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研究者情報

マスター

アカウント（マスター）

  • 氏名

    森　春英(モリ　ハルヒデ), モリ　ハルヒデ

所属（マスター）

  • 農学研究院　基盤研究部門　生物機能化学分野

所属（マスター）

  • 農学研究院　基盤研究部門　生物機能化学分野

独自項目

syllabus

  • 2021, 応用微生物学特論, Advanced Molecular Microbiology, 修士課程, 農学院, 微生物、微生物バイオテクノロジー、酵素、環境修復
  • 2021, 食品安全・機能性開発学特論, Advanced Safety and Function of Food, 修士課程, 農学院, 食品安全性、予測微生物、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
  • 2021, 食品安全・機能性開発学特論演習, Advanced Seminar on Safety and Function of Food, 修士課程, 農学院, 食品の安全、予測微生物学、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
  • 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 食品安全性、予測微生物、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
  • 2021, リーダーシップ学総論, Leadership Studies, 修士課程, 農学院, リーダーシップ、北海道大学、新渡戸稲造、科学者、イノベーション
  • 2021, 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), Freshman Seminar, 学士課程, 全学教育, 食料、安全、作物生産、放射能汚染、食品微生物、酵素利用技術、毒物、ガン、アレルギー、メタボリックシンドローム、腸内細菌叢、保健機能食品
  • 2021, 生物化学Ⅰ, Biochemistry I, 学士課程, 農学部, 生化学，生体成分，糖質，アミノ酸，タンパク質，酵素，補酵素，ビタミン，脂質，核酸
  • 2021, 生物化学Ⅱ, Biochemistry II, 学士課程, 農学部, 代謝，エネルギー，解糖系，トリカルボン酸回路，電子伝達系，酸化的リン酸化，糖新生，ペントースリン酸経路，脂質代謝，光合成，カルビンサイクル，窒素代謝，尿素サイクル
  • 2021, 生物化学Ⅲ, Biochemistry III, 学士課程, 農学部, タンパク質，酵素，応用糖質科学，糖質の構造，糖質の機能，糖質の代謝，糖質の合成，糖質の利用

PositionHistory

  • 大学院農学院副学院長, 2019年4月1日, 2021年3月31日
  • 大学院農学研究院副研究院長, 2019年4月1日, 2021年3月31日
  • 農学部副学部長, 2019年4月1日, 2021年3月31日
  • 評価室室員, 2016年7月1日, 2018年6月30日
  • 評価室室員, 2018年7月1日, 2020年6月30日

researchmap

プロフィール情報

学位

  • 博士（農学）（北海道大学）

プロフィール情報

  • 姓

    森, モリ

  • 名

    春英, ハルヒデ

  • ID各種

    200901035995397800

業績リスト

研究キーワード

  • 糖質加リン酸分解酵素   糖質異性化   マンノース含有オリゴ糖   オリゴ糖   α-グルコシグーゼ   酵素利用学   加水分解酵素   α-amylase   酵素反応機構   糖転移反応   酵素化学   イソマルトオリゴ糖   糖質関連酵素   多糖合成酵素   応用生物化学   Applied Biochemistry (6103)   

研究分野

  • ライフサイエンス / 応用生物化学

経歴

  • 2013年04月 - 現在 北海道大学 大学院農学研究院 教授
  • 2019年04月 - 2021年03月 北海道大学 大学院農学研究院・農学院・農学部 副研究院長・副学院長・副学部長
  • 2017年04月 - 2019年03月 北海道大学 大学院農学研究院 研究院長補佐
  • 2010年 - 2012年 北海道大学 (連合)農学研究科(研究院) (連合)農学研究科(研究院) 准教授
  • 2007年 - 大学院農学研究院応用生命科学研究部門応用分子生物学分野分子酵素学研究室 准教授

学歴

  •         - 1992年   北海道大学   農学研究科
  •         - 1992年   北海道大学

委員歴

  • 2023年09月 - 現在   日本応用糖質科学会   副会長
  • 2023年05月 - 現在   日本農芸化学会   北海道支部幹事（副支部長）
  • 2013年07月 - 現在   日本応用糖質科学会   評議員
  • 2023年05月 - 2026年02月   日本農芸化学会   大会実行委員会（副委員長）
  • 2021年09月 - 2023年09月   日本応用糖質科学会   監事
  • 2017年07月 - 2023年06月   日本応用糖質科学会   北海道支部長
  • 2021年08月 - 2021年09月   日本応用糖質科学会   2021年度大会実行委員長
  • 2014年09月 - 2021年09月   日本応用糖質科学会   理事
  • 2015年06月 - 2021年06月   北海道食の安全・安心委員会   委員・専門部会長
  • 2014年10月 - 2017年09月   日本応用糖質科学会   和文誌編集委員（委員長）
  • 2013年03月 - 2017年02月   日本農芸化学会   和文誌編集委員会
  • 2012年10月 - 2014年09月   日本応用糖質科学会   和文誌編集委員（副委員長）
  • 2009年07月 - 2013年06月   日本応用糖質科学会   北海道支部幹事長（事務局）   日本応用糖質科学会
  • 2011年04月 - 2013年02月   日本農芸化学会   広報委員会
  • 2010年 - 2011年   日本応用糖質科学会   H23大会実行委員会総務   日本応用糖質科学会
  • 2010年   日本応用糖質科学会   「応用糖質科学」1巻１号編集委員   日本応用糖質科学会

受賞

  • 2019年09月 日本応用糖質科学会 学会賞
     各種糖質加水分解酵素・加リン酸分解酵素・異性化酵素の機能と応用に関する研究 
    受賞者: 森 春英
  • 2007年 Best Poster Award
  • 2005年09月 日本応用糖質科学会 奨励賞
     植物α－アミラーゼの機能と構造に関する研究 
    受賞者: 森 春英
  • 2004年 ベストポスター賞
  • 2003年 B.B.B.論文賞
  • 2003年 Biosci Biotechnol Biochem Paper Award(JSBBA, 2003)

論文

  • Wataru Saburi, Takayoshi Tagami, Takuya Usui, Jian Yu, Toyoyuki Ose, Min Yao, Haruhide Mori
    Food Bioscience 61 104516 - 104516 2024年10月 [査読有り][通常論文]
  • Wataru Saburi, Haruhide Mori
    Bioscience, Biotechnology, and Biochemistry 88 10 1180 - 1187 2024年07月11日 
    Abstract Starch degradation in malted barley produces yeast-fermentable sugars. In this study, we compared the amylolytic enzymes and composition of the malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar established in Japan) and Kitanohoshi (the currently used cultivar for beer production). Hokudai 1 malt contained lower activity of amylolytic enzymes than Kitanohoshi malt, although these cultivars contained α-amylase AMY2 and β-amylase Bmy1 as the predominant enzymes. Malt starch hydrolysate of Hokudai 1 contained more limit dextrin and less yeast-fermentable sugars than that of Kitanohoshi. In mixed malt saccharification, a high Hokudai 1 malt ratio increased the limit dextrin levels and decreased the maltotriose and maltose levels. Even though Kitanohoshi malt contained more amylolytic enzymes than Hokudai 1 malt, addition of Kitanohoshi extract containing the amylolytic enzymes did not enhance malt starch degradation of Hokudai 1. Hokudai 1 malt starch was less degradable than Kitanohoshi malt starch.
  • Weeranuch Lang, Takayoshi Tagami, Yuya Kumagai, Seiya Tanaka, Hye-Jin Kang, Masayuki Okuyama, Wataru Saburi, Haruhide Mori, Tohru Hira, Chaehun Lee, Takuya Isono, Toshifumi Satoh, Hiroshi Hara, Takayuki Kurokawa, Nobuo Sakairi, Yoshiaki Yuguchi, Atsuo Kimura
    Carbohydrate Polymers 319 121185 - 121185 2023年11月 [査読有り][通常論文]
  • Tomoya Ota, Wataru Saburi, Takayoshi Tagami, Jian Yu, Shiro Komba, Linda Elizabeth Jewell, Tom Hsiang, Ryozo Imai, Min Yao, Haruhide Mori
    The Journal of biological chemistry 105294 - 105294 2023年09月27日 [査読有り][通常論文]
     
    The glycoside hydrolase family 55 (GH55) includes inverting exo-β-1,3-glucosidases and endo-β-1,3-glucanases, acting on laminarin, which is a β1-3/1-6-glucan consisting of a β1-3/1-6-linked main chain and β1-6-linked branches. Despite their different modes of action toward laminarin, endo-β-1,3-glucanases share with exo-β-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-β-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-β-1,3-glucanases, degraded internal β-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. β1-3-Glucans lacking β1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the non-reducing terminal β1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain β1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1mM-1) were much lower than to laminarin (5,920 s-1mM-1). These results indicate that β-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-β-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft structure. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases.
  • Yusuke Kido, Wataru Saburi, Taizo Nagura, Haruhide Mori
    Bioscience, biotechnology, and biochemistry 87 10 1169 - 1182 2023年09月21日 [査読有り][通常論文]
     
    Inulin, β-(2→1)-fructan, is a beneficial polysaccharide used as a functional food ingredient. Microbial inulosucrases (ISs), catalyzing β-(2→1)-transfructosylation, produce β-(2→1)-fructan from sucrose. In this study, we identified a new IS (NdIS) from the soil isolate, Neobacillus drentensis 57N. Sequence analysis revealed that, like other Bacillaceae ISs, NdIS consists of a glycoside hydrolase family 68 domain and shares most of the 1-kestose-binding residues of the archaeal IS, InuHj. Native and recombinant NdIS were characterized. NdIS is a homotetramer. It does not require calcium for activity. High performance liquid chromatography and 13C-nuclear magnetic resonance indicated that NdIS catalyzed the hydrolysis and β-(2→1)-transfructosylation of sucrose to synthesize β-(2→1)-fructan with chain lengths of 42 or more residues. The rate dependence on sucrose concentration followed hydrolysis-transglycosylation kinetics, and a 50% transglycosylation ratio was obtained at 344 m m sucrose. These results suggest that transfructosylation from sucrose to β-(2→1)-fructan occurs predominantly to elongate the fructan chain because sucrose is an unfavorable acceptor.
  • Tomoya Ota, Wataru Saburi, Shiro Komba, Haruhide Mori
    Bioscience, biotechnology, and biochemistry 2023年07月05日 [査読有り][通常論文]
     
    β1-3/1-6 Glucans, known for their diverse structures, comprise a β1-3-linked main chain and β1-6-linked short branches. Laminarin, a β1-3/1-6 glucan extracted from brown seaweed, for instance, includes β1-6 linkages even in the main chain. The diverse structures provide various beneficial functions of the glucan. To investigate the relationship between structure and functionality, and to enable the characterization of β1-3/1-6 glucan-metabolizing enzymes, oligosaccharides containing exact structures of β1-3/1-6 glucans are required. We synthesized the monomeric units for the synthesis of β1-3/1-6 mixed-linked glucooligosaccharides. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside served as an acceptor in the formation of β1-3 linkages. Phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldiphenylsilyl)-1-thio-β-d-glucopyranoside and phenyl 2,3-di-O-benzoyl-4,6-di-O-levulinyl-1-thio-β-d-glucopyranoside acted as donors, synthesizing acceptors suitable for the formation of β1-3- and β1-6-linkages, respectively. These were used to synthesize a derivative of Glcβ1-6Glcβ1-3Glcβ1-3Glc, demonstrating that the proposed route can be applied to synthesize the main chain of β-glucan, with the inclusion of both β1-3 and β1-6 linkages.
  • Hang Wang, Xiaomei Sun, Wataru Saburi, Saki Hashiguchi, Jian Yu, Toyoyuki Ose, Haruhide Mori, Min Yao
    Acta crystallographica. Section D, Structural biology 79 Pt 7 585 - 595 2023年07月01日 [査読有り][通常論文]
     
