Structural analysis of nylon oligomer hydrolase and application to enzymatic synthesis of unnatural amides

• Project/Area Number: 18360400
• 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 University of Hyogo, 大学院・工学研究科, 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) ; 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) ; NAKANO Hidehiko 兵庫県立大学, 大学院・工学研究科, 教授 (20047608)
• Project Period (FY): 2006 – 2009
• Project Status: Completed (Fiscal Year 2009)
• Budget Amount: ¥18,180,000 (Direct Cost: ¥15,300,000、Indirect Cost: ¥2,880,000); Fiscal Year 2009: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2008: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2007: ¥6,760,000 (Direct Cost: ¥5,200,000、Indirect Cost: ¥1,560,000); Fiscal Year 2006: ¥5,700,000 (Direct Cost: ¥5,700,000)
• Keywords: ナイロンオリゴマー / X線結晶構造解析 / 酵素進化 / βラクタマーゼ / カルボン酸エステル分解酵素 / Arthrobacter / 6-アミノカプロン酸 / 6-Aminohexanoate oligomer / ナイロンオリゴマー分解酵素 / アミド合成 / 耐熱性酵素 / N-terminal nucleophile hydrolase / maloamidase / 6-Aminohexanoate-cyclic dimer / Agromyces / Kokuria / 6-aminohexanoate-oligomer hydrolase / Ntn amidohydrolase

Research Abstract

Arthrobacter sp.strain KI72 produces NylB' carboxylesterase, which is 88% homologous to functional nylon oligomer hydrolase (NylB). Both NylB and NylB' utilize Ser112-Lys115-Tyr215 residues as catalytic triads. NylB' possesses approximately 0.5% of the level of the 6-aminohexanoate linear dimer (Ald)-hydrolytic activity of NylB. Three substitutions (G181D, H266N and D370Y) in NylB'-type carboxylesterase were sufficient to increase the level of the Ald-hydrolytic activity to that of the wild-type NylB enzyme. The triple mutant efficiently catalyzed the amide-synthetic reaction in organic solvent containing low concentration of water by reverse reaction of hydrolysis. We analyzed the structural requirements of the enzymes for effective amide-synthetic reaction.

Research Products

• [Journal Article] Enzymatic synthesis of nylon-6 units in organic solvents containing low concentrations of water. (2010)
  • Author(s): Kawashima, Y., Yasuhira, K., Shibata, N., Matsuura, Y., Tanaka, Y., Taniguchi, M., Miyoshi, Y., Takeo, M., Kato, D., Higuchi, Y., Negoro, S.
  • Journal Title: J. Mol. Cat. B: Enzymatic 64 Pages: 81-88
  • Peer Reviewed
• [Journal Article] X-ray crystallographic analysis of the 6-aminohexanoate cyclic dimer hydrolase: catalytic mechanism and evolution of an enzyme responsible for nylon-6 byproduct degradation. (2010)
  • Author(s): Yasuhira, K. Shibata, N., Mongami, G., Uedo, Y., Atsumi, Y., Kawashima, Y., Hibino, A, Tanaka, Y., Lee, Y-Ho, Kato, D., Takeo, M., Higuchi, Y., Negoro, S.
  • Journal Title: J. Biol. Chem. 285 Pages: 1239-1248
  • Peer Reviewed
• [Journal Article] Structural basis of the evolution of nylon-oligomer hydrolase NylB. (2010)
  • Author(s): Higuchi, Y., Shibata, N., Kawashima, Y., Ohki, T, Negoro, S.
  • Journal Title: Viva Origino. (in press)
  • NAID: 110007651043
  • Peer Reviewed
• [Journal Article] Enzymatic synthesis of nylon-6 units in organic solvents containing low concentrations of water (2010)
  • Author(s): Kawashima, Y., Yasuhira, K., Shibata, N., Matsuura Y., Tanaka, Y Taniguchi, M, Miyoshi, Y, Takeo, M Kato, D, Higuchi, Y, Negoro, S.
