Development of a technology to degrade, remove or process persistent proteins with enzymes

• Project/Area Number: 24380055
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Osaka University
• Principal Investigator: KANAYA SHIGENORI 大阪大学, 工学(系)研究科(研究院), 教授 (30273585)
• Co-Investigator(Renkei-kenkyūsha): KOGA Yuichi 大阪大学, 大学院工学研究科, 准教授 (30379119) ; CLEMENT Angkawidjaja 大阪大学, 大学院工学研究科, 特任助教 (20505987) ; KANAYA Eiko 大阪大学, 大学院工学研究科, 特任研究員 (80396217)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥17,940,000 (Direct Cost: ¥13,800,000、Indirect Cost: ¥4,140,000); Fiscal Year 2014: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2013: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2012: ¥8,060,000 (Direct Cost: ¥6,200,000、Indirect Cost: ¥1,860,000)
• Keywords: クチナーゼ / ポリエステル / サチライシン / プリオン / カルシウム結合部位 / メタゲノム / Ｘ線結晶構造解析 / セルラーゼ / 飽和変異 / セリンプロテアーゼ / 超好熱菌 / プリオン分解 / 速度論的安定性 / ジスルフィド結合 / 菌体外漏出 / 成熟化 / X線結晶構造解析

Outline of Final Research Achievements

The crystal structure of LC-cutinase with PET-degrading activity was determined. Then, the mutations were introduced into LC-cutinase and two proteases (Tk-subtilisin and Tk-SP) with abnormal prion-degrading activity based on their structures to improve their enzymatic properties. As a result, the LC-cutinase derivative with enhanced stability, the Tk-subtilisin derivative with increased maturation rate, and the Tk-SP derivative with high stability in the presence of EDTA were constructed. In addition, two thermostable cellulases were isolated from leaf-branch compost by a metagenomic approach and their crystal structures were determined.

Research Products

• [Journal Article] Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension. (2015)
  • Author(s): Okano, H., Hong, X.,, Kanaya, E., Angkawidjaja, C., and Kanaya, S.
  • Journal Title: Protein Sci. Volume: 24 Issue: 1 Pages: 93-1041
  • DOI: 10.1002/pro.2591
  • Peer Reviewed
• [Journal Article] Structure, activity and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain. (2015)
  • Author(s): Okano, H., Kanaya, E., Ozaki, M., Angkawidjaja, C., and Kanaya, S.
  • Journal Title: Protein Sci. Volume: 24 Issue: 3 Pages: 408-419
  • DOI: 10.1002/pro.2632
  • Peer Reviewed
• [Journal Article] Enzymatic activities of RNase H domains of HIV-1 reverse transcriptase with substrate binding domains of bacterial RNases H1 and H2. (2015)
  • Author(s): Permanasari, E.-D., Yasukawa, K., and Kanaya, S.
  • Journal Title: Mol. Biotechnol. Volume: - Issue: 6 Pages: 526-538
  • DOI: 10.1007/s12033-015-9846-5
• [Journal Article] Calcium-independent opening of lid1 of a family I.3 lipase by a single Asp to Arg mutation at the calcium-binding site. (2014)
  • Author(s): Cheng, M., Angkawidjaja, C., Koga, Y., and Kanaya, S
  • Journal Title: Protein Eng. Des. Sel. Volume: 27 Pages: 169-176
  • Peer Reviewed
• [Journal Article] Structural basis for salt-dependent folding of ribonuclease H1 from halophilic archaeon Halobacterium sp. NRC-1. (2014)
  • Author(s): You, D.-J., Jongruja, N., Tannous, E., Angkawidjaja, C., Koga, Y., and Kanaya, S.
  • Journal Title: J. Struct. Biol. Volume: 187 Issue: 2 Pages: 119-128
  • DOI: 10.1016/j.jsb.2014.06.005
  • Peer Reviewed
• [Journal Article] Divalent metal ion-induced folding mechanism of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1. (2014)
  • Author(s): Tannous, E., and Kanaya, S.
  • Journal Title: PLoS ONE Volume: 9 Issue: 9 Pages: e109016-e109016
  • DOI: 10.1371/journal.pone.0109016
  • Peer Reviewed / Open Access
• [Journal Article] Structure and stability of metagenome-derived glycoside hydrolase family 12 cellulase, LC-CelA, a homolog of Cel12A from Rhodothermus marinus. (2014)
  • Author(s): Okano, H., Ozaki, M., Kanaya, E., Kim, J.-J., Angkawidjaja, C., Koga, Y., and Kanaya, S.