    Mannose 2-epimerase (ME), a member of the acylglucosamine 2-epimerase (AGE) superfamily that catalyzes epimerization of D-mannose and D-glucose, has recently been characterized to have potential for D-mannose production. However, the substrate-recognition and catalytic mechanism of ME remains unknown. In this study, structures of Runella slithyformis ME (RsME) and its D254A mutant [RsME(D254A)] were determined in their apo forms and as intermediate-analog complexes [RsME-D-glucitol and RsME(D254A)-D-glucitol]. RsME possesses the (α/α)6-barrel of the AGE superfamily members but has a unique pocket-covering long loop (loopα7-α8). The RsME-D-glucitol structure showed that loopα7-α8 moves towards D-glucitol and closes the active pocket. Trp251 and Asp254 in loopα7-α8 are only conserved in MEs and interact with D-glucitol. Kinetic analyses of the mutants confirmed the importance of these residues for RsME activity. Moreover, the structures of RsME(D254A) and RsME(D254A)-D-glucitol revealed that Asp254 is vital for binding the ligand in a correct conformation and for active-pocket closure. Docking calculations and structural comparison with other 2-epimerases show that the longer loopα7-α8 in RsME causes steric hindrance upon binding to disaccharides. A detailed substrate-recognition and catalytic mechanism for monosaccharide-specific epimerization in RsME has been proposed.
  • Tomoya Ota, Wataru Saburi, Linda Elizabeth Jewell, Tom Hsiang, Ryozo Imai, Haruhide Mori
    Bioscience, biotechnology, and biochemistry 87 7 707 - 716 2023年06月23日 [査読有り][通常論文]
     
    Glycoside hydrolase family 3 (GH3) β-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its β-glucosidase has not been identified. In this study, a GH3 β-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl β-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis = 1.6 m m), and d-glucose caused competitive inhibition (Ki = 0.5 m m). MnBG3A acted on β-glucobioses with β1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to β1-6 linkage. MnBG3A has similar features to those of β-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.
  • Waraporn Auiewiriyanukul, Wataru Saburi, Tomoya Ota, Jian Yu, Koji Kato, Min Yao, Haruhide Mori
    Molecules 28 7 3109 - 3109 2023年03月30日 [査読有り][招待有り]
     
    α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on β→α loop 5 of the catalytic (β/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.
  • Saburi Wataru, Tomoya Ota, Koji Kato, Takayoshi Tagami, Keitaro Yamashita, Min Yao, Haruhide Mori
    Journal of Applied Glycoscience 70 2 43 - 52 2023年03月11日 [査読有り][通常論文]
  • Shu Horikoshi, Wataru Saburi, Jian Yu, Hideyuki Matsuura, James R Ketudat Cairns, Min Yao, Haruhide Mori
    Bioscience, biotechnology, and biochemistry 86 2 231 - 245 2022年01月24日 [査読有り]
     
    Plants possess many glycoside hydrolase family 1 (GH1) β-glucosidases, which physiologically function in cell wall metabolism and activation of bioactive substances, but most remain uncharacterized. One GH1 isoenzyme AtBGlu42 in Arabidopsis thaliana has been identified to hydrolyze scopolin using the gene deficient plants, but no enzymatic properties were obtained. Its sequence similarity to another functionally characterized enzyme Os1BGlu4 in rice suggests that AtBGlu42 also acts on oligosaccharides. Here, we show that the recombinant AtBGlu42 possesses high kcat/Km not only on scopolin, but also on various β-glucosides, cellooligosaccharides, and laminarioligosaccharides. Of the cellooligosaccharides, cellotriose was the most preferred. The crystal structure, determined at 1.7 Å resolution, suggests that Arg342 gives unfavorable binding to cellooligosaccharides at subsite +3. The mutants R342Y and R342A showed the highest preference on cellotetraose or cellopentaose with increased affinities at subsite +3, indicating that the residues at this position have an important role for chain length specificity.
  • Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Wataru Saburi, Rakrudee Sarnthima, Takayoshi Tagami, Masayuki Okuyama, Haruhide Mori, Nobuo Sakairi, Doman Kim, Atsuo Kimura
    Applied microbiology and biotechnology 106 2 689 - 698 2022年01月 [査読有り]
     
    Dextran dextrinase (DDase) catalyzes formation of the polysaccharide dextran from maltodextrin. During the synthesis of dextran, DDase also generates the beneficial material isomaltomegalosaccharide (IMS). The term megalosaccharide is used for a saccharide having DP = 10-100 or 10-200 (DP, degree of polymerization). IMS is a chimeric glucosaccharide comprising α-(1 → 6)- and α-(1 → 4)-linked portions at the nonreducing and reducing ends, respectively, in which the α-(1 → 4)-glucosyl portion originates from maltodextrin of the substrate. In this study, IMS was produced by a practical approach using extracellular DDase (DDext) or cell surface DDase (DDsur) of Gluconobacter oxydans ATCC 11894. DDsur was the original form, so we prepared DDext via secretion from intact cells by incubating with 0.5% G6/G7 (maltohexaose/maltoheptaose); this was followed by generation of IMS from various concentrations of G6/G7 substrate at different temperatures for 96 h. However, IMS synthesis by DDext was limited by insufficient formation of α-(1 → 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of α-(1 → 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 °C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP = 14.7) with 61% α-(1 → 6)-glucosyl content in 51% yield. Increases in substrate concentration and agitation efficiency were found to decrease dextran formation and increase IMS production, which improved the reaction conditions for DDext. Under modified conditions (20% G6/G7, agitation speed of 100 rpm at 45 °C), DDext produced IMS (average DP = 14.5) with 65% α-(1 → 6)-glucosyl content in a good yield of 87%. KEY POINTS: • Beneficial IMS was produced using thermostabilized DDase. • Optimum conditions for reduced dextran formation were successfully determined. • A practical approach was established to provide IMS with a great yield of 87%.
  • Wataru Saburi, Takanori Nihira, Hiroyuki Nakai, Motomitsu Kitaoka, Haruhide Mori
    Scientific Reports 12 1 2022年01月 [査読有り]
     
    AbstractGlycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized Paenibacillus borealis PBOR_28850 protein, belonging to glycoside hydrolase family 94. Screening of acceptor substrates for reverse phosphorolysis, in which α-d-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in Escherichia coli specifically utilized d-galactose as an acceptor and produced solabiose (β-d-Glcp-(1 → 3)-d-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to d-galactose and d-glucose by β-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.
  • Daisuke Tezuka, Hideyuki Matsuura, Wataru Saburi, Haruhide Mori, Ryozo Imai
    Plants 10 9 1875 - 1875 2021年09月10日 [査読有り]
     
    Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.
  • Kensuke Fukui, Wataru Saburi, Masahisa Ibuki, Kazunobu Tsumura, Haruhide Mori
    Food Science and Technology Research 27 2 249 - 257 2021年05月22日 [査読有り][通常論文]
  • 森 春英
    応用糖質科学 10 3 165 - 174 2020年08月20日 [査読有り][招待有り]
  • Yodai Taguchi, Wataru Saburi, Ryozo Imai, Haruhide Mori
    Carbohydrate research 488 107902 - 107902 2020年02月 [査読有り][通常論文]
     
    Trehalose 6-phosphate (Tre6P) is an important intermediate for trehalose biosynthesis. Recent researches have revealed that Tre6P is an endogenous signaling molecule that regulates plant development and stress responses. The necessity of Tre6P in physiological studies is expected to be increasing. To achieve the cost-effective production of Tre6P, a novel approach is required. In this study, we utilized trehalose 6-phosphate phosphorylase (TrePP) from Lactococcus lactis to produce Tre6P. In the reverse phosphorolysis by the TrePP, 91.9 mM Tre6P was produced from 100 mM β-glucose 1-phosphate (β-Glc1P) and 100 mM glucose 6-phosphate (Glc6P). The one-pot reaction of TrePP and maltose phosphorylase (MP) enabled production of 65 mM Tre6P from 100 mM maltose, 100 mM Glc6P, and 20 mM inorganic phosphate. Addition of β-phosphoglucomutase to this reaction produced Glc6P from β-Glc1P and thus reduced requirement of Glc6P as a starting material. Within the range of 20-469 mM inorganic phosphate tested, the 54 mM concentration yielded the highest amount of Tre6P (33 mM). Addition of yeast increased the yield because of its glucose consumption. Finally, from 100 mmol maltose and 60 mmol inorganic phosphate, we successfully achieved production of 37.5 mmol Tre6P in a one-pot reaction (100 mL), and 9.4 g Tre6P dipotassium salt was obtained.
  • Gao Y, Saburi W, Taguchi Y, Mori H
    Bioscience, biotechnology, and biochemistry 1 - 13 2019年07月 [査読有り][通常論文]
  • Saburi W, Sato S, Hashiguchi S, Muto H, Iizuka T, Mori H
    Applied microbiology and biotechnology 2019年06月 [査読有り][通常論文]
  • Tezuka D, Kawamata A, Kato H, Saburi W, Mori H, Imai R
    Plant Physiol Biochem 135 263 - 271 2019年02月 [査読有り][通常論文]
  • Mikiyasu Sakanaka, Shingo Nakakawaji, Shin Nakajima, Satoru Fukiya, Arisa Abe, Wataru Saburi, Haruhide Mori, Atsushi Yokota
    Applied and environmental microbiology 84 17 e00824-18  2018年09月01日 [査読有り][通常論文]
     
    Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for Bifidobacterium longum subsp. longum 105-A (JCM 31944) based on ISBlo11, a native IS3 family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the ISBlo11 transposase. An artificial transposon plasmid, pBFS12, in which ISBlo11 terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS3 elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >103 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in B. longum NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of B. longum subsp. longumIMPORTANCE Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for Bifidobacterium longum subsp. longum, a representative health-promoting Bifidobacterium species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.
  • Auiewiriyanukul W, Saburi W, Kato K, Yao M, Mori H
    FEBS Lett 592 13 2268 - 2281 2018年07月 [査読有り][通常論文]
  • Wataru Saburi, Nongluck Jaito, Koji Kato, Yuka Tanaka, Min Yao, Haruhide Mori
    Biochimie 144 63 - 73 2018年01月01日 [査読有り][通常論文]
     