  • Journal Title: Journal of Molecular Catalysis B : Enzymatic 64 Pages: 81-88
  • Peer Reviewed
• [Journal Article] Structural basis of the evolution of nylon-oligomer hydrolase, Ny1B (2010)
  • Author(s): Higuchi, Y, Shibata, N, Kawashima, Y, Ohki, T, Negoro, S.
  • Journal Title: Viva Origino (印刷中)
  • Peer Reviewed
• [Journal Article] X-ray crystallographic analysis of the 6-aminohexanoate cyclic dimer hydrolase : catalytic mechanism and evolution of an enzyme responsible for nylon-6 byproduct degradation (2010)
  • Author(s): Yasuhira, K, Shibata, N, Mongami, G Uedo, Y, Atsumi, Y, Kawashima, Y, Hibino, A, Tanaka, Y, Lee, Y-Ho, Kato, D, Takeo, M, Higuchi, Y, Negoro, S
  • Journal Title: Journal of Biological Chemistry 285 Pages: 1239-1248
  • Peer Reviewed
• [Journal Article] Molecular design of a nylon-6 byproduct-degrading enzyme from a carboxylesterase with a βlactamase fold. (2009)
  • Author(s): Kawashima Y. Ohki T. Shibata N. Higuchi Y, Takeo M, Kato D, Negoro S.
  • Journal Title: FEBS J. 276 Pages: 2547-2556
  • Peer Reviewed
• [Journal Article] Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a β-lactamase fold. (2009)
  • Author(s): Ohki T, Shibata N, Higuchi Y, Kawashima Y, Takeo M, Kato D, Negoro S
  • Journal Title: Protein Science 18 Pages: 1662-1673
  • Peer Reviewed
• [Journal Article] Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with b-lactamase fold : x-ray crystallo-graphic analysis of directly evolved 6-aminohexanoate-dimer hydrolase (2009)
  • Author(s): Ohki, T, Shibata, N, Higuchi, Y.Kawashima, Y, Takeo, M, Kato, D.Negoro, S.
  • Journal Title: Protein Science 18 Pages: 1662-1673
  • Peer Reviewed
• [Journal Article] Molecular design of a nylon-6 byproduct-degrading enzyme from a carboxylesterase with a β-lactamase fold (2009)
  • Author(s): Kawashima, Y.Ohki, T.Shibata, N Higuchi, Y.Wakitani, Y.Matsuura, Y.Nakata, Y, Takeo, M, Kato, D, Negoro, S.
  • Journal Title: FEBS Journal 276 Pages: 2547-2556
  • Peer Reviewed
• [Journal Article] Molecular design of a nylon-6 byproduct-degrading enzyme from a carboxylesterase with a βlactamase fold (2009)
  • Author(s): Kawashima Y. Ohki T. Shibata N. Higuchi Y, Takeo M, Kato D, Negoro S.
  • Journal Title: FEBS J. 276 Pages: 2547-2556
• [Journal Article] Two alternative modes for optimizing nylon-6 byproduct hydrolytic activity from a carboxylesterase with a β-lactamase fold (2009)
  • Author(s): Ohki T, Shibata N, Higuchi Y, Kawashima Y, Takeo M, Kato D, Negoro S
  • Journal Title: Protein Science (in press)
• [Journal Article] Molecular design of a nylon-6 byproduct-degrading enzyme from acarboxylesterase with a β-lactamase fold. (2009)
  • Author(s): Kawashima, Y. Shibata, N., Takeo, M., Kato, D. Negoro, S.
  • Journal Title: FEBS Journal 276 Pages: 2547-2556
  • Peer Reviewed
• [Journal Article] Mechanism of 4-nitrophenol oxidation in Rhodococcus sp. Strain PN1 : characterization of the two-component 4-nitrophenol hydroxylase and regulation of its expression (2008)
  • Author(s): Takeo, M., Murakami, M., Niihara, S., Yamamoto, K. Kato, D., Negoro, S.