  • Journal Title: FEBS Open Bio Volume: 4 Issue: 1 Pages: 936-946
  • DOI: 10.1016/j.fob.2014.10.013
  • Peer Reviewed / Open Access
• [Journal Article] Rational design of a glycosynthase by the crystal structure of β-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures. (2014)
  • Author(s): Henze, M., You, D.-J., Kamerke, C., Hoffmann, N., Angkawidjaja, C., Ernst, S., Pietruszka, J., Kanaya, S., and Elling, L.
  • Journal Title: J. Biotechnol. Volume: 191 Pages: 78-85
  • DOI: 10.1016/j.jbiotec.2014.07.003
  • Peer Reviewed
• [Journal Article] Structural and mechanistic insights into the kynurenine aminotransferase-mediated excretion of kynurenic acid. (2014)
  • Author(s): Okada, K., Angkawidjaja, C., Koga, Y., and Kanaya, S.
  • Journal Title: J. Struct. Biol. Volume: 185 Issue: 3 Pages: 257-266
  • DOI: 10.1016/j.jsb.2014.01.009
  • Peer Reviewed
• [Journal Article] Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1. (2014)
  • Author(s): Koga, Y., Sakudo, A., Aranishi, M., Tanaka, S., Hirata, A., Takano, K., Ikuta, K., and Kanaya, S.
  • Journal Title: Appl. Microbiol. Biotechnol. Volume: 98 Issue: 5 Pages: 2113-2120
  • DOI: 10.1007/s00253-013-5091-7
  • Peer Reviewed
• [Journal Article] Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase. (2014)
  • Author(s): Sulaiman, S., You, D.-J., Kanaya, E., Koga, Y., and Kanaya, S.
  • Journal Title: Biochemistry Volume: 53 Issue: 11 Pages: 1858-1869
  • DOI: 10.1021/bi401561p
  • Peer Reviewed
• [Journal Article] Investigating the structural dependence of protein stabilization by amino acid substitution. (2013)
  • Author(s): Tadokoro, T., Kazama, H., Koga, Y., Takano, K., and Kanaya, S.
  • Journal Title: Biochemistry Volume: 52 Issue: 16 Pages: 2839-2847
  • DOI: 10.1021/bi400076f
  • Peer Reviewed
• [Journal Article] Crystal structure of metagenome-derived LC9-RNase H1 with atypical DEDN active site motif. (2013)
  • Author(s): Nguyen, T.-N., You, D.-J., Kanaya, E., Koga, Y., and Kanaya, S.
  • Journal Title: FEBS Lett. Volume: 587 Pages: 1418-1423
  • Peer Reviewed
• [Journal Article] Crystal structure of metagenome-derived LC11-RNase H1 in complex with RNA/DNA hybrid. (2013)
  • Author(s): Nguyen, T.-N., You, D.-J., Matsumoto, H., Kanaya, E., Koga, Y., and Kanaya, S.
  • Journal Title: J. Struct. Biol. Volume: 182 Issue: 2 Pages: 144-154
  • DOI: 10.1016/j.jsb.2013.02.018
  • Peer Reviewed
• [Journal Article] Evolvability of thermophilic proteins from archaea and bacteria (2013)
  • Author(s): Takano, K., Aoi, A., Koga, Y., and Kanaya, S.
  • Journal Title: Biochemistry Volume: 52 Issue: 28 Pages: 4774-4780
  • DOI: 10.1021/bi400652c
  • Peer Reviewed
• [Journal Article] Role of N-terminal extension of Bacillus stearothermophilus RNase H2 and C-terminal extension of Thermotoga maritima RNase H2. (2013)
  • Author(s): Permanasari, E.-D., Angkawidjaja, C., Koga, Y., and Kanaya, S.
  • Journal Title: FEBS J. Volume: 280 Issue: 20 Pages: 5065-5079
  • DOI: 10.1111/febs.12479
  • Peer Reviewed
• [Journal Article] Increase in activation rate of Pro-Tk-subtilisin by a single nonpolar-to-polar amino acid substitution at the hydrophobic core of the propeptide domain. (2013)
  • Author(s): Yuzaki, K., Sanda, Y., You, D.-J., Uehara, R., Koga, Y., and Kanaya, S.
