Development of efficient method for producing optically pure alcohols with an asymmetric hydrogen-transfer biocatalysts and their library construction

• Project/Area Number: 17360402
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biofunction/Bioprocess
• Research Institution: Toyama Prefectural University
• Principal Investigator: ITOH Nobuya Toyama Prefectural University, 工学部, 教授 (90213066)
• Co-Investigator(Kenkyū-buntansha): 牧野 祥嗣 富山県立大学, 工学部, 講師 (20347355)
• Project Period (FY): 2005 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥10,440,000 (Direct Cost: ¥9,300,000、Indirect Cost: ¥1,140,000); Fiscal Year 2008: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000); Fiscal Year 2007: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000); Fiscal Year 2006: ¥2,300,000 (Direct Cost: ¥2,300,000); Fiscal Year 2005: ¥3,200,000 (Direct Cost: ¥3,200,000)
• Keywords: バイオ不斉還元 / 光学活性アルコール生産 / バイオリアクター / 進化分子工学 / 有機溶媒耐性酵素 / メタゲノム / 遺伝子ライブラリー / 進化分化工学

Research Abstract

フェニルアセトアルデヒド還元酵素(PAR)を進化分子工学的手法にて改良し、変異酵素Sar268/HAR1を取得した。これら酵素の機能解析を行った結果、明らかに極性溶媒中での活性が向上していた。また、同酵素を触媒とし、高濃度のケトン類(10-30%)から100%近い収率で目的の光学活性体を合成できた。さらに、Sar268およびLeifsonia由来アルコール脱水素酵素(LSADH)の発現菌体を固定化し、水溶性の高いケトンの還元反応を流動層型のリアクターで行う事に成功した。PAR/LSADHの類縁酵素遺伝子の土壌DNAからの分離を行い、PARとLSADHの基質特異性が異なる酵素ライブラリーの構築を目指した。PARについては、約60種のDNA断片の増幅に成功したが、活性を確認することができなかった。他方LSADHでは、生産菌の類縁微生物ゲノム情報を基に約30種の遺伝子を単離(LSと命名)し、その基質特異性、立体選択性を測定した。

