Promotion of reverse reaction of carbohydrolase and its applicationfor oligosaccharide synthesis

• Project/Area Number: 19580102
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Hokkaido University
• Principal Investigator: MORI Aruhide Hokkaido University, 大学院・農学研究院, 准教授 (80241363)
• Project Period (FY): 2007 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2008: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2007: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000)
• Keywords: 酵素利用学 / オリゴ糖 / 加水分解酵素 / 逆反応 / オリゴ糖合成 / アノマー反転型酵素

Research Abstract

トレハラーゼはトレハロースを加水分解する.本研究では, トレハラーゼ改変酵素と特殊化合物(βフッ化グルコース)を用いて, 加水分解の逆反応によりトレハロースを高効率で合成させることに成功した.変異酵素として, 塩基触媒変異体, および塩基触媒および加水分解の基質の水分子に影響を与えるアミノ酸残基変異体を用いた.何れも反応速度は野生型に比べ低下したが, 特に, 後者の合成効率が高く, 60%程度の収率を示した.

Research Products

• [Journal Article] Structural and Functional Analysis of aGlycoside Hydrolase Family 97 Enzyme fromBacteroides thetaiotaomicron (2008)
  • Author(s): Kitamura M, Okuyama M
  • Journal Title: J. Biol. Chem. 283(52) Pages: 6328-6337
  • Peer Reviewed
• [Journal Article] Structure-function relationship of substrate lengthspecificity of dextran glucosidase from Streptococcusmutants Biologia (2008)
  • Author(s): Saburi W, Hondoh H
  • Journal Title: 63(6) Pages: 1000-1005
  • Peer Reviewed
• [Journal Article] Substrate recognition mechanism ofα-1,6-glucosidic linkage hydrolyzing enzyme,dextran glucosidase from Streptococcus mutans (2008)
  • Author(s): Hondoh H, Saburi W, Mori H
  • Journal Title: J Mol Biol. 378(4) Pages: 911-920
  • Peer Reviewed
• [Journal Article] Structural and Functional Analysis of a Glycoside Hydrolase Family 97 Enzyme from Bacteroides thetaiotaomicron (2008)
  • Author(s): Kitamura M
  • Journal Title: J. Biol. Chem 283 Pages: 6328-6337
  • Peer Reviewed
• [Journal Article] A. Substrate recognitlon mechanism of α-1,6-glucosi4ic linkage hydrolyzing enzyme, dextran giucosidase from Streptococcus mutans (2008)
  • Author(s): Hondoh H
  • Journal Title: J. Mol. Biol 378 Pages: 911-920
  • Peer Reviewed
• [Journal Article] Aglycone specificity of Escherichia coliα-xylosidase investigated by transxylosylation (2007)
  • Author(s): Kang MS, Okuyama M
  • Journal Title: FEBS J. 274(23) Pages: 6074-6084
  • Peer Reviewed
• [Journal Article] Function-unknown glycoside hydrolase family31 proteins, mRNAs of which were expressed inrice ripening and germinating stages, areα-glucosidase and α-xylosidase. J Biochem (2007)
  • Author(s): Nakai H, Tanizawa S,
  • Journal Title: J Biochem. 142(4) Pages: 491-500
  • Peer Reviewed
• [Journal Article] Crystallization and preliminary X-ray analysis ofStreptococcus mutans dextran glucosidase (2007)
  • Author(s): Saburi W, Hondoh H
  • Journal Title: Acta Crystallogr Sect F Struct Biol CrystCommun 63(Pt9) Pages: 774-776
  • Peer Reviewed
• [Journal Article] Molecular Cloning of cDNA for Trehalase fromthe European Honeybee, Apis mellifera L., andIts Heterologous Expression in Pichia pastoris (2007)
  • Author(s): Lee, J.-H., Saito, S., Mori, H
  • Journal Title: Biosci. Biotechnol. Biochem 71(9) Pages: 2256-2265
  • Peer Reviewed
• [Journal Article] Molecular Cloning of cDNAs and Genes forThree α-Glucosidases from European Honeybees,Apis mellifera L., and Heterologous Productionof Recombinant Enzymes in Pichia pastoris (2007)
  • Author(s): Nishimoto M, Mori H
  • Journal Title: Biosci. Biotechnol. Biochem 71(7) Pages: 1703-1716
  • Peer Reviewed
• [Journal Article] Cloning of cDNA for Trehalase from the European Honeybee, Apis mellifera L., and Its Heterologous Expressiong in Pichia pastoris. (2007)