  • Journal Title: Protein Sci. Volume: 22 Issue: 12 Pages: 1711-1721
  • DOI: 10.1002/pro.2371
  • Peer Reviewed
• [Journal Article] Formation of the high-affinity calcium binding site in Pro-subtilisin E with the insertion sequence IS1 of Pro-Tk-subtilisin. (2013)
  • Author(s): Uehara, R., Angkawidjaja, C., Koga, Y., and Kanaya, S.
  • Journal Title: Biochemistry Volume: 52 Issue: 50 Pages: 9080-9088
  • DOI: 10.1021/bi401342k
  • Peer Reviewed
• [Journal Article] Accelerated maturation of Tk-subtilisin by a Leu→Pro mutation at the C-terminus of propeptide which reduces the binding of propeptide to Tk-subtilisin. (2013)
  • Author(s): Uehara, R., Ueda, Y., You, D-J., Koga, Y., and Kanaya, S.
  • Journal Title: FEBS Journal Volume: 280 Issue: 4 Pages: 994-1006
  • DOI: 10.1111/febs.12091
  • Peer Reviewed
• [Journal Article] Enzymatic activity of a subtilisin homo log, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein. (2013)
  • Author(s): Hirata, A., Hori, Y., Koga, Y., Sakudo, A., Ikuta, K., Kanaya, S.,
  • Journal Title: BMC Biotechnology Volume: 13 Issue: 1 Pages: 19-19
  • DOI: 10.1186/1472-6750-13-19
  • Peer Reviewed
• [Journal Article] Requirement of Ca2+ ions for the hyperthermostability of Tk-subtilisin from Thermococcus kodakarensis. (2012)
  • Author(s): Uehara, R., Takeuchi, Y., Tanaka, S., Takano, K., Koga. Y., and Kanaya S.
  • Journal Title: Biochemistry Volume: 51 Issue: 26 Pages: 5369-5378
  • DOI: 10.1021/bi300427u
  • Peer Reviewed
• [Journal Article] Structure and characterization of RNase H3 from Aquifex aeolicus. (2012)
  • Author(s): Jongruja, N., You, D-J., Angkawidjaja, C., Kanaya. E., Koga,
  • Journal Title: FEBS Journal Volume: 279 Issue: 15 Pages: 2737-2753
  • DOI: 10.1111/j.1742-4658.2012.08657.x
  • Peer Reviewed
• [Journal Article] Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures. (2012)
  • Author(s): Cheng, M., Angkawidjaja, C., Koga, Y., and Kanaya, S.
  • Journal Title: FEBS Journal Volume: 279 Issue: 19 Pages: 3727-3737
  • DOI: 10.1111/j.1742-4658.2012.08734.x
  • Peer Reviewed
• [Journal Article] Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyper thermophilic archaeon. (2012)
  • Author(s): Uehara, R., Tanaka, S., Takano, K., Koga, Y., and Kanaya, S.
  • Journal Title: Extremophiles Volume: 16 Issue: 6 Pages: 841-851
  • DOI: 10.1007/s00792-012-0479-3
  • Peer Reviewed
• [Journal Article] A dual role of divalent metal ions in catalysis and folding of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1. (2012)
  • Author(s): Tannous, E., Yokoyama, K., You, D-J., Koga, Y., and Kanaya, S.
  • Journal Title: FEBS Open Bio Volume: 2 Pages: 345-352
  • Peer Reviewed
• [Journal Article] Slow unfolding pathway of the hyper thermophilic Tk-RNase H2 examined by pulse proteolysis using the stable protease Tk-subtilisin. (2012)
  • Author(s): Okada, J., Koga, Y., Takano, K., and Kanaya, S.