    D-Mannose isomerase (MI) reversibly isomerizes D-mannose to D-fructose, and is attractive for producing D-mannose from inexpensive D-fructose. It belongs to the N-acylglucosamine 2-epimerase (AGE) superfamily along with AGE, cellobiose 2-epimerase (CE), and aldose-ketose isomerase (AKI). In this study, Marinomonas mediterranea Marme_2490, showing low sequence identity with any known enzymes, was found to isomerize D-mannose as its primary substrate. Marme_2490 also isomerized D-lyxose and 4-OH D-mannose derivatives (D-talose and 4-O-monosaccharyl-D-mannose). Its activity for D-lyxose is known in other D-mannose isomerizing enzymes, such as MI and AKI, but we identified, for the first time, its activity for 4-OH D-mannose derivatives. Marme_2490 did not isomerize D-glucose, as known MIs do not, while AKI isomerizes both D-mannose and D-glucose. Thus, Marme_2490 was concluded to be an MI. The initial and equilibrium reaction products were analyzed by NMR to illuminate mechanistic information regarding the Marme_2490 reaction. The analysis of the initial reaction product revealed that β-D-mannose was formed. In the analysis of the equilibrated reaction products in D2O, signals of 2-H of D-mannose and 1-H of D-fructose were clearly detected. This indicates that these protons are not substituted with deuterium from D2O and Marme_2490 catalyzes the intramolecular proton transfer between 1-C and 2-C. The crystal structure of Marme_2490 in a ligand-free form was determined and found that Marme_2490 is formed by an (α/α)6-barrel, which is commonly observed in AGE superfamily enzymes. Despite diverse reaction specificities, the orientations of residues involved in catalysis and substrate binding by Marme_2490 were similar to those in both AKI (Salmonella enterica AKI) and epimerase (Rhodothermus marinus CE). The Marme_2490 structure suggested that the α7→α8 and α11→α12 loops of the catalytic domain participated in the formation of an open substrate-binding site to provide sufficient space to bind 4-OH D-mannose derivatives.
  • Min Ma, Masayuki Okuyama, Megumi Sato, Takayoshi Tagami, Patcharapa Klahan, Yuya Kumagai, Haruhide Mori, Atsuo Kimura
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 101 16 6399 - 6408 2017年08月 [査読有り][通常論文]
     
    Aspergillus niger alpha-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of alpha-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of alpha-(1 -> 6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(alpha 1-4)Glc] yields both alpha-(1 -> 6)- and alpha-(1 -> 4)-glucosidic linkages, the latter constituting similar to 25% of the total transfer reaction product. The maltotriose [Glc(alpha 1-4)Glc(alpha 1-4)Glc], alpha-(1 -> 4)-glucosyl product disappears quickly, whereas the alpha-(1 -> 6)-glucosyl products panose [Glc(alpha 1-6)Glc(alpha 1-4)Glc], isomaltose [Glc(alpha 1-6)Glc], and isomaltotriose [Glc(alpha 1-6)Glc(alpha 1-6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the alpha-(1 -> 4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).
  • Ryo Matsui, Naruki Amano, Kosaku Takahashi, Yodai Taguchi, Wataru Saburi, Hideharu Mori, Norio Kondo, Kazuhiko Matsuda, Hideyuki Matsuura
    SCIENTIFIC REPORTS 7 1 6688  2017年07月 [査読有り][通常論文]
     
    In plants, cis-jasmone (CJ) is synthesized from a-linolenic acid (LA) via two biosynthetic pathways using jasmonic acid (JA) and iso-12-oxo-phytodienoic acid (iso-OPDA) as key intermediates. However, there have been no reports documenting CJ production by microorganisms. In the present study, the production of fungal-derived CJ by Lasiodiplodia theobromae was observed for the first time, although this production was not observed for Botrytis cinerea, Verticillium longisporum, Fusarium oxysporum, Gibberella fujikuroi, and Cochliobolus heterostrophus. To investigate the biosynthetic pathway of CJ in L. theobromae, administration experiments using [18,18,18-H-2(3), 17,17-H-2(2)] LA (LA-d5), [18,18,18-H-2(3), 17,17-H-2(2)]12-oxo-phytodienoic acid (cis-OPDA-d5), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 8:0 (OPC8-d6), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 6:0 (OPC6-d6), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 4:0 (OPC4-d6), and [11,11-H-2(2), 10,10-H-2(2), 8,8-H-2(2), 2,2-H-2(2)] methyl iso-12-oxo-phytodienoate (iso-MeOPDA-d8) were carried out, revealing that the fungus produced CJ through a single biosynthetic pathway via iso-OPDA. Interestingly, it was suggested that the previously predicted decarboxylation step of 3,7-didehydroJA to afford CJ might not be involved in CJ biosynthesis in L. theobromae.
  • β-マンナン分解に寄与するセロビオース２-エピメラーゼとβ-マンノシドホスホリラーゼの構造と機能
    佐分利 亘, 加藤 公児, 姚 閔, 松井 博和, 森 春英
    応用糖質科学 7 2 69 - 75 2017年05月 [査読有り][招待有り]
  • Masayuki Okuyama, Kana Matsunaga, Ken-ichi Watanabe, Keitaro Yamashita, Takayoshi Tagami, Asako Kikuchi, Min Ma, Patcharapa Klahan, Haruhide Mori, Min Yao, Atsuo Kimura
    FEBS J 284 5 766 - 783 2017年03月 [査読有り][通常論文]
     
    The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from alpha-glycosidases can give poor yields of desired products because they require generally unstable beta-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 alpha-galactosidase, using more stable beta-galactosyl azide and alpha-galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using beta-galactosyl azide and alpha-galactosyl transfer from alpha-galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, beta-galactosyl azide and acceptor. A three-dimensional structure of the mutant enzyme in complex with the transglycosylation product, beta-lactosyl alpha-D-galactoside, was solved to elucidate the ligand-binding aspects of the alpha-galactosidase. Subtle differences at the beta ->alpha loops 1, 2 and 3 of the catalytic TIM barrel of the alpha-galactosidase from those of a homologous GH97 alpha-glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in beta ->alpha loop 1 have separate roles. Trp312 of the alpha-galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the alpha-glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of alpha-galactoside recognition is conserved among GH27, 31, 36 and 97 alpha-galactosidases.
  • Yodai Taguchi, Wataru Saburi, Ryozo Imai, Haruhide Mori
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 8 1512 - 1519 2017年 [査読有り][通常論文]
     
    Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce beta-D-glucose 1-phosphate (beta-Glc1P) and D-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40 degrees C, and was stable in the pH range 5.0-8.0 and at up to 30 degrees C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, beta-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, D-mannose 6-phosphate (Man6P). From beta-Glc1P and Man6P, a novel sugar phosphate, alpha-D-Glcp-(1 <-> 1)-alpha-D-Manp6P, was synthesized with 51% yield.
  • Julan Liao, Masayuki Okuyama, Keigo Ishihara, Yoshinori Yamori, Shigeo Iki, Takayoshi Tagami, Haruhide Mori, Seiya Chiba, Atsuo Kimura
    Biosci Biotechnol Biochem 80 9 1747 - 1752 2016年09月 [査読有り][通常論文]
     
    The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl -galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k(cat)/K-m values for hydrolysis of the branched -galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched -galactosides, but not linear raffinose family oligosaccharides.
  • Okuyama M, Saburi W, Mori H, Kimura A
    Cell Mol Life Sci 73 14 2727 - 2751 2016年07月 [査読有り][通常論文]
     
    alpha-Glucosidases (AGases) and alpha-1,4-glucan lyases (GLases) catalyze the degradation of alpha-glucosidic linkages at the non-reducing ends of substrates to release alpha-glucose and anhydrofructose, respectively. The AGases belong to glycoside hydrolase (GH) families 13 and 31, and the GLases belong to GH31 and share the same structural fold with GH31 AGases. GH13 and GH31 AGases show diverse functions upon the hydrolysis of substrates, having linkage specificities and size preferences, as well as upon transglucosylation, forming specific alpha-glucosidic linkages. The crystal structures of both enzymes were determined using free and ligand-bound forms, which enabled us to understand the important structural elements responsible for the diverse functions. A series of mutational approaches revealed features of the structural elements. In particular, amino-acid residues in plus subsites are of significance, because they regulate transglucosylation, which is used in the production of industrially valuable oligosaccharides. The recently solved three-dimensional structure of GLase from red seaweed revealed the amino-acid residues essential for lyase activity and the strict recognition of the alpha-(1 -> 4)-glucosidic substrate linkage. The former was introduced to the GH31 AGase, and the resultant mutant displayed GLase activity. GH13 and GH31 AGases hydrate anhydrofructose to produce glucose, suggesting that AGases are involved in the catabolic pathway used to salvage unutilized anhydrofructose.
  • Yuxin Ye, Wataru Saburi, Rei Odaka, Koji Kato, Naofumi Sakurai, Keisuke Komoda, Mamoru Nishimoto, Motomitsu Kitaoka, Haruhide Mori, Min Yao
    FEBS LETTERS 590 6 828 - 837 2016年03月 [査読有り][通常論文]
     
    In Ruminococcus albus, 4-Omicron-beta-D-mannosyl-D-glucose phosphorylase (RaMP1) and beta-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze beta-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-Omicron-beta-D-mannosyl-D-glucose and RaMP2 with/without beta-(1 -> 4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.
  • Masuda Y, Okuyama M, Iizuka T, Nakai H, Saburi W, Fukukawa T, Maneesan J, Tagami T, Naraoka T, Mori H, Kimura A
    Biosci Biotechnol Biochem 80 3 479 - 485 2016年03月 [査読有り][通常論文]
     
    Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an -glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 degrees C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest V-max/K-m value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.
  • Structural and biochemical studies of plant α-glucosidases with a series of long-chain inhibitors.
    Tagami T, Yamashita K, Okuyama M, Mori H, Yao M, Kimura A
    Bull Appl Glycosci 6 2 103 - 108 2016年 [査読有り][通常論文]
  • Yuki Murakami, Teruyo Ojima-Kato, Wataru Saburi, Haruhide Mori, Hirokazu Matsui, Soichi Tanabe, Takuya Suzuki
    BRITISH JOURNAL OF NUTRITION 114 11 1774 - 1783 2015年12月 [査読有り][通常論文]
     
    Obesity is one of the major health problems throughout the world. The present study investigated the preventive effect of epilactose - a rare non-digestible disaccharide - on obesity and metabolic disorders in mice fed high-fat (HF) diets. Feeding with HF diets increased body weight gain, fat pad weight and adipocyte size in mice (P<0.01), and these increases were effectively prevented by the use of supplemental epilactose without influencing food intake (P<0.01). Caecal pools of SCFA such as acetic and propionic acids in mice fed epilactose were higher compared with mice not receiving epilactose. Supplemental epilactose increased the expression of uncoupling protein (UCP)-1, which enhances energy expenditure, to 2-fold in the gastrocnemius muscle (P=0.04) and to 1.3-fold in the brown adipose tissue (P=0.02) in mice fed HF diets. Feeding HF diets induced pro-inflammatory macrophage infiltration into white adipose tissue, as indicated by the increased expression of monocyte chemotactic protein-1, TNF-alpha and F4/80, and these increases were attenuated by supplemental epilactose. In differentiated myogenic-like C2C12 cells, propionic acid, but not acetic or n-butyric acids, directly enhanced UCP-1 expression by approximately 2-fold (P<0.01). Taken together, these findings indicate that the epilactose-mediated increase in UCP-1 in the skeletal muscle and brown adipose tissue can enhance whole-body energy expenditure, leading to effective prevention of obesity and metabolic disorders in mice fed HF diets. It is suggested that propionic acid - a bacterial metabolite - acts as a mediator to induce UCP-1 expression in skeletal muscles.
  • Hua Y, Ekkhara W, Sansenya S, Srisomsap C, Roytrakul S, Saburi W, Takeda R, Matsuura H, Mori H, Ketudat Cairns JR
    Arch Biochem Biophys 583 36 - 46 2015年10月 [査読有り][通常論文]
     