Research Products

• [Journal Article] 有機溶媒耐性酵素の開発と医薬品中間体製造 (2009)
  • Author(s): 伊藤 伸哉
  • Journal Title: バイオインダストリ 4 Pages: 34-44
• [Journal Article] Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity (2008)
  • Author(s): H. Asako, M. Shimizu, N. Itoh
  • Journal Title: Appl. Microbiol. Biotechnol. 80 Pages: 803-812
  • Peer Reviewed
• [Journal Article] Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction (2008)
  • Author(s): N. Itoh, M. Nakamura, K. Inoue, Y. Makino
  • Journal Title: Appl. Microbiol.Biotechnol. 75 Pages: 1249-812
  • Peer Reviewed
• [Journal Article] Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity. (2008)
  • Author(s): H. Asako, M. Shimizu, N. Itoh
  • Journal Title: Appl. Microbiol. Biotechnol. 80 Pages: 803-812
  • Peer Reviewed
• [Journal Article] 水素移動型バイオ不斉還元による光学活性アルコールの効率的生産 (2008)
  • Author(s): 伊藤 伸哉
  • Journal Title: ファインケミカル 37 Pages: 44-48
• [Journal Article] 酵素触媒の極性有機溶媒耐性と不斉還元バイオプロセスへの応用 (2007)
  • Author(s): 伊藤伸哉, 牧野祥嗣
  • Journal Title: 酵素工学ニュース 58 Pages: 27-34
• [Journal Article] Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol (2007)
  • Author(s): Y. Makino, T. Dairi, N. Itoh
  • Journal Title: Appl. Microbiol.Biotechnol. 77 Pages: 833-843
  • Peer Reviewed
• [Journal Article] Continuous production of chiral 1,3-butanediol using immobilized bioca talysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction (2007)
  • Author(s): Nobuya Itoh, Masatoshi Nakamura, Kousuke Inoue, Yoshihide Makino
  • Journal Title: Applied Microbiology and Biotechnology 75 Pages: 1249-1256
  • Peer Reviewed
• [Journal Article] (2007)
  • Author(s): 伊藤 伸哉、牧野 祥嗣
  • Journal Title: 酵素工学ニュース 58 Pages: 27-34
• [Journal Article] Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor : promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction (2007)
  • Author(s): Nobuya Itoh, Masatoshi Nakamura, Kousuke Inoue, Yoshihide Makino
  • Journal Title: Applied Microbiology and Biotechnology Online first(未定)
• [Journal Article] Gene Cloning and expression of Leifsonia alcohol dehydrogenase (LSADH) involved in asymmetric hydrogen-transfer bioreduction to produce (R)-form chiral alcohols. (2006)
  • Author(s): K.Inoue, Y. Makino, T. Dairi, N. Itoh
  • Journal Title: Biosci. Biotechnol. Biochem. 70 Pages: 418-426
  • Peer Reviewed
• [Journal Article] Gene Cloning and Expression of Leifsonia Alcohol Dehydrogenase (LSADH) Involved in Asymmetric Hydrogen- Transfer Bioreduction to Produce (R)-Form Chiral Alcohols (2006)
  • Author(s): Kousuke Inoue, Yoshihide Makino, Tohru Dairi, Nobuya Itoh
  • Journal Title: Biosci.Biotechnol.Biochem. 70 Pages: 418-426
• [Journal Article] Purification and cDNA cloning of NADPH-dependent aldoketoreductase, involved in asymmetric reduction of methyl 4-bromo-3-oxobutyrate, from Penicillium citrinum IFO463 (2005)
  • Author(s): H. Asako, R. Wakita, K. Matsumura, M. Shimizu, J. Sakai, N. Itoh
  • Journal Title: Appl.Environ. Microbiol. 71 Pages: 1101-1104
  • Peer Reviewed
• [Journal Article] Production of (R)-chiral alcohols by a hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol dehydrogenase (LSADH) (2005)
  • Author(s): K. Inoue, Y. Makino, N. Itoh
  • Journal Title: Tetrahedron:Asymmetry 16 Pages: 2539-2549
  • Peer Reviewed
• [Journal Article] Purification and characterization of a novel alcohol dehydrogenase from Leifsonia sp. strain S749: a promising biocatalyst for an asymmetric hydrogen transferbioreduction (2005)
  • Author(s): K. Inoue, Y. Makino, N. Itoh
  • Journal Title: Environ. Microbiol. 71 Pages: 3633-3641
  • Peer Reviewed
• [Journal Article] Engineering of phenylacetaldehyde reductase for efficient substrateconversion in concentrated 2-propanol (2005)
  • Author(s): Y. Makino, K. Inoue, T. Dairi, N. Itoh
  • Journal Title: Appl. Environ. Microbiol. 71 Pages: 4713-4720
  • Peer Reviewed
• [Journal Article] Purification and Characterization of a Novel Alcohol Dehydrogenase (LSADH) from Leifsonia sp. S749 : a Promising Biocatalyst for an Asymmetric Hydrogen Transfer Bioreduction (2005)