  • Journal Title: Biochemistry Volume: 51 Issue: 45 Pages: 9178-9191
  • DOI: 10.1021/bi300973n
  • Peer Reviewed
• [Presentation] Structure, activity and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain (2015)
  • Author(s): Hiroyuki Okano, Eiko kanaya, Masashi Ozaki, Clement Angkawidjaja, and Shigenori Kanaya
  • Organizer: JSPS Symposium “Biotechnology and Chemistry for Green Growth”
  • Place of Presentation: 千里ライフサイエンスセンター、大阪
  • Year and Date: 2015-03-09 – 2015-03-11
• [Presentation] Crystal structure of glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain isolated from leaf-branch compost (2015)
  • Author(s): Hiroyuki Okano, Eiko kanaya, Masashi Ozaki, Clement Angkawidjaja, Yuichi Koga, and Shigenori Kanaya
  • Organizer: 59th Annual Meeting of Biophysical Society
  • Place of Presentation: ボルティモア、アメリカ
  • Year and Date: 2015-02-07 – 2015-02-11
• [Presentation] Proteolysis of abnormal prion protein with a thermostable protease from a hyper-thermophilic archaeon Thermococcus kodakarensis KOD1 (2015)
  • Author(s): Yuichi Koga, Nami Shimizu, Akikazu Sakudo, and Shigenori Kanaya
  • Organizer: 59th Annual Meeting of Biophysical Society
  • Place of Presentation: ボルティモア、アメリカ
  • Year and Date: 2015-02-07 – 2015-02-11
• [Presentation] Secretion of metagenome-derived LC-cutinase with PET degrading activity to extracellular medium of E. coli cells (2014)
  • Author(s): Sintawee Sulaiman, Eiko Kanaya, and Shigenori Kanaya
  • Organizer: 6th Annual PEGS Europe
  • Place of Presentation: リスボン、ポルトガル
  • Year and Date: 2014-11-03 – 2014-11-07
• [Presentation] 枝葉コンポストから単離したグリコシドヒドロラーゼファミリー12セルラーゼの結晶構造と熱安定化機構に関する研究 (2014)
  • Author(s): 岡野啓志, 金谷栄子, 尾崎昌史, Angkawidjaja Clement, 古賀雄一, 金谷茂則
  • Organizer: 第87回日本生化学会
  • Place of Presentation: 国立京都国際会館、京都
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] Ca2+結合部位の変異による超好熱菌由来セリンプロテアーゼTk-SPの安定化 (2014)
  • Author(s): 三浦 明子、洪 遜、阿部 一勝、Tita Foophow、古賀 雄一、金谷 茂則
  • Organizer: 第87回日本生化学会
  • Place of Presentation: 国立京都国際会館、京都
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] 超好熱菌由来プロテアーゼによるプリオン蛋白質分解の評価 (2014)
  • Author(s): 清水七海, 古賀雄一, 作道章一, 原 英之, 坂口末廣, 金谷茂則
  • Organizer: 第87回日本生化学会
  • Place of Presentation: 国立京都国際会館、京都
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] ベトナムDongNai 川流域のメタゲノムライブラリからの酵素探索 (2014)
  • Author(s): 藤澤 裕紀、古賀 雄一、Le Phi Nga、金谷 茂則
  • Organizer: 第87回日本生化学会
  • Place of Presentation: 国立京都国際会館、京都
  • Year and Date: 2014-10-15 – 2014-10-18
• [Presentation] 超好熱菌由来プロテアーゼによるプリオンタンパク質分解の評価 (2014)
  • Author(s): 古賀雄一, 清水七海, 作道章一, 原 英之, 坂口末廣, 金谷茂則
  • Organizer: 第66回日本生物工会
  • Place of Presentation: 札幌コンベンションセンター、札幌
  • Year and Date: 2014-09-09 – 2014-09-11
• [Presentation] Characterization of novel metagenome-derived esterase and cutinase from leaf- branch compost (2013)
  • Author(s): Hiroyuki Okano, Sintawee Sulaiman, Eiko kanaya, Dong-Ju You, Yuichi Koga, and Shigenori Kanaya
  • Organizer: 5th Congress of the European Microbiologists (FEMS 2013)
  • Place of Presentation: ライプチヒ、ドイツ
• [Presentation] Isolation of novel cellulolytic enzymes from leaf-branch compost by a metagenomic approach (2013)
  • Author(s): Masashi Ozaki, Eiko kanaya, Hiroyuki Okano, Yuichi Koga, and Shigenori Kanaya
  • Organizer: 5th Congress of the European Microbiologists (FEMS 2013)