  • Journal Title: Journal of Bacteriology 190 Pages: 7367-7374
  • Peer Reviewed
• [Journal Article] Nylon-oligomer degrading enzyme/substrate complex. (2007)
  • Author(s): Negoro S, Shibata N, Kato D, Takeo M, Higuchi Y
  • Journal Title: J. Mol. Biol. 370 Pages: 142-156
  • Peer Reviewed
• [Journal Article] 6-Amino-hexanoate oligomer hydrolases from the alkalophilic bacteria Agromyces sp. strain KY5R and Kocuria sp. strain KY2. (2007)
  • Author(s): Yasuhira K, Tanaka Y, Shibata H, Kato D, Takeo M, Negoro S
  • Journal Title: Appl. Environ. Microbiol. 73 Pages: 7099-7102
  • Peer Reviewed
• [Journal Article] Genetic organization of nylon-oligomer-degrading enzymes from an alkalophilic bacterium Agromyces sp. KY5R. (2007)
  • Author(s): Yasuhira K, Uedo Y, Takeo M, Kato D, Negoro S
  • Journal Title: J. Biosci. Bioeng. 104 Pages: 521-524
  • NAID: 110006533091
• [Journal Article] Nylon-oligomer degrading enzyme/substrate complex (2007)
  • Author(s): Negoro S, Shibata N, Kato D, Takeo M, Higuchi Y
  • Journal Title: J. Mol. Biol. 370 Pages: 142-156
• [Journal Article] 6-Amino- hexanoate oligomer hydrolases from the alkalophilic bacteria Agromyces sp. strain KY5R and Kocuria sp. strain KY2 (2007)
  • Author(s): Yasuhira K, Tanaka Y, Shibata H, Kato D, Takeo M, Negoro S
  • Journal Title: Appl. Environ. Microbiol. 73 Pages: 7099-7102
• [Journal Article] Genetic organization of nylon-oligomer-degrading enzymes from an alkalophilic bacterium Agromyces sp. KY5R (2007)
  • Author(s): Yasuhira K, Uedo Y, Takeo M, Kato D, Negoro S
  • Journal Title: J. Biosci. Bioeng. 104 Pages: 521-524
  • NAID: 110006533091
• [Journal Article] Nylon-oligomen degrading enzyme/substrate complex:catalytic mechanism of 6-aminohexanoate-dimer hydrolase (2007)
  • Author(s): S.Negoro, T.Ohki, N.Shibata, H.Hayashi, H.Nakano, K.Yasuhira, D.Kato, M.Takeo & Y.Higuchi
  • Journal Title: Journal of Molecular Biology 370 Pages: 142-156
  • Peer Reviewed
• [Journal Article] 6-Aminohexanoate-oligomer hydrolase from the alkalophilic bacteria Agromyces sp. KY5R and Kocuria sp.KY2 (2007)
  • Author(s): K.Yasuhira, Y.Tanaka, H.Shibata, D.Kato, M.Takeo, and S.Negoro.
  • Journal Title: Applied and Environmental Microbiology 73 Pages: 7099-7102
  • Peer Reviewed
• [Journal Article] Purification and characterization of alkylcatechol 2,3-dioxygenase from butylphenol degradation pathway of Pseudomonas putida MT4. (2007)
  • Author(s): T.Takeo, M.Nishimura, H.Takahashi, C.Kitamura, D.Kato, and S.Negoro.
  • Journal Title: J.Biosci.Bioeng. 104 Pages: 309-314
  • NAID: 110006437013
  • Peer Reviewed
• [Journal Article] Enzyme-linked immunosorbent assay of nonylphenol using antibody-bound microfluid filters in certical fluidic operation. (2007)
  • Author(s): K.Matsui, I.Kawaji, Y.Utsumi, Y.Ukita, M.Takeo, D.Kato, and S.Negoro.