    Gibberellin 1-O-beta-D-glucose ester hydrolysis activity has been detected in rice seedling extracts, but no enzyme responsible for this activity has ever been purified and identified. Therefore, gibberellin A4 glucosyl ester (GA(4)-GE) beta-D-glucosidase activity was purified from ten-day rice seedling stems and leaves. The family 1 glycoside hydrolase Os4BGlu13 was identified in the final purification fraction. The Os4BGlu13 cDNA was amplified from rice seedlings and expressed as an N-terminal thioredoxin-tagged fusion protein in Escherichia coll. The purified recombinant Os4BGlu13 protein (rOs4BGlu13) had an optimum pH of 4.5, for hydrolysis of p-nitrophenyl beta-D-glucopyranoside (pNPGlc), which was the best substrate identified, with a k(cat)/K-m of 637 mM(-1) s(-1). rOs4BGlu13 hydrolyzed helicin best among natural glycosides tested (K-cat/K-m, of 74.4 mM(-1) s(-1)). Os4BGlu13 was previously designated tuberonic acid glucoside (TAG) beta-glucosidase (TAGG), and here the k(cat)/K-m of rOsBGlu13 for TAG was 6.68 mM(-1) s(-1), while that for GA4-GE was 3.63 mM(-1) s(-1) and for salicylic acid glucoside (SAG) is 0.88 mM(-1) s(-1). rOs4BGlu13 also hydrolyzed oligosaccharides, with preference for short beta-(1 -> 3)-linked over beta-(1 -> 4)linked glucooligosaccharides. The enzymatic data suggests that Os4BGlu13 may contribute to TAG, SAG, oligosaccharide and GA4-GE hydrolysis in the rice plant, although helicin or a similar compound may be its primary target. (C) 2015 Elsevier Inc. All rights reserved.
  • Jang SJ, Sato M, Sato K, Jitsuyama Y, Fujino K, Mori H, Takahashi R, Benitez ER, Liu B, Yamada T, Abe J
    PLoS One 10 6 e0128527  2015年06月 [査読有り][通常論文]
     
    Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L) Merr.), we developed a near-isogenic line (NIL) of a permeable (soft-seeded) cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja) introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of beta-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-beta-glucanase. A single-nucleotide polymorphism (SNP) introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded) NIL into the permeable cultivar Kariyutaka resulted in accumulation of beta-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of beta-1,4-glucan derivatives such as xyloglucan and/or beta-(1,3)(1,4)-glucan that reinforce the impermeability of seed coats in soybean.
  • Saburi W, Tanaka Y, Muto H, Inoue S, Odaka R, Nishimoto M, Kitaoka M, Mori H
    Biosci Biotechnol Biochem 79 6 969 - 977 2015年06月 [査読有り][通常論文]
     
    The aerobic soil bacterium Cellvibrio vulgaris has a beta-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting d-mannose to d-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-beta-d-mannosyl-d-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and beta-(1 -> 4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for beta-(1 -> 4)-mannobiose was 5.5x10(4)-fold higher than it was for d-mannose. Recombinant UnkA phosphorolyzed beta-d-mannosyl-(1 -> 4)-d-glucose and specifically utilized d-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-beta-d-mannosyl-d-glucose phosphorylase.
  • Xing Shen, Wataru Saburi, Zuoqi Gai, Koji Kato, Teruyo Ojima-Kato, Jian Yu, Keisuke Komoda, Yusuke Kido, Hirokazu Matsui, Haruhide Mori, Min Yao
    Acta Crystallogr D Biol Crystallogr 71 Pt 6 1382 - 1391 2015年06月 [査読有り][通常論文]
     
    alpha-Glucosidases, which catalyze the hydrolysis of the alpha-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of alpha-glucosides. Halomonas sp. H11 alpha-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the alpha-(1 -> 4)-linked disaccharide maltose among naturally occurring substrates. Although several three- dimensional structures of GH13 members have been solved, the disaccharide specificity and alpha-(1 -> 4) recognition mechanism of alpha-glucosidase are unclear owing to a lack of corresponding substrate- bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl- enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with d- glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long beta ->alpha loop 4 which exists in alpha-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite + 1. Furthermore, an explanation of the alpha-glucosidase reaction mechanism is proposed based on the glucosyl- enzyme intermediate structure.
  • Wataru Saburi, Hiroaki Rachi-Otsuka, Hironori Hondoh, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    FEBS LETTERS 589 7 865 - 869 2015年03月 [査読有り][通常論文]
     
    Glycoside hydrolase family 13 contains exo-glucosidases specific for alpha-(1 -> 4)- and alpha-(1 -> 6)-linkages including alpha-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The alpha-(1 -> 6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to alpha-(1 -> 4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher k(cat)/K-m for maltose than wild-type, but its k(cat)/K-m for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher k(cat)/K-m for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the alpha-(1 -> 6)-glucosidic linkage. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Momoko Kobayashi, Wataru Saburi, Daichi Nakatsuka, Hironori Hondoh, Koji Kato, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Min Yao
    FEBS LETTERS 589 4 484 - 489 2015年02月 [査読有り][通常論文]
     
    Streptococcus mutans dextran glucosidase (SmDG) belongs to glycoside hydrolase family 13, and catalyzes both the hydrolysis of substrates such as isomaltooligosaccharides and subsequent transglucosylation to form alpha-(1 -> 6)-glucosidic linkage at the substrate non-reducing ends. Here, we report the 2.4 angstrom resolution crystal structure of glucosyl-enzyme intermediate of SmDG. In the obtained structure, the Trp238 side-chain that constitutes the substrate-binding site turned away from the active pocket, concurrently with conformational changes of the nucleophile and the acid/base residues. Different conformations of Trp238 in each reaction stage indicated its flexibility. Considering the results of kinetic analyses, such flexibility may reflect a requirement for the reaction mechanism of SmDG. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • 竹松 知紀, 瀬戸 義哉, 宮澤 吉郎, 和久田 真司, 荻原 毅, 佐分利 亘, 森 春英, 高橋 公咲, 松浦 英幸
    天然有機化合物討論会講演要旨集 57 Oral14  天然有機化合物討論会実行委員会 2015年 

    【目 的】

    植物は大地に根を張り移動が困難である事から、自己の被る様々な環境要因，ストレスに適応して生活環を終結させなくてはならない。よって、植物は独自の環境応答機構を有している。この応答機構を司る生理活性物質として植物ホルモンと呼称される一群の化合物が知られ、多くの研究がなされてきた。我々の研究室では長年、植物ホルモンの一種であるジャスモン酸(JA)について、鋭意研究を行っている。JA類は植物の傷害応答シグナル物質として必須であり、他の興味ある生理活性としてバレイショ塊茎の誘導活性、植物の就眠運動における就眠誘導、花粉管伸長促進などが知られている。近年、JA類の生理活性の制御の観点から、配糖化、チトクロームP450による酸化などに注目した報告がある。

    我々はJA類の配糖体化に関して研究を進め、イネに含まれるOsSGTを12-hydroxyJA(12-OHJA)配糖化酵素として報告した[1]。OsSGT は糖供与体としてUDP-Glcを用いるが、12-OHJAよりサリチル酸(SA)に対してより高い糖転位活性を示した。しかしながら、本酵素の探索過程において、SAに対してほとんど配糖化活性を示さず、12-OHJAに特異性の高いUDP-Glc非依存性の配糖化酵素の存在も明らかとした。現在のところ、UDP-Glc非依存性の植物2次代謝産物を標的とした配糖化酵素に関しては松葉らの報告[2]があるのみであり、植物ホルモンの配糖体化に関してUDP-Glc非依存性の糖転位酵素が関与しているとの報告は一切無い。

    上記の興味ある発見の詳細を明らかとすべく我々は、1）イネカルスに含まれるUDP-Glc非依存性の配糖化酵素の精製、2）イネカルスに含まれる本配糖化酵素の糖供与体の単離精製、構造決定,3）本配糖化酵素の諸性質の究明を進めたところ、本配糖化酵素はb−glucosydase, OsBGlu1 （accession number: AK100165）であると突き止め、イネにおいてサリチル酸グルコシド(SAG)が糖供与体として働くことを明らかとした(図1)。本酵素の生理学的意義は水酸化JA類を配糖化しJAの活性調節に寄与していると示唆できた事から、本成果について報告する。

    【研究方法および結果】

    1. UDP-Glc非依存性配糖化酵素の発見

    イネカルス粗酵素溶液にUDP-Glcを糖供与体としない12-OHJA 選択的な配糖化酵素が存在する事を、市販の糖供与体を用い、以下の実験より発見した。糖転位反応液としてイネカルスより抽出した粗酵素溶液、糖受容体としてJA水酸化体の一種である12-OHJA、糖供与体として市販のGlc 1-phosphate, TDP-Glc, octyl-Glc, UDP-Glcを用いて実験を行った。その結果、octyl-Glc はUDP-Glcよりも12-OHJAにより選択的に糖を供与し、SAはほとんど配糖化されなかった。

    2. イネ由来のUDP-Glc非依存性配糖化酵素の同定

    12-OHJA選択的糖転位活性を指標に、イネカルス粗酵素溶液に含まれる糖転位酵素の同定を試みた。糖供与体としてoctyl-Glc、糖受容体として12-OHJAを用い、種々の精製操作の後、得られた精製タンパク質はペプチドマスフィンガープ

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  • Sadahiro J, Mori H, Saburi W, Okuyama M, Kimura A
    Biochem Biophys Res Commun 456 1 500 - 505 2015年 [査読有り][通常論文]
     
    Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DDext) and an intracellular DDase (DDint), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DDext and DDint were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DDint was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DDext), and the cell-associated form (DDint) appeared after depletion of glucose from the medium. (C) 2014 Elsevier Inc. All rights reserved.
  • Wataru Saburi, Masayuki Okuyama, Yuya Kumagai, Atsuo Kimura, Haruhide Mori
    BIOCHIMIE 108 140 - 148 2015年01月 [査読有り][通常論文]
     
    alpha-Glucosidases are ubiquitous enzymes that hydrolyze the alpha-glucosidic linkage at the non-reducing end of substrates. In this study, we characterized an alpha-glucosidase (BspAG31A) belonging to glycoside hydrolase family 31 from Bacillus sp. AHU 2001. Recombinant B5pAG31A, produced in Escherichia colt, had high hydrolytic activity toward maltooligosaccharides, kojibiose, nigerose, and neotrehalose. This is the first report of an alpha-glucosidase with high activity toward neotrehalose. The transglucosylation products, nigerose, kojibiose, isomaltose, and neotrehalose, were generated from 440 mm maltose. Substitution of Tyr268, situated on the beta -> alpha loop 1 of B5pAG31A, with Trp increased hydrolytic activity toward isomaltose. This mutation reduced the hydrolytic activity toward maltooligosaccharides more than toward kojibiose, nigerose, and neotrehalose. Analysis of the Y173A mutant of B5pAG31A showed that Tyr173, situated on the N-terminal domain loop, is associated with the formation of subsite +2. In Y173A, the k(cad)/K-m for maltooligosaccharides slightly decreased with an increasing degree of polymerization compared with wild type. Among the amino acid residues surrounding the substrate binding site, Va1543 and Glu545 of B5pAG31A were different from the corresponding residues of Bacillus thermoamyloliquefaciens alpha-glucosidase II, which has higher activity toward isomaltose than B5pAG31A. The E545G mutation slightly enhanced isomaltase activity without a large reduction of hydrolytic activities toward other substrates. V543A showed 1.8-3.5-fold higher hydrolytic activities toward all substrates other than neotrehalose compared with wild type, although its preference for isomaltose was unchanged. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.
  • Tagami T, Yamashita K, Okuyama M, Mori H, Yao M, Kimura A
    J Biol Chem 290 3 1796 - 1803 2015年01月 [査読有り][通常論文]
     
