicals Symposium of JPI 4th KSIEC-JPI Joint Presentation
  • Int'l Joint Research / Invited
• [Book] Catalysis and the mechanism of methane conversion to chemicals: C-C and C-O bonds formation using heterogeneous, homogenous, and biological catalysts (2020)
  • Author(s): Toshihide Baba, Akimitsu Miyaji
  • Total Pages: 220
  • Publisher: Springer, Singapore
  • ISBN: 9789811541322