  • Journal Title: J.Biosci.Bioeng. 104 Pages: 347-350
  • Peer Reviewed
• [Journal Article] 多機能フィルタを用いた垂直流体操作の挙動予測と検証 (2007)
  • Author(s): 藤原 邦代、浮田 芳昭、浅野 豪文、松井 勝弘、武尾 正弘、根来 誠司、内海 裕一
  • Journal Title: 電気学会論文誌(C) 127 Pages: 210-216
  • NAID: 10017641280
  • Peer Reviewed
• [Journal Article] 積層型マイクロリアクターを用いた酵素免疫測定法による生体物質測定系の開発 (2007)
  • Author(s): 松井 勝弘、森本 祥平、浅野 豪文、加藤 太一郎、武尾 正弘、内海 裕一、根来 誠司
  • Journal Title: 電気学会論文誌(C) 127 Pages: 204-209
  • NAID: 10018480527
  • Peer Reviewed
• [Journal Article] New application of firefly luciferase:it can catalyze the enantioselective thioester formation of 2-arylpropanoic acid (2007)
  • Author(s): D.Kato, K.,Teruya H. Yoshida M.Takeo, S.Negoro, and H.Ohta.
  • Journal Title: FEBS Journal 274 Pages: 3877-3885
  • Peer Reviewed
• [Journal Article] Purification and characterization of catechol 2,3-dioxygenase from the aniline degradation pathwary from Acinetobacter sp.YAA and its mutant enzyme,which resist substrate inhibition. (2007)
  • Author(s): T.Takeo, M.Nishimura, M.Shirai, H.Takahashi, and S.Negoro.
  • Journal Title: Bioscience Biotechnology Biochemistry 71 Pages: 1668-1675
  • Peer Reviewed
• [Journal Article] Immunoassay usingmicrfluid filters constructed by deep X-ray lithography. (2007)
  • Author(s): K.Matsui, Y.Ustumi, Y.Ukita, M.Takeo, D.Kato, and S.Negoro
  • Journal Title: Bioscience Biotechnology Biochemistry 71 Pages: 3098-3101
  • Peer Reviewed
• [Journal Article] Mutational analysis of 6-amino-hexanoate-dimer hydrolase. (2006)
  • Author(s): Ohki T, Wakitani Y, Takeo M, Yasuhira K, Shibata N, Higuchi Y, Negoro S
  • Journal Title: FEBS Lett 580 Pages: 5054-5058
  • Peer Reviewed
• [Journal Article] Crystallization and X-ray diffraction analysis of 6-aminohexanoate-cyclic dimer hydro-lase from Arthrobacter sp. KI72. (2006)
  • Author(s): Yasuhira K, Shibata N, Negoro S, Takeo M, Higuchi Y.
  • Journal Title: Acta Cryst. F62 Pages: 1209-1211
  • Peer Reviewed
• [Journal Article] Mutational analysis of 6-amino- hexanoate-dimer hydrolase (2006)
  • Author(s): Ohki T, Wakitani Y, Takeo M, Yasuhira K, Shibata N, Higuchi Y, Negoro S
  • Journal Title: FEBS Lett 580 Pages: 5054-5058
• [Journal Article] Crystallization and X-ray diffraction analysis of 6-aminohexanoate-cyclic dimer hydro- lase from Arthrobacter sp. KI72 (2006)
  • Author(s): Yasuhira K, Shibata N, Negoro S, Takeo M, Higuchi Y.
  • Journal Title: Acta Cryst. F62 Pages: 1209-1211
• [Journal Article] Mutational analysis of 6-aminohexanoate-dimer hydrolase : relationship between nylon oligomer hydrolytic and esterolytic activities (2006)
  • Author(s): T.Ohki, Y.Wakitani, M.Takeo, K.Yasuhira, N.Shibata, Y.Higuchi, S.Negoro.