    The alpha-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower K-m and higher k(cat)/K-m for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligo-saccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of alpha-glycosidases. The clear correlation obtained between log K-i of the acarviosyl-maltooligosaccharides and log(K-m/k(cat)) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the K-m for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.
  • Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Janjira Maneesan, Masayuki Okuyama, Haruhide Mori, Nobuo Sakairi, Atsuo Kimura
    BIORESOURCE TECHNOLOGY 169 518 - 524 2014年10月 [査読有り][通常論文]
     
    Intermolecular interaction of linear-type alpha-(1 -> 6)-glucosyl megalosaccharide rich (L-IMS) and water-insoluble anionic ethyl red was firstly characterized in a comparison with inclusion complexation by cyclodextrins (CDs) to overcome the problem of poor solubility and bioavailability. Phase solubility studies indicated an enhancement of 3- and 9-fold over the solubility in water upon the presence of L-IMS and beta-CD, respectively. H-1 NMR and circular dichrosim spectra revealed the dye forms consisted of 1:1 stoichiometric inclusion complex within the beta-CD cavity, whereas they exhibited non-specific hydrophobic interaction, identified by solvent polarity changes, with L-IMS. The inclusion complex delivered by beta-CD showed an uncompetitive inhibitory-type effect to azoreductase, particularly with high water content that did not promote dye liberation. Addition of the solid dye dispersed into coupled-enzyme reaction system supplied by L-IMS as the dye solubilizer provided usual degradation rate. The dye intermission in series exhibited successful removal with at least 5 cycles was economically feasible. (C) 2014 Elsevier Ltd. All rights reserved.
  • Masayuki Okuyama, Takuya Yoshida, Hironori Hondoh, Haruhide Mori, Min Yao, Atsuo Kimura
    FEBS LETTERS 588 17 3213 - 3217 2014年08月 [査読有り][通常論文]
     
    The role of calcium ion in the active site of the inverting glycoside hydrolase family 97 enzyme, BtGH97a, was investigated through structural and kinetic studies. The calcium ion was likely directly involved in the catalytic reaction. The pH dependence of k(cat)/K-m values in the presence or absence of calcium ion indicated that the calcium ion lowered the pK(a) of the base catalyst. The significant decreases in k(cat)/K-m for hydrolysis of substrates with basic leaving groups in the absence of calcium ion confirmed that the calcium ion facilitated the leaving group departure. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Naoya Tamamura, Wataru Saburi, Atsushi Mukai, Naoki Morimoto, Toshihiko Takehana, Seiji Koike, Hirokazu Matsui, Haruhide Mori
    BIOCHEMICAL ENGINEERING JOURNAL 86 8 - 15 2014年05月 [査読有り][通常論文]
     
    The hydrolytic activity of a thermophilic alkalophilic alpha-amylase from Bacillus sp. AAH-31 (AmyL) toward soluble starch was enhanced through optimization of amino acid (aa) residues situated near the substrate binding site. Twenty-four selected aa residues were replaced with Ala, and Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of AmyL's aa sequence with related enzymes. Y426A, H431A,I509A, and K549A showed notably higher activity than the wild type at 162-254% of wildtype activity. Tyr426, His431, and Ile509 were predicted to be located near subsite -2, while Lys549 was near subsite +2. Ser, Ala, Ala, and Met were found to be the best aa residues for the positions of Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single mutations at distant positions were effective in enhancing catalytic activity. The double-mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of the selected single mutations, showed 340%, 252%, and 271% of wild type activity, respectively. Triple and quadruple-mutant enzymes of the selected mutations did not show higher activity than the best double-mutant, Y426S/K549M. (C) 2014 Elsevier B.V. All rights reserved.
  • Shen X, Saburi W, Gai ZQ, Komoda K, Yu J, Ojima-Kato T, Kido Y, Matsui H, Mori H, Yao M
    Acta crystallographica. Section F, Structural biology communications 70 Pt 4 464 - 466 2014年04月 [査読有り][通常論文]
     
    The α-glucosidase HaG from the halophilic bacterium Halomonas sp. strain H11 catalyzes the hydrolysis of the glucosidic linkage at the nonreducing end of α-glucosides, such as maltose and sucrose, to release α-glucose. Based on its amino-acid sequence, this enzyme is classified as a member of glycoside hydrolase family 13. HaG has three unique characteristics: (i) a very narrow substrate specificity, almost exclusively hydrolyzing disaccharides; (ii) activation by monovalent cations, such as K(+), Rb(+), Cs(+) and NH4(+); and (iii) high transfer activity of the glucose moiety to the OH group of low-molecular-weight compounds, including glycerol and 6-gingerol. Crystallographic studies have been performed in order to understand these special features. An expression vector was constructed and recombinant HaG protein was overexpressed, purified and crystallized. A data set to 2.15 Å resolution was collected and processed. The crystal belonged to space group P212121, with unit-cell parameters a = 60.2, b = 119.2, c = 177.2 Å. The structure has been determined by molecular replacement using the isomaltulose synthase PalI as the search model (PDB entry 1m53).
  • Fujiwara T, Saburi W, Matsui H, Mori H, Yao M
    The Journal of biological chemistry 289 6 3405 - 3415 American Society for Biochemistry and Molecular Biology (ASBMB) 2014年02月 [査読有り][通常論文]
     
    Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of β1,4-mannobiose to 4-O-β-d-mannosyl-d-glucose in mannan metabolism. However, the detailed catalytic mechanism of CE is unclear due to the lack of structural data in complex with ligands. We determined the crystal structures of halothermophile Rhodothermus marinus CE (RmCE) in complex with substrates/products or intermediate analogs, and its apo form. The structures in complex with the substrates/products indicated that the residues in the β5-β6 loop as well as those in the inner six helices form the catalytic site. Trp-322 and Trp-385 interact with reducing and non-reducing end parts of these ligands, respectively, by stacking interactions. The architecture of the catalytic site also provided insights into the mechanism of reversible epimerization. His-259 abstracts the H2 proton of the d-mannose residue at the reducing end, and consistently forms the cis-enediol intermediate by facilitated depolarization of the 2-OH group mediated by hydrogen bonding interaction with His-200. His-390 subsequently donates the proton to the C2 atom of the intermediate to form a d-glucose residue. The reverse reaction is mediated by these three histidines with the inverse roles of acid/base catalysts. The conformation of cellobiitol demonstrated that the deprotonation/reprotonation step is coupled with rotation of the C2-C3 bond of the open form of the ligand. Moreover, it is postulated that His-390 is closely related to ring opening/closure by transferring a proton between the O5 and O1 atoms of the ligand.
  • Maneesan J, Matsuura H, Tagami T, Mori H, Kimura A
    Biosci Biotechnol Biochem 78 12 2064 - 2068 2014年 [査読有り][通常論文]
  • Nongluck Jaito, Wataru Saburi, Rei Odaka, Yusuke Kido, Ken Hamura, Mamoru Nishimoto, Motomitsu Kitaoka, Hirokazu Matsui, Haruhide Mori
    Bioscience, Biotechnology and Biochemistry 78 2 263 - 270 Taylor & Francis 2014年01月01日 [査読無し][通常論文]
     
    © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry.4-O-β-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-β-D-mannosyl-D-glucose (Man-Glc) to α-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts β-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-β-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75°C, and it was stable between pH 5.5-8.3 and below 80°C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-D-glucose and D-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl β-D-glucoside and 1,5-anhydro-D-glucitol as acceptor substrates.
  • Weeranuch Lang, Sarote Sirisansaneeyakul, Ligia O. Martins, Lukana Ngiwsara, Nobuo Sakairi, Wasu Pathom-aree, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    JOURNAL OF ENVIRONMENTAL MANAGEMENT 132 155 - 164 2014年01月 [査読有り][通常論文]
     
    This study reports the characterization of the ability of Dermacoccus spp. isolated from the deepest point of the world's oceans for azo dye decolorization. A detailed investigation of Dermacoccus abyssi MT1.1(T) with respect to the azoreductase activity and enzymatic mechanism as well as the potential role of the bacterial strain for biocleaning of industrial dye baths is reported. Resting cells with oxygen-insensitive azoreductase resulted in the rapid decolorization of the polysulfonated dye Brilliant Black BN (BBN) which is a common food colorant. The highest specific decolorization rate (nu(s)) was found at 50 degrees C with a moderately thermal tolerance for over 1 h. Kinetic analysis showed the high rates and strong affinity of the enzymatic system for the dye with a V-max = 137 mg/g cell/h and a Km = 19 mg/L. The degradation of BBN produces an initial orange intermediate, 8-amino-5-((4-sulfonatophenyl)diazenyl)naphthalene-2-sulfonic acid, identified by mass spectrometry which is later converted to 4-aminobenzene sulfonic acid. Nearly 80% of the maximum nu(s) is possible achieved in resting cell treatment with the salinity increased up to 5.0% NaCl in reaction media. Therefore, this bacterial system has potential for dye decolorization bioprocesses occurring at high temperature and salt concentrations e.g. for cleaning dye-containing saline wastewaters. (C) 2013 Elsevier Ltd. All rights reserved.
  • Wataru Saburi, Momoko Kobayashi, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 44 31670 - 31677 2013年11月 [査読有り][通常論文]
     
    Dextran glucosidase from Streptococcus mutans (SmDG) catalyzes the hydrolysis of an alpha-1,6-glucosidic linkage at the non-reducing end of isomaltooligosaccharides and dextran. This enzyme has an Asp-194 catalytic nucleophile and two catalytically unrelated Cys residues, Cys-129 and Cys-532. Cys-free SmDG was constructed by replacement with Ser (C129S/C532S (2CS), the activity of which was the same as that of the wild type, SmDG). The nucleophile mutant of 2CS was generated by substitution of Asp-194 with Cys (D194C-2CS). The hydrolytic activity of D194C-2CS was 8.1 x 10(-4) % of 2CS. KI-associated oxidation of D194C-2CS increased the activity up to 0.27% of 2CS, which was 330 times higher than D194C-2CS. Peptide-mapping mass analysis of the oxidized D194C-2CS (Ox-D194C-2CS) revealed that Cys-194 was converted into cysteine sulfinate. Ox-D194C-2CS and 2CS shared the same properties (optimum pH, pI, and substrate specificity), whereas Ox-D194C-2CS had much higher transglucosylation activity than 2CS. This is the first study indicating that a more acidic nucleophile (-SOO-) enhances transglycosylation. The introduction of cysteine sulfinate as a catalytic nucleophile could be a novel approach to enhance transglycosylation.
  • Sawano T, Saburi W, Hamura K, Matsui H, Mori H
    The FEBS journal 280 18 4463 - 4473 Wiley-Blackwell 2013年09月 [査読有り][通常論文]
     
