Development of glycosides synthesis system using a novel enzymatic activation of carbohydrates with enzymes

• Project/Area Number: 26450114
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Hokkaido University
• Principal Investigator: Okuyama Masayuki 北海道大学, 農学研究院, 講師 (00344490)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 糖転移反応 / フラクトシル化 / スクロース / 糖質のフラクトシル化 / グリコシダーゼ / 糖転移酵素 / スクロースアナログ / β-フルクトフラノシダーゼ / スクロース類似体 / レバンスクラーゼ / スクロースシンターゼ

Outline of Final Research Achievements

The purpose of this study is to make donor substrates for transglycosylation, which glycosidases catalyze. Using β-fructofranosidase and levansucrase, I succeeded in transferring a fructosyl group of sucrose to each saccharide, such as xylose, galactose, mannose, isomaltooligosaccharides, maltooligosaccharides, and glucuronic acid. The reaction product can be a donor substrate of the transglycosylation by glycosidases. Furthermore, I investigated the transglycosylation properties of several glycosidases and modified their specificity through molecular analyses. In addition, nucleotide sugar was successfully synthesized by the reaction of sucrose synthase with fructosyl saccharide as substrate.

Research Products

• [Journal Article] Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b) (2016)
  • Author(s): Masayuki Okuyama, Kana Matsunaga, Ken-ichi Watanabe, Keitaro Yamashita, Takayoshi Tagami, Asako Kikuchi, Min Ma, Patcharapa Klahan, Haruhide Mori, Min Yao, Atsuo Kimura
  • Journal Title: FEBS Journal Volume: 284 Issue: 5 Pages: 766-783
  • DOI: 10.1111/febs.14018
  • NAID: 120006401450
  • Peer Reviewed / Open Access
• [Journal Article] Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger (2016)
  • Author(s): Julan Liao, Masayuki Okuyama, Keigo Ishihara, Yoshinori Yamori, Shigeo Iki, Takayoshi Tagami, Haruhide Mori, Seiya Chiaba, Atsuo Kimura
  • Journal Title: Bioscience, Biotechnology, and Biochemistry Volume: 80 Issue: 9 Pages: 1747-1752
  • DOI: 10.1080/09168451.2015.1136884
  • NAID: 120005955471
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis) (2016)
  • Author(s): Masuda Y, Okuyama M, Iizuka T, Nakai H, Saburi W, Fukukawa T, Maneesan J, Tagami T, Naraoka T, Mori H & Kimura A
  • Journal Title: Biosci Biotechnol Biochem Volume: 80 Issue: 3 Pages: 479-485
  • DOI: 10.1080/09168451.2015.1116926
  • Peer Reviewed / Open Access
• [Journal Article] Heat treatment of curdlan enhances the enzymatic production of biologically active β-(1,3)-glucan oligosaccharides (2016)
  • Author(s): Kumagai Y, Okuyama M, Kimura A
  • Journal Title: Carbohtdr. Polym. Volume: - Pages: 396-401
  • DOI: 10.1016/j.carbpol.2016.03.066
  • Peer Reviewed
• [Journal Article] The loop structure of Actinomycete glycoside hydrolase family 5 mannanases governs substrate recognition (2015)
  • Author(s): Kumagai Y, Yamashita K, Tagami T, Uraji M, Wan K, Okuyama M, Yao M, Kimura A, Hatanaka T
  • Journal Title: FEBS J. Volume: 282 Issue: 20 Pages: 4001-4014
  • DOI: 10.1111/febs.13401
  • NAID: 120005906892
  • Peer Reviewed / Open Access
• [Journal Article] Structural elements responsible for the glucosidic linkage-selectivity of a glycoside hydrolase family 13 exo-glucosidase (2015)
  • Author(s): Wataru Saburi, Hiroaki Rachi-Otsuka, Hironori Hondoh, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
  • Journal Title: FEBS letters Volume: 589 Issue: 7 Pages: 865-869
  • DOI: 10.1016/j.febslet.2015.02.023
  • NAID: 120005725141
  • Peer Reviewed
• [Journal Article] Structural insights into catalytic reaction that is involved in the reorientation of Trp238 at the substrate-binding site in GH13 dextran glucosidase (2015)