  • Journal Title: FEBS Letters 580 Pages: 5054-5058
• [Journal Article] Characterization of the alkylphenol degradation gene cluster in Pseudomonas putida MT4 and evidence of oxidation of alkylphebnols and alkylcatechols with a medium-length alkyl chain. (2006)
  • Author(s): M.Takeo, S.K.Prabu, C.Kitamura, M.Hirai, H.Takahashi, D.Kato, S.Negoro
  • Journal Title: J.Biochem.Bioeng. 102(4) Pages: 352-361
• [Journal Article] アルカリ性ナイロンオリゴマー分解菌ノ分離 : 6-アミノカプロン酸オリゴマー加水分解酵素遺伝子のクローニングと配列分析 (2006)
  • Author(s): 安平健吾, 武尾正弘, 根来誠司
  • Journal Title: 日本水処理生物学会誌 42(4) Pages: 199-205
• [Journal Article] 積層型マイクロ流体デバイスの製作と内分泌攪乱物質分析への応用 (2006)
  • Author(s): 松井勝弘, 大村貴士, 森本祥平, 浅野豪文, 浮田芳昭, 内海裕一, 武尾正弘, 根来誠司
  • Journal Title: 水処理技術 47 Pages: 151-156
• [Journal Article] Crystallization and X-ray diffraction analysis of 6-aminohezanoate-cyclic dimer hydrolase from Arthrobacter sp.K172 (2006)
  • Author(s): K.Yasuhira, Y.Uedo, N.Shibata, S.Negoro, M.Takeo, Y.Higuchi.
  • Journal Title: Acta Crystallographica F62 Pages: 1209-1211
• [Presentation] エコ・ナノ・バイオ境界領域の開拓を目指して (2009)
  • Author(s): 根来誠司
  • Organizer: 日本材料学会 UFA研究分科会 第10回講演会
  • Place of Presentation: 大阪大学
  • Year and Date: 2009-12-08
• [Presentation] 加水分解酵素の逆反応によるアミド合成:高効率触媒の形成要件 (2009)
  • Author(s): 河島康之, 松浦悠介, 柴田直樹, 田中優佑, 小林達也, 武尾正弘, 加藤太一郎, 樋口芳樹, 根来誠司
  • Organizer: 日本生物工学会 第61回大会
  • Place of Presentation: 名古屋大学
  • Year and Date: 2009-09-24
• [Presentation] N-terminal nucleophile(Ntn)hydrolase superfamily(Ny1C)の耐熱化機構 (2009)
  • Author(s): 柴田浩, 田中優佑, 橋本悠, 松浦悠介, 柴田直樹, 李映昊, 櫻井一正, 加藤太一郎, 武尾正弘, 樋口芳樹, 後藤祐児, 根来誠司
  • Organizer: 日本生物工学会 第61回大会
  • Place of Presentation: 名古屋大学
  • Year and Date: 2009-09-24
• [Presentation] 6-アミノヘキサン酸環状2量体加水分解酵素(Ny1A)の触媒機構 (2009)
  • Author(s): 安平健吾, 柴田直樹, 門上剛, 厚海悠, 日比野敦, 吉野拓真, 河島康之, 加藤太一郎, 武尾正弘, 樋口芳樹, 根来誠司
  • Organizer: 日本生物工学会 第61回大会
  • Place of Presentation: 名古屋大学
  • Year and Date: 2009-09-24
• [Presentation] ナイロンオリゴマー分解酵素(Ny1C)の安定性と触媒機能に及ぼすアミノ酸置換効果 (2009)
  • Author(s): 柴田浩, 熊谷尚也, 松浦悠介, 田中優佑, 橋本悠, 加藤太一郎, 柴田直樹, 武尾正弘, 樋口芳樹, 根来誠司
  • Organizer: 日本生物工学会 第61回大会
  • Place of Presentation: 名古屋大学
  • Year and Date: 2009-09-24