  • Author(s): Kousuke Inoue, Yoshihide Makino, Nobuya Itoh
  • Journal Title: Appl.Environ.Microbiol. 71 Pages: 3633-3641
• [Journal Article] Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol (2005)
  • Author(s): Yoshihide Makino, Kosuke Inoue, Tohru Dairi, Nobuya Itoh
  • Journal Title: Appl.Environ.Microbiol. 71 Pages: 4713-4720
• [Journal Article] Production of (R)-chiral alcohols by a hydrogen-transfer bioreduction with NADH-dependent Leifsonia alcohol dehydrogenase (LSADH) (2005)
  • Author(s): Kousuke Inoue, Yoshihide Makino, Nobuya Ltoh
  • Journal Title: Tetrahedron : Asymmetry 16 Pages: 2539-2549
• [Presentation] メタゲノムを用いた酸化還元酵素遺伝子ライブラリーの構築とその評価 (2009)
  • Author(s): 磯谷、石母田、加藤、牧野、朝子、伊藤
  • Organizer: 日本農芸化学会大会
  • Place of Presentation: 福岡
  • Year and Date: 2009-03-28
• [Presentation] メタゲノムを用いた酸化還元酵素遺伝子ライブラリーの構築とその評価 (2009)
  • Author(s): 磯谷, 石母田, 加藤, 牧野, 朝子, 伊藤
  • Organizer: 日本農芸化学会大会
  • Place of Presentation: 福岡国際会議場
  • Year and Date: 2009-03-28
• [Presentation] 進化分子工学による極性有機溶媒耐性酵素の創製とそのメカニズム (2008)
  • Author(s): 伊藤伸哉, 牧野祥嗣
  • Organizer: 第11回生体触媒化学シンポジウム
  • Place of Presentation: 鳥取
  • Year and Date: 2008-01-24
• [Presentation] Efficient method for producing optically pure alcohols with an asymmetrichydrogen-transfer biocatalysis and evolutionary mechanism of alcoholdehydrogenase (ADH) to increase the activity in polar organic solvent. (2007)
  • Author(s): N.Itoh, Y. Makino, T. Dairi, K. Inoue, M. Nakamura
  • Organizer: Biotrans 2007
  • Place of Presentation: Oviedo, Spain
  • Year and Date: 2007-07-10
• [Presentation] Efficient method for producing optically pure alcohols with an asymmetric hydrogen-transfer biocatalysis and evolutionary mechanism of alcohol dehydrogenase(ADH)to increase the activity in polar organic solvent (2007)
  • Author(s): N. Itoh, Y. Makino, T. Dairi, K. Inoue, M. Nakamura
  • Organizer: Biotrans 2007
  • Place of Presentation: Oviedo, Spain
  • Year and Date: 2007-07-10
• [Presentation] 不斉還元バイオ触媒を用いる高効率な光学活性アルコール生産法の開発 (2006)
  • Author(s): 伊藤伸哉
  • Organizer: グリーン・サステイナブルケミストリーシンポジウム
  • Place of Presentation: 東京
  • Year and Date: 2006-03-08
• [Presentation] Asymmetric hydrogen-transfer bioreduction by Leifsonia alcohol dehydrogenase (LSADH) to produce chiral alcohols. (2005)
  • Author(s): N.Itoh, Y. Makino, T. Dairi, K. Inoue
  • Organizer: Biotrans 2005
  • Place of Presentation: Delft, The Netherlands
  • Year and Date: 2005-07-07
• [Book] 有機溶媒耐性酵素の開発と医薬品中間体製造 (2009)
  • Author(s): 伊藤伸哉
  • Publisher: バイオインダストリー4
• [Book] 水素移動型バイオ不斉還元による光学活性アルコールの効率的生産 (2008)
  • Author(s): 伊藤伸哉
  • Publisher: ファインケミカル 37
• [Book] 有機溶媒耐性酵素, 酵素開発・利用の最新技術(今中忠行監修) (2006)
  • Author(s): 伊藤伸哉, 牧野祥嗣
  • Publisher: シーエムシー出版
• [Book] 有機溶媒耐性酵素(酵素開発・利用の最新技術 の第1編 第4章) (2006)
  • Author(s): 伊藤 伸哉, 牧野 祥嗣
  • Total Pages: 10
  • Publisher: シーエムシー出版
• [Remarks]
  • URL: http://www.pu-toyama.ac.jp/BR/itoh/
• [Remarks]
  • URL: http://www.pu-toyama.ac.jp/BR/itoh/
• [Remarks]
  • URL: http://www.pu-toyama.ac.jp/BR/itoh/
• [Patent(Industrial Property Rights)] 還元酵素及びその利用 (2008)
  • Inventor(s): 朝子弘之, 伊藤伸哉, 牧野祥嗣,大利徹
  • Industrial Property Rights Holder: 住友化学, 伊藤伸哉
  • Industrial Property Number: 2008-078602
  • Filing Date: 2008-03-25
• [Patent(Industrial Property Rights)] 還元酵素遺伝子の取得方法、及び、還元酵素遺伝子 (2008)
  • Inventor(s): 伊藤伸哉、牧野祥嗣, 朝子弘之
  • Industrial Property Rights Holder: 住友化学、伊藤伸哉
  • Industrial Property Number: 2008-291407
  • Filing Date: 2008-11-13
• [Patent(Industrial Property Rights)] 還元酵素遺伝子の取得方法、及び、還元酵素遺伝子 (2008)
  • Inventor(s): 伊藤, 牧野朝子
  • Industrial Property Rights Holder: 住友化学, 伊藤伸哉
  • Industrial Property Number: 2008-291407
  • Filing Date: 2008-11-13
• [Patent(Industrial Property Rights)] 還元酵素遺伝子を含む発現ベクター (2006)
  • Inventor(s): 伊藤伸哉, 牧野祥嗣, 大利徹
  • Industrial Property Rights Holder: 住友化学, 伊藤伸哉
  • Patent Publication Number: 2008-017773
  • Filing Date: 2006-07-13
• [Patent(Industrial Property Rights)] 還元酵素及びその利用 (2005)
  • Inventor(s): 伊藤伸哉, 牧野祥嗣, 大利徹
  • Industrial Property Rights Holder: 住友化学, 伊藤伸哉
  • Patent Publication Number: 2007-061065
  • Filing Date: 2005-09-02