    Ruminococcus albus has the ability to intracellularly degrade cello-oligosaccharides primarily via phosphorolysis. In this study, the enzymatic characteristics of R. albus cellodextrin phosphorylase (RaCDP), which is a member of glycoside hydrolase family 94, was investigated. RaCDP catalyzes the phosphorolysis of cellotriose through an ordered 'bi bi' mechanism in which cellotriose binds to RaCDP before inorganic phosphate, and then cellobiose and glucose 1-phosphate (Glc1P) are released in that order. Among the cello-oligosaccharides tested, RaCDP had the highest phosphorolytic and synthetic activities towards cellohexaose and cellopentaose, respectively. RaCDP successively transferred glucosyl residues from Glc1P to the growing cello-oligosaccharide chain, and insoluble cello-oligosaccharides comprising a mean of eight residues were produced. Sophorose, laminaribiose, β-1,4-xylobiose, β-1,4-mannobiose and cellobiitol served as acceptors for RaCDP. RaCDP had very low affinity for phosphate groups in both the phosphorolysis and synthesis directions. A sequence comparison revealed that RaCDP has Gln at position 646 where His is normally conserved in the phosphate binding sites of related enzymes. A Q646H mutant showed approximately twofold lower apparent Km values for inorganic phosphate and Glc1P than the wild-type. RaCDP has Phe at position 633 corresponding to Tyr and Val in the +1 subsites of cellobiose phosphorylase and N,N′-diacetylchitobiose phosphorylase, respectively. A F633Y mutant showed higher preference for cellobiose over β-1,4-mannobiose as an acceptor substrate in the synthetic reaction than the wild-type. Furthermore, the F633Y mutant showed 75- and 1100-fold lower apparent Km values for inorganic phosphate and Glc1P, respectively, in phosphorolysis and synthesis of cellotriose.
  • Hamura K, Saburi W, Matsui H, Mori H
    Carbohydrate research 379 21 - 25 Elsevier sci ltd 2013年09月 [査読有り][通常論文]
     
    Cellobiose phosphorylase (EC 2.4.1.20, CBP) catalyzes the reversible phosphorolysis of cellobiose to alpha-D-glucose 1-phosphate (Glc1P) and D-glucose. Cys485, Tyr648, and Glu653 of CBP from Ruminococcus albus, situated at the +1 subsite, were mutated to modulate acceptor specificity. C485A, Y648F, and Y648V were active enough for analysis. Their acceptor specificities were compared with the wild type based on the apparent kinetic parameters determined in the presence of 10 mM Glc1P. C485A showed higher preference for D-glucosamine than the wild type. Apparent k(cat)/K-m values of Y648F for D-mannose and 2-deoxy-D-glucose were 8.2- and 4.0-fold higher than those of the wild type, respectively. Y648V had synthetic activity toward N-acetyl-D-glucosamine, while the other variants did not. The oligosaccharide production in the presence of the same concentrations of wild type and each mutant was compared. C485A produced 4-O-beta-D-glucopyranosyl-D-glucosamine from 10 mM Glc1P and D-glucosamine at a rate similar to the wild type. Y648F and Y648V produced 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine much more rapidly than the wild type when D-mannose and N-acetyl-D-glucosamine were used as acceptors, respectively. After a 4 h reaction, the amounts of 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively. (C) 2013 Elsevier Ltd. All rights reserved.
  • Takatsugu Miyazaki, Megumi Ichikawa, Gaku Yokoi, Motomitsu Kitaoka, Haruhide Mori, Yoshikazu Kitano, Atsushi Nishikawa, Takashi Tonozuka
    FEBS Journal 280 4560 - 4571 2013年09月01日 [査読無し][通常論文]
     
    Proteins belonging to glycoside hydrolase family 63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing α-glucosidase I, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichia coli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with β-d-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O-α-d-glucopyranosyl-α-d-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK. Database The coordinates and structure factors for E727A-Glc12Gal and D324N-melibiose have been deposited in the Protein Data Bank under accession numbers 3W7W and 3W7X, respectively We converted a glycoside hydrolase family 63 enzyme, Escherichia coli YgjK, to a glycosynthase to probe its substrate specificity. The reactions with β-d-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A indicated that the disaccharide bound at the active site was 2-O-α-d-glucopyranosyl-d-galactopyranose. © 2013 FEBS.
  • Kyung-Mo Song, Masayuki Okuyama, Mariko Nishimura, Takayoshi Tagami, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 8 1759 - 1765 2013年08月 [査読有り][通常論文]
     
    The specificity for the alpha-1,4- and alpha-1,6-glucosidic linkages varies among glycoside hydrolase family 31 alpha-glucosidases. This difference in substrate specificity has been considered to be due to the difference in an aromatic residue on beta ->alpha loop 1 in the catalytic domain with a (beta/alpha)(8) barrel fold; i.e., the enzymes having Tyr and Trp on beta ->alpha loop 1 were respectively described as alpha-1,4-specific and alpha-1,6-specific alpha-glucosidases. Schwanniomyees oceidentalis alpha-glucosidase, however, prefers the alpha-1,4-glucosidic linkage, although the enzyme possesses Trp324 at the corresponding position. The mutation of Trp324 to Tyr decreased the ability for hydrolysis of the alpha-1,6-glucosidic linkage and formation of the alpha-1,6-glucosidic linkage in transglycosylation, indicating Trp324 to be closely associated with alpha-1,6 specificity, even if the enzyme preferred the alpha-1,4-glucosidic linkage. The mutant enzyme was found to catalyze the production of the branched oligosaccharide, 2,4-di-O-(alpha-D-glucopyranosyl)-D-glucopyranose, more efficiently than the wild-type enzyme.
  • Nami Himeno, Wataru Saburi, Shinji Wakuta, Ryosuke Takeda, Hideyuki Matsuura, Kensuke Nabeta, Sompong Sansenya, James R, Ketudat Cairns, Haruhide Mori, Ryozo Imai, Hirokazu Matsui
    Bioscience, Biotechnology and Biochemistry 77 5 934 - 939 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2013年06月12日 [査読有り][通常論文]
     
    β-Glucosidases (EC 3.2.1.21) split β-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release β-D-glucose. One of the important functions of plant β-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonaterelated compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid β-D-glucoside in rice (Oryza sativa L.). The expression of OsTAGG2 is upregulated by wounding and by methyl jasmonate, suggesting that this isozyme is involved in responses to biotic stresses and wounding, but the physiological substrate of OsTAGG2 remains ambiguous. In this study, we produced recombinant OsTAGG2 in Pichia pastoris (rOsTAGG2P), and investigated its substrate specificity in detail. From 1L of culture medium, 2.1mg of purified recombinant enzyme was obtained by ammonium sulfate precipitation and Ni-chelating column chromatography. The specific activity of rOsTAGG2P (182 U/mg) was close to that of the native enzyme (171 U/mg), unlike recombinant OsTAGG2 produced in Escherichia coli, which had approximately 3-fold lower specific activity than the native enzyme. The optimum pH and temperature for rOsTAGG2P were pH 3.4 and 60 βC. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4-9.8 and below 55 βC. Native OsTAGG2 and rOsTAGG2P showed 4.5-4.7-fold higher activities towards salicylic acid β-D-glucoside, an inactive storageform of salicylic acid, than towards tuberonic acid β-Dglucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity.
  • Takayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 26 19296 - 19303 2013年06月 [査読有り][通常論文]
     
    Sugar beet alpha-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher k(cat)/K-m values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific alpha-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 angstrom resolution. The active site pocket of SBG is formed by a (beta/alpha)(8) barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (beta/alpha)(8) barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.
  • Fujiwara T, Saburi W, Inoue S, Mori H, Matsui H, Tanaka I, Yao M
    FEBS letters 587 7 840 - 846 2013年04月 [査読有り][通常論文]
  • Takayoshi Tagami, Yoshiyuki Tanaka, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 2 312 - 319 2013年02月 [査読有り][通常論文]
     
    Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrolases. Its derivatives, acarviosyl-maltooligosaccharides, which have longer maltooligosaccharide parts than the maltose unit of acarbose, were synthesized using a disproportionating enzyme partially purified from adzuki cotyledons. The enzyme was identified as a typical type-1 disproportionating enzyme (DPE1) by primary structure analysis. It produced six compounds from 100 mm acarbose and 7.5% (w/v) of maltotetraose-rich syrup. The masses of the six products were confirmed to accord with acarviosyl-maltooligosaccharides with the degrees of polymerization = 5-10 (AC5-AC10) by electrospray ionization mass spectrometry. H-1 and C-13 NMR spectra indicated that AC5-AC10 were alpha-acarviosyl-(1 -> 4)maltooligosaccharide, which have maltotriose-maltooctaose respectively in the maltooligosaccharide part. A predominance of AC7 in the products at the early stage of the reaction indicated that DPE1 catalyzes the transfer of the acarviosyl-glucose moiety from acarbose to the acceptors. ACn can be useful tools as new inhibitors of glycoside hydrolases.
  • Ojima T, Saburi W, Yamamoto T, Mori H, Matsui H
    Bioscience, biotechnology, and biochemistry 77 1 189 - 193 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2013年 [査読有り][通常論文]
     
    Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of β-1,4-linked oligosaccharides to D-mannose residues. In this study, we characterized CE-like proteins from various aerobes (Flavobacterium johnsoniae NBRC 14942, Pedobacter heparinus NBRC 12017, Dyadobacter fermentans ATCC 700827, Herpetosiphon aurantiacus ATCC 23779, Saccharophagus degradans ATCC 43961, Spirosoma linguale ATCC 33905, and Teredinibacter turnerae ATCC 39867), because aerobes, more easily cultured on a large scale than anaerobes, are applicable in industrial processes. The recombinant CE-like proteins produced in Escherichia coli catalyzed epimerization at the C2 position of cellobiose, lactose, epilactose, and β-1,4-mannobiose, whereas N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, D-glucose, and D-mannose were inert as substrates. All the CEs, except for P. heparinus CE, the optimum pH of which was 6.3, showed highest activity at weakly alkaline pH. CEs from D. fermentans, H. aurantiacus, and S. linguale showed higher optimum temperatures and thermostability than the other enzymes analyzed. The enzymes from D. fermentans, S. linguale, and T. turnerae showed significantly high k cat and K m values towards cellobiose and lactose. Especially, T. turnerae CE showed a very high k cat value towards lactose, an attractive property for the industrial production of epilactose, which is carried out at high substrate concentrations.
  • Saburi W, Morimoto N, Mukai A, Kim DH, Takehana T, Koike S, Matsui H, Mori H
    Bioscience, biotechnology, and biochemistry 77 9 1867 - 1873 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2013年 [査読有り][通常論文]
     
    α-Amylases (EC 3.2.1.1) hydrolyze internal α-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic α-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial α-amylases. In this study, the AmyL gene was cloned to determine its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. AmyL shows no hydrolytic activity towards pullulan, but the central region of AmyL (Gly395-Asp684) was similar to neopullulanase-like α-amylases. In contrast to known neopullulanase-like α-amylases, the N-terminal region (Gln29-Phe102) of AmyL was similar to carbohydrate-binding module family 20 (CBM20), which is involved in the binding of enzymes to starch granules. Recombinant AmyL showed more than 95% of its maximum activity in a pH range of 8.2-10.5, and was stable below 65 °C and from pH 6.4 to 11.9. The kcat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s(-1), 67.6 s(-1), and 5.33 s(-1), respectively, and the Km values were 0.100 mg/mL, 0.348 mM, and 2.06 mM, respectively. Recombinant AmyL did not bind to starch granules. But the substitution of Trp45 and Trp84, conserved in site 1 of CBM20, with Ala reduced affinity to soluble starch, while the mutations did not affect affinity for oligosaccharides. Substitution of Trp61, conserved in site 2 of CBM20, with Ala enhanced hydrolytic activity towards soluble starch, indicating that site 2 of AmyL does not contribute to binding to soluble long-chain substrates.
  • Aki Shinoki, Weeranuch Lang, Charin Thawornkuno, Hee-Kwon Kang, Yuya Kumagai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka, Hiroshi Hara
    FOOD CHEMISTRY 136 2 293 - 296 2013年01月 [査読有り][通常論文]
     