  • Author(s): Kobayashi M, Saburi W, Nakatsuka D, Hondoh H, Kato K, Okuyama M, Mori H, Kimura A, and Yao M
  • Journal Title: FEBS Letters Volume: 589 Issue: 4 Pages: 484-489
  • DOI: 10.1016/j.febslet.2015.01.005
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural advantage of sugar beet α-glucosidase to stabilize the Michaelis complex with long-chain substrate (2015)
  • Author(s): Tagami T, Yamashita K, Okuyama M, Mori H, Yao M, Kimura A
  • Journal Title: J. Biol. Chem. Volume: 290 Issue: 3 Pages: 1796-1803
  • DOI: 10.1074/jbc.m114.606939
  • Peer Reviewed
• [Journal Article] Biochemical properties and substrate recognition mechanism of GH31 α-glucosidase from Bacillus sp. AHU 2001 with broad substrate specificity (2015)
  • Author(s): Wataru Saburi, Masayuki Okuyama, Yuya Kumagai, Atsuo Kimura, Haruhide Mori
  • Journal Title: Biochimie Volume: 108 Pages: 140-148
  • DOI: 10.1016/j.biochi.2014.11.010
  • Peer Reviewed
• [Journal Article] 糖質関連酵素の最近の進歩(4) 糖質加水分解酵素ファミリー内の機能の保存性と多様性 (2015)
  • Author(s): 奥山 正幸
  • Journal Title: 化学と生物: 日本農芸化学会会誌: 生命・食・環境 Volume: 53 Pages: 120-126
  • Peer Reviewed
• [Journal Article] Catalytic role of the calcium ion in GH97 inverting glycoside hydrolase (2014)
  • Author(s): Masayuki Okuyama, Takuya Yoshida, Hironori Hondoh, Haruhide Mori, Min Yao, Atsuo Kimura
  • Journal Title: FEBS letters Volume: 588 Issue: 17 Pages: 3213-3217
  • DOI: 10.1016/j.febslet.2014.07.002
  • Peer Reviewed
• [Journal Article] Different molecular complexity of linear-isomaltomegalosaccharides and β-cyclodextrin on enhancing solubility of azo dye ethyl red: Towards dye biodegradation (2014)
  • Author(s): Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Janjira Maneesan, Masayuki Okuyama, Haruhide Mori, Nobuo Sakairi, Atsuo Kimura
  • Journal Title: Bioresource Technology Volume: 169 Pages: 518-524
  • DOI: 10.1016/j.biortech.2014.07.025
  • Peer Reviewed
• [Journal Article] Biodecolorization of a food azo dye by the deep sea Dermacoccus abyssi MT1.1(T) strain from the Mariana Trench (2014)
  • Author(s): Lang W, Sirisansaneeyakul S, Martins, L O, Ngiwsara L, Sakairi N, Pathom-aree W, Okuyama M, Mori H, Kimura, A
  • Journal Title: J. Environ. Manage. Volume: 132 Pages: 155-164
  • DOI: 10.1016/j.jenvman.2013.11.002
  • Peer Reviewed
• [Journal Article] Identity of the two dextran dextrinases produced by Gluconobacter oxydans ATCC 11894 and its localization change depending on the cell growth (2014)
  • Author(s): Juri Sadahiro, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 456 Issue: 1 Pages: 500-505
  • DOI: 10.1016/j.bbrc.2014.11.115
  • Peer Reviewed
• [Presentation] Characterization of thermostable α-glucosidase from Geobacillus caldoxylosilyticus NBRC 107762, which displays remarkable transglucosylation (2017)
  • Author(s): Min MA, Masayuki OKUYAMA, Takayoshi TAGAMI, Atsuo KIMURA
  • Organizer: 日本農芸化学会
  • Place of Presentation: 京都女子大学・京都市・京都
  • Year and Date: 2017-03-17
• [Presentation] Bacteroides thetaiotaomicron β-L-arabinopyranosidase の立体構造解析 (2016)
  • Author(s): 菊池麻子，加藤公児，奥山正幸，大嵜翔平，姚閔，木村淳夫
  • Organizer: 日本応用糖質科学会
  • Place of Presentation: 福山大学・福山市・広島
  • Year and Date: 2016-09-14
• [Presentation] GH97 α-galactosidase求核触媒変異酵素が触媒する糖転移反応の反応機構 (2016)
  • Author(s): 松永夏奈，奥山正幸，渡辺健一，田上貴祥，山下恵太郎，姚閔，森春英，木村淳夫
  • Organizer: 日本応用糖質科学会
  • Place of Presentation: 福山大学・福山市・広島
  • Year and Date: 2016-09-14
• [Presentation] Analysis of amino acid residues relating to specificity of endodextranase from Streptococcus mutans (2016)
  • Author(s): Klahan P., Okuyama M., Mori H., Kimura A.