  • Place of Presentation: ライプチヒ、ドイツ
• [Presentation] Evolvability of thermophilic proteins from archaea and bacteria (2013)
  • Author(s): Kazufumi Takano, Atsushi Aoi, Yuichi Koga, Shigenori Kanaya
  • Organizer: Thermophiles 2013
  • Place of Presentation: レゲンスブルグ、ドイツ
• [Presentation] Thermal adaptation of Tk-subtilisin from a hyperthermophilic archaeon (2013)
  • Author(s): Ryo Uehara, Yuichi Koga, Shigenori Kanaya
  • Organizer: SeleCa Symposium
  • Place of Presentation: アーヘン、ドイツ
• [Presentation] アミノ酸変異によるタンパク質安定化の部位依存性の評価 (2013)
  • Author(s): 田所高志、風間洋隆、古賀雄一、高野和文、金谷茂則
  • Organizer: 第１３回日本蛋白質科学会
  • Place of Presentation: とりぎん文化会館、鳥取
• [Presentation] 超好熱菌由来セリンプロテアーゼTk-SPの安定性：活性部位の水素結合の重要性 (2013)
  • Author(s): 阿部一勝、古賀雄一、金谷茂則
  • Organizer: 第36回日本分子生物会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] Ca2+結合部位の変異による超好熱菌由来セリンプロテアーゼTk-SPの安定化 (2013)
  • Author(s): 三浦明子、洪 遜、阿部一勝、Tita Foophow、古賀雄一、金谷茂則
  • Organizer: 第36回日本分子生物会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] Role of disulfide bond in stability and activity of metagenome-derived LC-cutinase with PET degrading activity (2013)
  • Author(s): Sintawee Sulaiman, Dong Ju You, Eiko Kanaya, Yuichi Koga, and Shigenori Kanaya
  • Organizer: 第36回日本分子生物会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] メタゲノム法により枝葉コンポストから単離した新規セルラーゼの構造機能解析 (2013)
  • Author(s): Hiroyuki Okano, Eiko Kanaya, Masashi Ozaki, Clement Angkawidjaja, Yuichi Koga, Shigenori Kanaya
  • Organizer: 第36回日本分子生物会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] 超好熱古細菌由来Tk-subtilisin の高効率成熟化法の開発 (2013)
  • Author(s): 勇崎孝太、上原了、古賀雄一、金谷茂則
  • Organizer: 第36回日本分子生物会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] 基質結合部位への変異導入によるLC-cutinaseの機能改変 (2013)
  • Author(s): 森数渉、Sintawee Sulaiman、金谷栄子、古賀雄一、金谷茂則
  • Organizer: 第36回日本分子生物会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] Degradation of abnormal prion protein by a hyper-stable protease (2012)
  • Author(s): Yuki Hori, Azumi Hirata, Yuichi Koga, Akikazu Sakudo, Kazuyoshi Ikuta, Shigenori Kanaya, and Kazufumi Takano
  • Organizer: Prion 2012
  • Place of Presentation: アムステルダム、オランダ
• [Presentation] Proteolysis of PrPSc with a thermostable protease and the analysis of its infectivity (2012)
  • Author(s): Yuichi Koga, Shunichi Tanaka, Akikazu Sakudo, Azumi Hirata, Kazufumi Takano, Kazuyoshi Ikuta, Shigenori Kanaya
  • Organizer: Asian Pacific Prion Symposium (APPS) 2012
  • Place of Presentation: パシフィコ横浜、横浜
• [Presentation] Tk-subtilisinによる異常型プリオンタンパク質分解効果の生物学的検討 (2012)
  • Author(s): 平田あずみ、古賀雄一、田中俊一、作道章一、生田和良、金谷茂則、高野和文
  • Organizer: 第23回日本生体防御学会総会
  • Place of Presentation: 品川区総合区民会館、東京
• [Presentation] 超好熱菌由来プロテアーゼTk-subtilisinのCa2+依存性耐熱化機構 (2012)
  • Author(s): 上原 了、竹内勇希、田中俊一、高野和文、古賀雄一、金谷茂則
  • Organizer: 第64回日本生物工学会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] プロペプチドの改変によるTk-subtilisinの成熟化速度の促進 (2012)
  • Author(s): 勇崎孝太、上原 了、三田雄大、古賀雄一、金谷茂則
  • Organizer: 第64回日本生物工学会
  • Place of Presentation: 神戸国際会議場、神戸
• [Presentation] Isolation of LC-cutinase with PET-Degrading Activity from Leaf-branch Compost using a Metagenomic Approach (2012)
  • Author(s): Sintawee SVLAIMAN, Saya YAMATO, Dong-Ju YOU, Eiko KANAYA, Yuichi KOGA, and Shigenori KANAYA
  • Organizer: 第64回日本生物工学会
  • Place of Presentation: 神戸国際会議場、神戸