    The presence of an alpha-1,6-glucosaccharide enhances absorption of water-soluble quercetin glycosides, a mixture of quercetin-3-O-beta-D-glucoside (Q3G, 31.8%), mono (23.3%), di (20.3%) and more D-glucose adducts with alpha-1,4-linkage to a D-glucose moiety of Q3G, in a ligated small intestinal loop of anesthetized rats. We prepared alpha-1,6-glucosaccharides with different degrees of polymerization (DP) enzymatically and separated them into a megalo-isomaltosaccharide-containing fraction (M-IM, average DP = 11.0) and an oligo-isomaltosaccharide-containing fraction (O-IM, average DP = 3.6). Luminal injection of either saccharide fraction promoted the absorption of total quercetin-derivatives from the small intestinal segment and this effect was greater for M-IM than O-IM addition. M-IM also increased Q3G, but not the quercetin aglycone, concentration in the water-phase of the luminal contents more strongly than O-IM. The enhancement of Q3G solubilization in the luminal contents may be responsible for the increases in the quercetin glucoside absorption promoted by alpha-1,6-glucosaccharides, especially that by M-IM. These results suggest that the ingestion of alpha-1,6-glucosaccharides promotes Q3G bioavailability. (C) 2012 Elsevier Ltd. All rights reserved.
  • TAGAMI Takayoshi, OKUYAMA Masayuki, NAKAI Hiroyuki, KIM Young-min, MORI Haruhide, TAGUCHI Kazunori, SVENSSON Birte, KIMURA Atsuo
    Biochimica et Biophysica Acta 1834 1 329 - 335 2013年01月 [査読有り][通常論文]
     
    Glycoside hydrolase family 31 alpha-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger alpha-glucosidase (ANG) is specific for short-chain substrates with the highest k(cat)/K-m for maltotriose, while sugar beet alpha-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k(cat)/K-m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K-m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K-m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AG5. (C) 2012 Elsevier B.V. All rights reserved.
  • Kyung-Mo Song, Masayuki Okuyama, Kazuyuki Kobayashi, Haruhide Mori, Atsuo Kimura
    Bioscience, Biotechnology and Biochemistry 77 10 2117 - 2124 2013年 [査読有り][通常論文]
     
    For Podospora anserina, several studies of cellulolytic enzymes have been established, but characteristics of amylolytic enzymes are not well understood. When P. anserina grew in starch as carbon source, it accumulated glucose, nigerose, and maltose in the culture supernatant. At the same time, the fungus secreted α-glucosidase (PAG). PAG was purified from the culture supernatant, and was found to convert soluble starch to nigerose and maltose. The recombinant enzyme with C-terminal His-tag (rPAG) was produced with Pichia pastoris. Most rPAG produced under standard conditions lost its affinity for nickel-chelating resin, but the affinity was improved by the use of a buffered medium (pH 8.0) supplemented with casamino acid and a reduction of the cultivation time. rPAG suffered limited proteolysis at the same site as the original PAG. A site-directed mutagenesis study indicated that proteolysis had no effect on enzyme characteristics. A kinetic study indicated that the PAG possessed significant transglycosylation activity.
  • Kawahara R, Saburi W, Odaka R, Taguchi H, Ito S, Mori H, Matsui H
    The Journal of biological chemistry 287 50 42389 - 42399 50 2012年12月 [査読有り][通常論文]
     
    Ruminococcus albus is a typical ruminal bacterium digesting cellulose and hemicellulose. Cellobiose 2-epimerase (EC 5.1.3.11, CE), which converts cellobiose to 4-O-β-D-glucosyl-D-mannose, is a particularly unique enzyme in R. albus, but its physiological function is unclear. Recently, a new metabolic pathway of mannan involving CE was postulated for another CE producing bacterium, Bacteroides fragilis. In this pathway, β-1,4-mannobiose is epimerized to 4-O-β-D-mannosyl-D-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to α-D-mannosyl 1-phosphate (Man1P) and D-glucose by Man-Glc phosphorylase (EC 2.4.1.281, MP). Ruminococcus albus NE1 showed intracellular MP activity, and two MP isozymes, RaMP1 and RaMP2, were obtained from the cell-free extract. These enzymes were highly specific for the mannosyl residue at the non-reducing end of the substrate and catalyzed the phosphorolysis and synthesis of Man-Glc through a sequential bi bi mechanism. In a synthetic reaction, RaMP1 showed high activity only towards D-glucose and 6-deoxy-D-glucose in the presence of Man1P, while RaMP2 showed acceptor specificity significantly different from RaMP1. RaMP2 acted on D-glucose derivatives at the C2- and C3-positions including deoxy- and deoxyfluoro-analogues and epimers, but not on those substituted at the C6-position. Furthermore, RaMP2 had high synthetic activity toward the following oligosaccharides: β-linked glucobioses, maltose, N, N'-diacetylchitobiose, and β-1,4-mannooligosaccharides. Particularly, β-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than D-glucose. In the phosphorolytic reactions, RaMP2 had weak activity towards β-1,4-mannobiose but efficiently degraded β-1,4-mannooligosaccharides longer than β-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of β-1,4-mannooligosaccharides longer than β-1,4-mannobiose to produce Man1P and β-1,4-mannobiose.
  • Hitoshi Iwaya, Jae-Sung Lee, Shinya Yamagishi, Aki Shinoki, Weeranuch Lang, Charin Thawornkuno, Hee-Kwon Kang, Yuya Kumagai, Shiho Suzuki, Shinichi Kitamura, Hiroshi Hara, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka
    PLOS ONE 7 11 11 - e50658 2012年11月 [査読有り][通常論文]
     
    Background: Isomaltosyloligosaccharides (IMO) and dextran (Dex) are hardly digestible in the small intestine and thus influence the luminal environment and affect the maintenance of health. There is wide variation in the degree of polymerization (DP) in Dex and IMO (short-sized IMO, S-IMO; long-sized IMO, L-IMO), and the physiological influence of these compounds may be dependent on their DP. Methodology/Principal Findings: Five-week-old male Wistar rats were given a semi-purified diet with or without 30 g/kg diet of the S-IMO (DP = 3.3), L-IMO (DP = 8.4), or Dex (DP = 1230) for two weeks. Dextran sulfate sodium (DSS) was administered to the rats for one week to induce experimental colitis. We evaluated the clinical symptoms during the DSS treatment period by scoring the body weight loss, stool consistency, and rectal bleeding. The development of colitis induced by DSS was delayed in the rats fed S-IMO and Dex diets. The DSS treatment promoted an accumulation of neutrophils in the colonic mucosa in the rats fed the control, S-IMO, and L-IMO diets, as assessed by a measurement of myeloperoxidase (MPO) activity. In contrast, no increase in MPO activity was observed in the Dex-diet-fed rats even with DSS treatment. Immune cell populations in peripheral blood were also modified by the DP of ingested saccharides. Dietary S-IMO increased the concentration of n-butyric acid in the cecal contents and the levels of glucagon-like peptide-2 in the colonic mucosa. Conclusion/Significance: Our study provided evidence that the physiological effects of alpha-glucosaccharides on colitis depend on their DP, linkage type, and digestibility. Citation: Iwaya H, Lee J-S, Yamagishi S, Shinoki A, Lang W, et al. (2012) The Delay in the Development of Experimental Colitis from Isomaltosyloligosaccharides in Rats Is Dependent on the Degree of Polymerization. PLoS ONE 7(11): e50658. doi: 10.1371/journal.pone.0050658
  • Lukana Ngiwsara, Gaku Iwai, Takayoshi Tagami, Natsuko Sato, Hiroyuki Nakai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 10 1967 - 1974 2012年10月 [査読有り][通常論文]
     
    Honeybees, Apis mellifera, possess three alpha-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-M preferred sucrose to maltose as substrate and formed only alpha-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the alpha-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) alpha-glucosidases and related enzymes.
  • Young-Min Kim, Eiji Yamamoto, Min-Sun Kang, Hiroyuki Nakai, Wataru Saburi, Masayuki Okuyama, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Zui Fujimoto, Mikihiko Kobayashi, Doman Kim, Atsuo Kimura
    FEBS JOURNAL 279 17 3185 - 3191 2012年09月 [査読有り][通常論文]
     
    Bacteroides thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family 66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482) in Escherichia coli and determined that recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization > 4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase 66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases.
  • KIM Dae Hoon, MORIMOTO Naoki, SABURI Wataru, MUKAI Atsushi, IMOTO Koji, TAKEHANA Toshihiko, KOIKE Seiji, MORI Haruhide, MATSUI Hirokazu
    Bioscience, Biotechnology, and Biochemistry 76 7 1378 - 1383 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2012年07月 [査読無し][通常論文]
     
    α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimu...
  • Nobuhiro Suzuki, Young-Min Kim, Zui Fujimoto, Mitsuru Momma, Masayuki Okuyama, Haruhide Mori, Kazumi Funane, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 24 19916 - 19926 2012年06月 [査読有り][通常論文]
     
    Dextranase is an enzyme that hydrolyzes dextran alpha-1,6 linkages. Streptococcus mutans dextranase belongs to glycoside hydrolase family 66, producing isomaltooligosaccharides of various sizes and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln(100) to Ile(732) of S. mutans dextranase, devoid of its N- and C-terminal variable regions, was determined at 1.6 angstrom resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like beta-sandwich fold; domain A contained the enzyme's catalytic module, comprising a (beta/alpha)(8)-barrel; and domain C formed a beta-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined, and, in the enzyme-isomaltotriose complex structure, the bound isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites, -4 to -1, in the catalytic cleft. The complexed structure with 4',5'-epoxypentyl-alpha-D-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp(385) side chain. These structures collectively indicated that Asp(385) was the catalytic nucleophile and that Glu(453) was the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme's catalytic machinery.
  • Young-Min Kim, Yoshiaki Kiso, Tomoe Muraki, Min-Sun Kang, Hiroyuki Nakai, Wataru Saburi, Weeranuch Lang, Hee-Kwon Kang, Masayuki Okuyama, Haruhide Mori, Ryuichiro Suzuki, Kazumi Funane, Nobuhiro Suzuki, Mitsuru Momma, Zui Fujimoto, Tetsuya Oguma, Mikihiko Kobayashi, Doman Kim, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 24 19927 - 19935 2012年06月 [査読有り][通常論文]
     