  • Organizer: International Carbohydrate Symposium
  • Place of Presentation: New Orleans, LA, USA
  • Year and Date: 2016-07-17
  • Int'l Joint Research
• [Presentation] Purification and characterization of a-1,3-glucosidase from Aspergillus niger (2016)
  • Author(s): Min Ma, Takayoshi Tagami, Masayuki Okuyama and Atsuo Kimura
  • Organizer: International Carbohydrate Symposium
  • Place of Presentation: New Orleans, LA, USA
  • Year and Date: 2016-07-17
  • Int'l Joint Research
• [Presentation] Zymomonas mobilis 由来levansucraseとinvertaseの反応特異性の解析 (2016)
  • Author(s): 芹沢 領、田沼 正成、奥山 正幸、木村 淳夫
  • Organizer: 日本農芸化学会2016年度大会
  • Place of Presentation: 札幌コンベンションセンター （北海道・札幌市）
  • Year and Date: 2016-03-26
• [Presentation] Solubilizing ability of double-anchor type isomaltomegalosaccharide for drug delivery of ibuprofen (2016)
  • Author(s): Sota UEKUSA, Weeranuch Lang, Yuya Kumagai, Masayuki Okuyama, Atsuo Kimura
  • Organizer: 日本農芸化学会2016年度大会
  • Place of Presentation: 札幌コンベンションセンター （北海道・札幌市）
  • Year and Date: 2016-03-26
• [Presentation] Kribbella flavida由来isomaltosyltransferaseの機能解析 (2016)
  • Author(s): 宮野 江梨、田上 貴祥、奥山 正幸、木村 淳夫
  • Organizer: 日本農芸化学会2016年度大会
  • Place of Presentation: 札幌コンベンションセンター （北海道・札幌市）
  • Year and Date: 2016-03-26
• [Presentation] Efficient production and characterization of α-1,3-glucosidase from Aspergillus niger (2016)
  • Author(s): Min MA, Takayoshi TAGAMI, Masayuki OKUYAMA, Atsuo KIMURA
  • Organizer: 日本農芸化学会2016年度大会
  • Place of Presentation: 札幌コンベンションセンター （北海道・札幌市）
  • Year and Date: 2016-03-26
• [Presentation] 環状四糖[cyclo-(1→6)-(α-nigerosyl-nigerosyl)]の分解を担う2種の糖質加水分解酵素の発見 (2016)
  • Author(s): 田上 貴祥、貞廣 樹里、奥山 正幸、木村 淳夫、岩崎 智仁
  • Organizer: 日本農芸化学会2016年度大会
  • Place of Presentation: 札幌コンベンションセンター （北海道・札幌市）
  • Year and Date: 2016-03-26
• [Presentation] Gluconobacter oxydans由来の菌体外酵素dextran dextrinaseは自らの生成物デキストランによって細胞表層から遊離する (2016)
  • Author(s): 貞廣 樹里、森 春英、佐分利 亘、奥山 正幸、木村 淳夫
  • Organizer: 日本農芸化学会2016年度大会
  • Place of Presentation: 札幌コンベンションセンター （北海道・札幌市）
  • Year and Date: 2016-03-26
• [Presentation] Zymomonas mobilis由来levansucraseとinvertaseの生成物特異性に関与する構造因子の推定 (2016)
  • Author(s): 芹沢領，奥山正幸，木村淳夫
  • Organizer: 日本農芸化学会北海道支部例会
  • Place of Presentation: 北海道大学・札幌市・北海道
• [Presentation] Isomaltooligosaccharide 6-α-glucosyltransferaseの構造と触媒部位の機能性アミノ酸残基の同定 (2015)
  • Author(s): 川田恭平、藤本 瑞、鐘ヶ江倫世、西村崇志、奥山正幸、森 春英、木村淳夫
  • Organizer: 日本応用糖質科学会平成27年度大会
  • Place of Presentation: 奈良春日野国際フォーラム 甍～I・RA・KA～ (奈良県・奈良市)
  • Year and Date: 2015-09-16
• [Presentation] Podospora anserina α-glucosidaseの高い糖転移能に関わる構造因子 (2015)
  • Author(s): 福本健太、Song Kyung-Mo、奥山正幸、木村淳夫
  • Organizer: 日本応用糖質科学会平成27年度大会
  • Place of Presentation: 奈良春日野国際フォーラム 甍～I・RA・KA～ (奈良県・奈良市)
  • Year and Date: 2015-09-16
• [Presentation] Enzymatic synthesis of functional linear isomaltomegalosaccharide by Gluconobacter oxydans dextran dextrinase (2015)
  • Author(s): Yuya Kumagai, Weeranuch Lang, Juri Sadahiro, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
  • Organizer: 11th Carbohydrate Bioengineering Meeting
