Molecular design of nylon oligomer degradation enzyme and its application to bioproduction

2013 Fiscal Year Final Research Report

• Project/Area Number: 22360350
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: University of Hyogo
• Principal Investigator: NEGORO Seiji 兵庫県立大学, 工学(系)研究科(研究院), 教授 (90156159)
• Co-Investigator(Kenkyū-buntansha): TAKEO Masahiro 兵庫県立大学, 大学院・工学研究科, 准教授 (40236443) ; KATO Dai-ichiro 兵庫県立大学, 大学院・工学研究科, 助教 (60423901)
• Co-Investigator(Renkei-kenkyūsha): HIGUCHI Yoshiki 兵庫県立大学, 大学院・生命理学研究科, 教授 (90183574) ; SHIBATA Naoki 兵庫県立大学, 大学院・生命理学研究科, 准教授 (30295753) ; GOTO Yuji 大阪大学, 蛋白質研究所, 教授 (40153770) ; IKEGAMI Takahisa 大阪大学, 蛋白質研究所, 准教授 (20283939) ; SHIGETA Yasuteru 筑波大学, 数理物質科学研究科, 教授 (80376483) ; KAMIYA Katsumasa 神奈川工科大学, 准教授 (60436243)
• Project Period (FY): 2010-04-01 – 2014-03-31
• Keywords: ナイロン / 結晶構造解析 / 耐熱性 / 生分解 / 加水分解

Research Abstract

Nylon hydrolase is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of nylon-6. X-ray crystallographic analysis revealed that the enzyme constitute four identical heterodimers, which resulted from the autoprocessing of the precursor protein (36 kDa). The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC enhanced the protein thermostability by 36 oC.

Research Products

• [Journal Article] Enzymatic hydrolysis of nylons : quantification of the reaction rate of nylon hydrolase for thin-layered nylons (2014)
  • Author(s): Nagai, K. Iida, K. Shimizu, K. Kinugasa, R. Izumi, M. Kato, D. Takeo, M. Mochiji, K. & Negoro S.
  • Journal Title: Appl. Microbiol. Biotechnol Volume: (in press)
  • Peer Reviewed
• [Journal Article] Nylonoligomer hydrolase promoting cleavage reactions in unnatural amide compounds (2014)
  • Author(s): Kamiya, K., Baba, T., Boero, M., Matsui, T., Negoro, S., & Shigeta. Y.
  • Journal Title: J. Phys. Chem. Lett. Volume: 5 Pages: 1210-1216
  • DOI: 10.1021/jz500323y
  • Peer Reviewed
• [Journal Article] 根来誠司「高分子ナイロンを加水分解する酵素(NylC)の発見」(総説) (2014)
  • Author(s): 加藤太一郎, 武尾正弘
  • Journal Title: 生物工学会誌 Volume: 92巻特集(掲載決定)
  • Peer Reviewed
• [Journal Article] Crystallization and X-ray diffraction analysis of nylon hydrolase (NylC) from Arthrobacter sp (2013)
  • Author(s): Nagai, K., Yasuhira, K., Tanaka, Y., Kato, D., Takeo, M., Higuchi, Y., Negoro, S., & Shibata N.
  • Journal Title: KI72 Acta Cryst. Volume: F69 Pages: 1151-1154
  • DOI: 10.1107/S1744309113024263
  • Peer Reviewed
• [Journal Article] 樋口芳樹「立体構造を基盤としたナイロン加水分解酵素の開発と耐熱化機構」 (2013)
  • Author(s): 根来誠司, 永井圭介, 大島 祥平, 飯田一希, 加藤太一郎, 武尾正弘,持地広造, 柴田直樹
  • Journal Title: (総説)酵素工学ニュース Volume: 69号 Pages: 12-18
  • Peer Reviewed
• [Journal Article] Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis (2012)
  • Author(s): Negoro, S., Shibata, N., Tanaka, Y.,Yasuhira, K., Shibata, H., Hashimoto, H., Lee, Y-H., Oshima, S., Santa,R., Oshima,S., Mochiji, K., Goto,Y., Ikegami,T., Nagai,K., Kato, D., Takeo, M., & Higuchi, Y.
  • Journal Title: J. Biol. Chem. Volume: 287 Pages: 5079-5090
  • DOI: 10.1074/jbc.M111.321992
  • Peer Reviewed
• [Journal Article] Crystallization and X-ray diffraction analysis of nylon oligomer hydrolase (NylC) from Agromyces sp (2011)
  • Author(s): Yasuhira, K., Shibata, N., Tanaka, Y., Kumagai, N., Tanaka, Y., Nagai, K., Kato, D., Takeo, M., Negoro, S., Higuchi, Y
  • Journal Title: KY5R.Acta Cryst Volume: F67 Pages: 892-895
  • DOI: 10.1107/S1744309111022858
  • Peer Reviewed
• [Journal Article] Molecular dynamics studies on the mutational structures of a nylon-6 byproductdegrading enzyme (2011)
  • Author(s): Baba, T., Kamiya K., Matsui, T., Shibata, N., Higuchi, Y., Kobayashi, T., Negoro, S., & Shigeta Y
  • Journal Title: Chem. Phys. Lett Volume: 507 Pages: 157-161
  • DOI: 10.1016/j.cplett.2011.03.046
  • Peer Reviewed
• [Presentation] 高解像度X 線結晶構造解析を基盤としたナイロン加水分解酵素(NylC)の耐熱化機構の解明 (2013)
  • Author(s): 衣笠 凌, 永井圭介, 三田隆二, 篠田昌宜, 柴田 直樹, 樋口芳樹, 李 映昊, 後藤祐 児, 加藤太一郎, 武尾正弘, 根来誠司
  • Organizer: 第65回日本生物工学会大会
  • Place of Presentation: 広島国際会議場
  • Year and Date: 2013-09-19
• [Presentation] 分子動力学シミュレーションによるナイロン加水分解酵素(NylC)の自己分断機構の解析 (2013)
  • Author(s): 永井圭介, 大島祥平, 柴田直樹, 樋口芳樹, 馬場 剛史, 重田育照, 加藤太一郎, 武尾 正弘, 根来誠司
  • Organizer: 第65回日本生物工学会大会
  • Place of Presentation: 広島国際会議場
  • Year and Date: 2013-09-19
• [Presentation] 「ナイロン加水分解酵素の分子設計と生分解性ポリアミド開発への展望」(シンポジウム) (2013)
  • Author(s): 根来誠司
  • Organizer: 2013年度日本農芸化学会大会
  • Place of Presentation: 東北大学・川内キャンパス
  • Year and Date: 2013-03-28
  • Invited
• [Remarks]
  • URL: http://www.eng.u-hyogo.ac.jp/msc/msc3/