  • Author(s): Lee, J. -H.
  • Journal Title: Biosci. Biotechnol. Biochem. 71 Pages: 2256-2265
  • Peer Reviewed
• [Presentation] 加水分解反応の抑制によりトレハロースを縮合蓄積させるトレハラーゼ変異酵素 (2009)
  • Author(s): 牧 孝多朗
  • Organizer: 日本農芸化学会大会
  • Place of Presentation: 福岡
  • Year and Date: 2009-03-28
• [Presentation] Isomaltooligosaccharide 6-α-glucosyltransferase(I6GT)のイソマルトオリゴ糖への作用と転移に関する構造因子 (2009)
  • Author(s): 西村崇志
  • Organizer: 日本農芸化学会大会
  • Place of Presentation: 福岡
  • Year and Date: 2009-03-28
• [Presentation] 加水分解反応の抑制によりトレハロースを縮合蓄積させるトレハラーゼ変異酵素 (2009)
  • Author(s): 牧孝太朗
  • Organizer: 日本農芸化学会大会
  • Place of Presentation: マリンメッセ(福岡)
  • Year and Date: 2009-03-28
• [Presentation] 大腸菌trehalase の触媒アミノ酸残基の決定とglycosynthase 反応 (2008)
  • Author(s): 森春英
  • Organizer: 日本応用糖質科学会大会
  • Place of Presentation: 沖縄
  • Year and Date: 2008-09-18
• [Presentation] デキストリンデキストラナーゼ遺伝子の異種宿主発現および組換え酵素の解析 (2008)
  • Author(s): 貞廣樹里
  • Place of Presentation: 沖縄
  • Year and Date: 2008-09-18
• [Presentation] 大腸菌trehalaseの触媒アミノ酸残基の決定とglycosynthase反応 (2008)
  • Author(s): 森春英
  • Organizer: 日本応用糖質科学会大会
  • Place of Presentation: 琉球大(那覇・沖縄)
  • Year and Date: 2008-09-18
• [Presentation] 新規酵素isomaltooligosaccharide 6-α-glucosyltransferase(I6GT)の機能解析 (2008)
  • Author(s): 鐘ヶ江倫世
  • Organizer: 日本農芸化学会北海道支部学術合同講演会
  • Place of Presentation: 札幌
  • Year and Date: 2008-08-08
• [Presentation] デキストリンデキストラナーゼの一次構造決定および組換え酵素の解析 (2008)
  • Author(s): 貞廣樹里
  • Organizer: 日本農芸化学会北海道支部講演会
  • Place of Presentation: 北海道大学(札幌)
  • Year and Date: 2008-08-08
• [Presentation] Identification of essential catalyticresidues in an Escherichia coli trehalase and aninverting glycosynthase reaction catalyzed by thecatalytic base-mutated enzyme (2008)
  • Author(s): Mori H
  • Organizer: XXIVInternational Carbohydrate Symposium
  • Place of Presentation: オスロ大学
  • Year and Date: 2008-07-28
• [Presentation] Identification of essential catalytic residues in an Escherichia coli trehalase and an inverting glycosynthase reaction catalyzed by the catalytic base-mutated enzyme (2008)
  • Author(s): 森春英
  • Organizer: International Carbohydrate Symposium(国際炭水化物シンポジウム)
  • Place of Presentation: ホスロ大学(オスロ,ノルウェー)
  • Year and Date: 2008-07-28
• [Presentation] Functional and structural analysis of glycoside hydrolase family 97 enzyme from Bacteroides thetaiotaomicron (2008)
  • Author(s): 奥山正幸
  • Organizer: International Carbohydrate Symposium(国際炭水化物シンポジウム)
  • Place of Presentation: オスロ大学(オスロ,ノルウェー)
  • Year and Date: 2008-07-28
• [Presentation] 新しく見出されたイソマルトオリゴ糖不均化酵素の遺伝子クローニングと異種宿主発現 (2008)
  • Author(s): 鐘ケ江 倫世
  • Organizer: 日本農芸化学会大会
  • Place of Presentation: 名古屋(名城大)
  • Year and Date: 2008-03-28
• [Presentation] Functional and structural analysisof glycoside hydrolase family 97 enzyme fromBacteroides thetaiotaomicron (2008)
  • Author(s): Okuyama M
  • Organizer: XXIV InternationalCarbohydrate Symposium
  • Place of Presentation: オスロ大学
• [Presentation] Substrate recognition mechanism ofdextran glucosidase from Streptococcus mutans(DexB) (2007)
  • Author(s): Hondoh H
  • Organizer: Carbohydrate Bioengineering Meeting 7
  • Place of Presentation: ドイツ/ブラウンシュヴァイ
• [Book] Molecular mechanism ofα-glucosidase In Carbohydrate-active enzymes:structure, function, and applications (2008)
  • Author(s): Okuyama M, Mori H, Hondoh H, Nakai H, SaburiW, Kang MS, Kim YM, Nishimoto M, WongchawalitJ, Yamamoto T, Son M, Lee JH, Mar SS, Fukuda K,Chiba S, Kimura A
  • Publisher: Woodhead Publishing Ltd.,Cambridge, UK