  • Place of Presentation: Espoo (Finland)
  • Year and Date: 2015-05-10
  • Int'l Joint Research
• [Presentation] A transglycosylation of catalytic nucleophile mutant of GH97 a-galactosidase with an external nuclephile (2015)
  • Author(s): Masayuki Okuyama, Kana Matsunaga, Ken-ichi Watanabe, Tokayoshi Tagami, Keitaro Yamashita, Haruhide Mori, Min Yao, Atsuo Kimura
  • Organizer: 11th Carbohydrate Bioengineering Meeting
  • Place of Presentation: Espoo (Finland)
  • Year and Date: 2015-05-10
  • Int'l Joint Research
• [Presentation] Structural and biochemical studies of sugar beet a-glucosidase exhibiting high specificity for long-chain substrates (2015)
  • Author(s): Tokayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura
  • Organizer: 11th Carbohydrate Bioengineering Meeting
  • Place of Presentation: Espoo (Finland)
  • Year and Date: 2015-05-10
  • Int'l Joint Research
• [Presentation] 生理活性を持つβ-1,3-グルカンオリゴ糖の酵素的調製法の検討 (2015)
  • Author(s): 熊谷祐也, 奥山正幸, 木村淳夫
  • Organizer: 日本農芸化学会2015年度大会
  • Place of Presentation: 岡山市 岡山大学
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Bacillus subtilis由来levansucraseによるsucrose類似体の合成および受容体特異性に関する研究 (2015)
  • Author(s): 渡辺基志、木村淳夫、奥山正幸
  • Organizer: 日本農芸化学会2015年度大会
  • Place of Presentation: 岡山市 岡山大学
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Bacteroides thetaiotaomicron由来GH97 β-L-アラビノピラノシダーゼの機能解析 (2015)
  • Author(s): 大嵜 翔平、松永 夏奈、奥山 正幸、木村 淳夫
  • Organizer: 日本農芸化学会2015年度大会
  • Place of Presentation: 岡山市 岡山大学
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] 糖質加水分解酵素ファミリー97における触媒機構と多様性 (2015)
  • Author(s): 奥山 正幸
  • Organizer: 日本農芸化学会2015年度大会シンポジウム
  • Place of Presentation: 岡山市 岡山大学
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Bacillus sp. AHU 2001由来GH31 α-グルコシダーゼの生化学的諸性質と基質認識に重要な構造因子の解析 (2015)
  • Author(s): 佐分利 亘、奥山 正幸、熊谷 祐也、木村 淳夫、森 春英
  • Organizer: 日本農芸化学会2015年度大会
  • Place of Presentation: 岡山市 岡山大学
  • Year and Date: 2015-03-26 – 2015-03-29
• [Presentation] Dextran dextrinaseのPhe602の生成物特異性に関する研究 (2014)
  • Author(s): 坂谷 敦、熊谷祐也、貞廣樹里、Lang Weeranuch、奥山正幸、森 春英、木村淳夫
  • Organizer: 日本応用糖質科学会 平成25年度大会
  • Place of Presentation: 新潟市 朱鷺メッセ
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Amphiphilic function of linear-isomaltooligosaccharides (L-IMS) on ethyl red (ER) solublity (2014)
  • Author(s): Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Masayuki Okuyama, Haruhide Mori, Nobuo Sakairi, Atuso Kimura
  • Organizer: 日本応用糖質科学会 平成25年度大会
  • Place of Presentation: 新潟市 朱鷺メッセ
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] 放線菌マンナナーゼの基質特異性に関するループ7の解析 (2014)
  • Author(s): 熊谷祐也, 裏地美杉, 奥山正幸, 木村淳夫, 畑中唯史
  • Organizer: 日本応用糖質科学会 平成25年度大会
  • Place of Presentation: 新潟市 朱鷺メッセ
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] 放線菌マンナナーゼの分岐オリゴ糖に対する基質特異性に関する解析 (2014)
  • Author(s): 熊谷祐也, 裏地美杉, 奥山正幸, 木村淳夫, 畑中唯史
  • Organizer: 第66回 日本生物工学会大会
  • Place of Presentation: 札幌市 札幌コンベンションセンター
  • Year and Date: 2014-09-09 – 2014-09-11
• [Remarks] 北海道大学大学院農学研究院分子酵素学研究室 研究概要
  • URL: http://www.agr.hokudai.ac.jp/molenzlab/research/