Distinct way to prepare the hyper-stable and stress-tolerant enzymes-halophilic enzyme engineering and hyper-functional cell production

• Project/Area Number: 20580372
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied molecular and cellular biology
• Research Institution: Kagoshima University
• Principal Investigator: TOKUNAGA Masao 鹿児島大学, 農学部, 教授 (20112782)
• Co-Investigator(Kenkyū-buntansha): ISHIBASHI Matsujiro 鹿児島大学, 農学部, 准教授 (20305163) ; TOKUNAGA Hiroko 鹿児島大学, 農学部, 技能補佐員 (60381191)
• Project Period (FY): 2008 – 2010
• Project Status: Completed (Fiscal Year 2010)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2010: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2009: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2008: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 蛋白質・糖鎖工学 / 極限環境微生物 / 蛋白質発現 / 高機能化 / 好塩性酵素 / 酸性アミノ酸 / 構造可逆性 / 好塩性α-アミラーゼ / 融合蛋白質 / 可溶性 / 封入体 / 大腸菌発現系

Research Abstract

The amino acid composition of halophilic enzymes is in general characterized by an abundant content of acidic amino acid leading to high aqueous solubility without aggregation in both native and denatured states and hence allowing efficient renaturation of halophilic proteins after heat or denaturant treatment. The replacement of neutral or basic amino acid residues in non-halophilic proteins with acidic amino acid residues converted non-halophilic to halophilic proteins which show higher aqueous solubility and stability.

Research Products

• [Journal Article] Halophilic beta-lactamase as a new solubilityand folding-enhancing tag protein : production of native human interleukin 1alpha and human neutrophil alpha-defensin (2010)
  • Author(s): Tokunaga H, Saito S, Sakai K, Yamaguchi R, Katsuyama I, Arakawa T, Onozaki K, Arakawa T, Tokunaga M
  • Journal Title: Appl Microbiol Biotechnol Volume: 86(2) Pages: 649-658
  • Peer Reviewed
• [Journal Article] Dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacterium Chromohalobacter salexigens DSM3043 : both residues 134 and 146 are critical for the tetrameric assembly (2010)
  • Author(s): H. Tokunaga, K. Izutsu, S. Arai, Y. Yonezawa, R. Kuroki, T. Arakwa, and M. Tokunaga.
  • Journal Title: Enz. Microbial Technol Volume: 46 Pages: 129-135
  • Peer Reviewed
• [Journal Article] Salt-dependent thermo-reversible α-amylase : cloning and characterization of halophilic α-amylase from moderately halophilic bacterium, Kocuria varians. (2010)
  • Author(s): Yamaguchi R, Tokunaga H, Ishibashi M, Arakawa T, Tokunaga M.
  • Journal Title: Appl Microbiol Biotechnol. Volume: 89 Pages: 673-684
  • Peer Reviewed
• [Journal Article] High solubility supports efficient refolding of thermally unfolded β-lactamase (2010)
  • Author(s): Arakawa T, Tokunaga H, Yamaguchi R, Tokunaga M.
  • Journal Title: Int J Biol Macromol. Volume: 47 Pages: 706-709
  • Peer Reviewed
• [Journal Article] Halophilic β-lactamase as a new solubility-and folding-enhancing tag protein : Production of native human interleukin 1α and human neutrophil α-defensin (2010)
  • Author(s): H.Tokunaga, S.Saito, K.Sakai, R.Yamaguchi, I.Katsuyama, T.Arakawa, K.Onozaki, T.Arakawa, M.Tokunaga
  • Journal Title: Applied Microbiology and Biotechnology 86 Pages: 649-658
  • Peer Reviewed
• [Journal Article] Engineering of halophilic enzymes : two acidic amino acid residues at the carboxy-terminal region confer halophilic characterstics to Halomonas and Pseudomonas nucleoside diphosphate kinases (2008)
  • Author(s): Tokunaga, H., Arakawa, T., Tokunaga, M.
  • Journal Title: Protein Sciences Volume: 17 Pages: 1603-1610
  • Peer Reviewed
• [Journal Article] Engineering of halophilic enzymes : two acidic amino acid residues at the carboxy-terminal region confer halophilic characterstics to Halomonas and Pseudomonas nucleoside diphosphate kinases. (2008)
  • Author(s): Tokunaga, H., Arakawa, T., Tokunaga, M.
  • Journal Title: Protein Sciences 17 Pages: 1603-1610
  • Peer Reviewed
• [Presentation] 塩依存的にrefoldingする中度好塩菌Kocuria varians由来α-amylaseの性質検討とcloning及び発現 (2011)
  • Author(s): 山口類、石橋松二郎、徳永廣子、荒川力、徳永正雄
  • Organizer: 日本農芸化学会2011年大会
  • Place of Presentation: 京都女子大学
  • Year and Date: 2011-03-26
• [Presentation] 好塩性β-ラクタマーゼを可溶性タグとした融合蛋白質発現系の開発 (2010)
  • Author(s): 山口類、徳永廣子、斎藤尚子、逆井一樹、勝山巌、荒川友博、小野崎菊夫、荒川力、徳永正雄
  • Organizer: 2010日本農芸化学会大会
  • Place of Presentation: 東京
  • Year and Date: 2010-03-29
• [Presentation] 高度好塩菌由来nucleoside diphosphate kinase変異体G114Rの解析 (2010)
  • Author(s): 石橋松二郎、岩佐達也、徳永正雄
  • Organizer: 2010日本農芸化学会大会
  • Place of Presentation: 東京
  • Year and Date: 2010-03-28
• [Presentation] 好塩性付与蛋白質の分子育種 (2009)
  • Author(s): 徳永廣子、荒川力、山口類、徳永正雄
  • Organizer: 2009年日本農芸化学会大会
  • Place of Presentation: 福岡
  • Year and Date: 2009-03-28
• [Presentation] 1アミノ酸(134th)置換による好塩性nueleoside diphosphate kinaseの2量体-4量体変換 (2008)
  • Author(s): 徳永廣子, 石橋松二郎, 有坂文雄, 新井栄揮, 黒木良太, 山口類, 荒川力, 徳永正雄
  • Organizer: 2008年日本蛋白質科学会年会
  • Place of Presentation: 東京タワーホール船堀
• [Remarks] 鹿児島大学農学部生物資源化学科応用分子微生物学研究室
  • URL: http://chem.agri.kagoshima-u.ac.jp/Oyoseika/oubi/HP060703/top-040621.html