• [Presentation] ナイロンオリゴマー分解酵素の実験室内進化 : 触媒中心形成の構造基盤 (2008)
  • Author(s): 大木卓, 柴田直樹, 武尾正弘, 加藤太一郎, 樋口芳樹, 根来誠司
  • Organizer: 第60回日本生物工学会大会
  • Place of Presentation: 東北学院大学
• [Presentation] ナイロンオリゴマー分解酵素によるアミド合成・分解機能とアミノ酸置換効果 (2008)
  • Author(s): 河島康之, 松浦悠介, 柴田直樹, 武尾正弘, 加藤太一郎, 樋口芳樹, 根来誠司
  • Organizer: 第60回日本生物工学会大会
  • Place of Presentation: 東北学院大学
• [Presentation] Rhodococcus sp. PN1 株の4-ニトロフェノール分解における二つの酸化酵素系の役割 (2008)
  • Author(s): 山本健太, 貞弘建, 加藤太一郎, 武尾正弘, 根来誠司
  • Organizer: 第60回日本生物工学会大会
  • Place of Presentation: 東北学院大学
• [Presentation] ホタルルシフェラーゼの発光活性と2-アリールプロパン酸に対する立体選択的なチオェステル化活性の関係 (2008)
  • Author(s): 加藤太一郎, 横山敬佑, 吉田裕充, 番匠亜沙美, 武尾正弘, 根来誠司
  • Organizer: 第60回日本生物工学会大会
  • Place of Presentation: 東北学院大学
• [Presentation] 非常に簡単なアミノアシル-tRNAの作り方 (2008)
  • Author(s): 加藤太一郎, 大迫淳一, 松本剛, 武尾正弘, 根来誠司
  • Organizer: 第12回生体触媒化学シンポジウム
  • Place of Presentation: 東邦大学
• [Presentation] 6-アミノカプロン酸環状2量体加水分解酵素・基質複合体のX線結晶構造解析 (2007)
  • Author(s): 安平 健吾、柴田 直樹、門上 剛、樋口 芳樹、加藤 太一郎、武尾 正弘、根来 誠司
  • Organizer: 日本生物工学会
  • Place of Presentation: 広島大学
  • Year and Date: 2007-09-25
• [Presentation] エンド型6-アミノカプロン酸オリゴマー加水分解酵素(E3)のX線結晶構造解析 (2007)
  • Author(s): 安平 健吾, 柴田 直樹, 柴田 浩, 田中 康仁, 樋口 芳樹, 加藤 太一郎, 武尾 正弘, 根来 誠司
  • Organizer: 日本生物工学会
  • Place of Presentation: 広島大学
  • Year and Date: 2007-09-25
• [Remarks] ホームページ
  • URL: http://www.eng.u-hyogo.ac.jp/msc/msc3/
• [Remarks]
  • URL: http://www.eng.u-hyogo.ac.jp/msc/msc3/top.html
• [Patent(Industrial Property Rights)] 遊離カルボン酸・アミンの直接縮合反応を触媒する酵素の設計方法及びナイロンオリゴマーの酵素的製造方法 (2006)
  • Inventor(s): 根来誠司、武尾正弘、加藤太一郎、樋口芳樹、柴田直樹
  • Industrial Property Number: 2006-239119
  • Filing Date: 2006-09-04
  • Acquisition Date: 2006-09-04
• [Patent(Industrial Property Rights)] 遊離カルボン酸・アミンの直接縮合反応を触媒する酵素の設計方法及びナイロンオリゴマーの酵素的製造方法 (2006)
  • Inventor(s): 根来誠司, 武尾正弘, 加藤太一郎, 樋口芳樹, 柴田直樹
  • Industrial Property Rights Holder: 兵庫県
  • Industrial Property Number: 2006-239119
  • Filing Date: 2006-09-04
• [Patent(Industrial Property Rights)] 遊離カルボン酸・アミンの直接縮合反 応を触媒する酵素の設計方法及びナイロンオリゴマーの酵素的製造方法
  • Inventor(s): 根来誠司, 武尾正弘, 加藤太一郎, 樋口芳樹, 柴田直樹
  • Industrial Property Number: 2006-239119