    A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val(837)-Met(932) for PsDex and Tyr(404)-Tyr(492) for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala(39)-Ser(1304)) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp(189), Asp(340), Glu(412), and Asp(1254) of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1,500 to 1/40,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using alpha-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a beta- or alpha-isomaltotetraosyl azide, respectively, strongly indicating Asp(340) and Glu(412) as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from alpha-isomaltotetraosyl fluoride in the presence of NaN3.
  • Young-Min Kim, Wataru Saburi, Shukun Yu, Hiroyuki Nakai, Janjira Maneesan, Min-Sun Kang, Seiya Chiba, Doman Kim, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 287 27 22441 - 22444 2012年06月 [査読有り][通常論文]
     
    alpha-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form alpha-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for alpha-glucosidases. alpha-Glucosidase-catalyzed hydration produced alpha-glucose from AF, which plateaued with time. Combined reaction with alpha-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger alpha-glucosidase (31AG), which is stable in organic solvent, produced ethyl alpha-glucoside from AF in 80% ethanol. The findings indicate that alpha-glucosidases catalyze trans-addition. This is the first report of alpha-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.
  • Ken Hamura, Wataru Saburi, Shotaro Abe, Naoki Morimoto, Hidenori Taguchi, Haruhide Mori, Hirokazu Matsui
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 4 812 - 818 2012年04月 [査読無し][通常論文]
     
    Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to produce alpha-D-glucopyranosyl phosphate (Glc1P) and D-glucose. It is an essential enzyme for the metabolism of cello-oligosaccharides in a ruminal bacterium, Ruminococcus albus. In this study, recombinant R. albus CBP (RaCBP) produced in Escherichia coli was characterized. It showed highest activity at pH 6.2 at 50 degrees C, and was stable in a pH range of 5.5-8.8 and at below 40 degrees C. It phosphorolyzed only cellobiose efficiently, and the reaction proceeded through a random-ordered bi hi mechanism, by which inorganic phosphate and cellobiose bind in random order and D-glucose is released before Glc1P. In the synthetic reaction, RaCBP showed highest activity to D-glucose, followed by 6-deoxy-D-glucose. D-Mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, 1,5-anhydro-D-glucitol, and gentiobiose also served as acceptors, although the activities for them were much lower than for D-glucose. D-Glucose acted as a competitive-uncompetitive inhibitor of the reverse synthetic reaction, which bound not only the Glc1P site (competitive) but also the ternary enzyme-Glc1P-D-glucose complex (uncompetitive).
  • Atip Chantarudee, Preecha Phuwapraisirisan, Kiyoshi Kimura, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Chanpen Chanchao
    BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 12 45  2012年04月 [査読有り][通常論文]
     
    Background: Bee pollen is composed of floral pollen mixed with nectar and bee secretion that is collected by foraging honey (Apis sp.) and stingless bees. It is rich in nutrients, such as sugars, proteins, lipids, vitamins and flavonoids, and has been ascribed antiproliferative, anti-allergenic, anti-angiogenic and free radical scavenging activities. This research aimed at a preliminary investigation of the chemical constituents and free radical scavenging activity in A. mellifera bee pollen. Methods: Bee pollen was directly collected from A. mellifera colonies in Nan province, Thailand, in June, 2010, whilst floral corn (Zea mays L.) pollen was collected from the nearby corn fields. The pollen was then sequentially extracted with methanol, dichloromethane (DCM) and hexane, and each crude extract was tested for free radical scavenging activity using the DPPH assay, evaluating the percentage scavenging activity and the effective concentration at 50% (EC50). The most active crude fraction from the bee pollen was then further enriched for bioactive components by silica gel 60 quick and adsorption or Sephadex LH-20 size exclusion chromatography. The purity of all fractions in each step was observed by thin layer chromatography and the bioactivity assessed by the DPPH assay. The chemical structures of the most active fractions were analyzed by nuclear magnetic resonance. Results: The crude DCM extract of both the bee corn pollen and floral corn pollen provided the highest active free radical scavenging activity of the three solvent extracts, but it was significantly (over 28-fold) higher in the bee corn pollen (EC50 = 7.42 +/- 0.12 mu g/ml), than the floral corn pollen (EC50 = 212 +/- 13.6% mu g/ml). After fractionation to homogeneity, the phenolic hydroquinone and the flavone 7-O-R-apigenin were found as the minor and major bioactive compounds, respectively. Bee corn pollen contained a reasonably diverse array of nutritional components, including biotin (56.7 mu g/100 g), invert sugar (19.9 g/100 g), vitamin A and beta carotene (1.53 mg/100 g). Conclusions: Bee pollen derived from corn (Z. mays), a non-toxic or edible plant, provided a better free radical scavenging activity than floral corn pollen.
  • Dungporn Teerasripreecha, Preecha Phuwapraisirisan, Songchan Puthong, Kiyoshi Kimura, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Chanpen Chanchao
    BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 12 27  2012年03月 [査読有り][通常論文]
     
    Background: Propolis is a complex resinous honeybee product. It is reported to display diverse bioactivities, such as antimicrobial, anti-inflammatory and anti-tumor properties, which are mainly due to phenolic compounds, and especially flavonoids. The diversity of bioactive compounds depends on the geography and climate, since these factors affect the floral diversity. Here, Apis mellifera propolis from Nan province, Thailand, was evaluated for potential anti-cancer activity. Methods: Propolis was sequentially extracted with methanol, dichloromethane and hexane and the cytotoxic activity of each crude extract was assayed for antiproliferative/cytotoxic activity in vitro against five human cell lines derived from duet carcinoma (BT474), undifferentiated lung (Chaco), liver hepatoblastoma (Hep-G(2)), gastric carcinoma (KATO-III) and colon adenocarcinoma (SW620) cancers. The human foreskin fibroblast cell line (Hs27) was used as a non-transformed control. Those crude extracts that displayed antiproliferative/cytotoxic activity were then further fractionated by column chromatography using TLC-pattern and MTT-cytotoxicity bioassay guided selection of the fractions. The chemical structure of each enriched bioactive compound was analyzed by nuclear magnetic resonance and mass spectroscopy. Results: The crude hexane and dichloromethane extracts of propolis displayed antiproliferative/cytotoxic activities with IC50 values across the five cancer cell lines ranging from 41.3 to 52.4 mu g/ml and from 43.8 to 53.5 mu g/ml, respectively. Two main bioactive components were isolated, one cardanol and one cardol, with broadly similar in vitro antiproliferation/cytotoxicity IC50 values across the five cancer cell lines and the control Hs27 cell line, ranging from 10.8 to 29.3 mu g/ml for the cardanol and < 3.13 to 5.97 mu g/ml (6.82 - 13.0 mu M) for the cardol. Moreover, both compounds induced cytotoxicity and cell death without DNA fragmentation in the cancer cells, but only an antiproliferation response in the control Hs27 cells However, these two compounds did not account for the net antiproliferation/cytotoxic activity of the crude extracts suggesting the existence of other potent compounds or synergistic interactions in the propolis extracts. Conclusion: This is the first report that Thai A. mellifera propolis contains at least two potentially new compounds (a cardanol and a cardol) with potential anti-cancer bioactivity. Both could be alternative antiproliferative agents for future development as anti-cancer drugs.
  • Sato H, Saburi W, Ojima T, Taguchi H, Mori H, Matsui H
    Bioscience, biotechnology, and biochemistry 76 8 1584 - 1587 8 2012年 [査読有り][通常論文]
     
    Cellobiose 2-epimerase (CE) efficiently forms epilactose which has several beneficial biological functions. A thermostable CE from Rhodothermus marinus was immobilized on Duolite A568 and packed into a column. Lactose (100 g/L) was supplied to the reactor, kept at 50 °C at a space velocity of 8 h−1. The epilactose concentration of the resulting eluate was 30 g/L, and this was maintained for 13 d.
  • Degree of polymerization in dietary α-1,6-gluco¬sac¬cha¬rides modulates symptom of experimental colitis in rats.
    Iwaya H, Lee JS, Yamagishi S, Shinoki A, Lang W, Kang HK, Okuyama M, Mori H, Hara H, Kimura A, Ishizuka S
    Plos One 7 11 e50658 - e50658 2012年 [査読有り][通常論文]
  • Characterization of some enzymatic properties of recombinant α-glucosidase III from the Thai honeybee, Apis cerana indica Fabricus.
    Kaewmuangmoon J, Yoshiyama M, Kimura K, Okuyama M, Mori H, Kimura A, Chanchao C
    Afr J Biotechnol 11 96 16220 - 16232 2012年 [査読有り][通常論文]
  • Nobuhiro Suzuki, Young-Min Kim, Zui Fujimoto, Mitsuru Momma, Hee-Kwon Kang, Kazumi Funane, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 67 12 1542 - 1544 2011年12月 [査読無し][通常論文]
     
    Streptococcus mutans dextranase hydrolyzes the internal a-1,6-linkages of dextran and belongs to glycoside hydrolase family 66. An N- and C-terminal deletion mutant of S. mutans dextranase was crystallized by the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.6 angstrom and belonged to space group P21, with unit-cell parameters a = 53.2, b = 89.7, c = 63.3 angstrom, beta = 102.3 degrees. Assuming that the asymmetric unit of the crystal contained one molecule, the Matthews coefficient was calculated to be 4.07 angstrom 3 Da-1; assuming the presence of two molecules in the asymmetric unit it was calculated to be 2.03 angstrom 3 Da-1.
  • Teruyo Ojima, Wataru Saburi, Hiroki Sato, Takeshi Yamamoto, Haruhide Mori, Hirokazu Matsui
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 11 2162 - 2168 2011年11月 [査読無し][通常論文]
     
    Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of beta-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinas DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 degrees C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 degrees C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.
  • Janos A. Motyan, Erika Fazekas, Haruhide Mori, Birte Svensson, Peter Bagossi, Lili Kandra, Gyoengyi Gyemant
    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC 72 3-4 229 - 237 2011年11月 [査読有り][通常論文]
     
    The transglycosylation activity of barley alpha-amylase 1 (AMY1) and active site AMY1 subsite mutant enzymes was investigated. We report here the transferase ability of the V47A, V47F, V47D and S48Y single mutants and V47K/S48G and V47G/S48D double mutant AMY1 enzymes in which the replaced amino acids play important role in substrate binding at subsites at -3 through -5. Although mutation increases the transglycosylation activity of enzymes, in the presence of acceptors the difference between wild type and mutants is not so significant. Oligomer transfer reactions of AMY1 wild type and its mutants were studied using maltoheptaose and maltopentaose donors and different chromophore containing acceptors. The conditions for the chemoenzymatic synthesis of 4-methylumbelliferyl-alpha-D-maltooligosaccharides (MU-alpha-D-MOSs) were optimized using 4-methylumbelliferyl-beta-D-glucoside as acceptor and maltoheptaose as donor. 4-Methylumbelliferyl-alpha-D-maltoside, -maltotrioside, maltotetraoside and -maltopentaoside have been synthesized. Products were identified by MALDI-TOF MS. H-1 and C-13 NMR analyses showed that AMY1 V47F preserved the stereo- and regioselectivity. The produced MU-alpha-D-MOSs of degree of polymerization DP 2, DP 3 and DP 5 were successfully applied to detect activity of Bacillus stearothermophilus maltogenic alpha-amylase, human salivary alpha-amylase and Bacillus licheniformis alpha-amylase, respectively in a fast and simple fluorometric assay. (C) 2011 Elsevier B.V. All rights reserved.
  • Momoko Kobayashi, Hironori Hondoh, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 8 1557 - 1563 2011年08月 [査読無し][通常論文]
     
    Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl2 increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl2 also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca2+, were considered to stabilize SmDG. On the other hand, CaCl2 had no significant effect on catalytic reaction. The enhanced stability by Ca2+ was probably related to calcium binding in the beta -> alpha loop 1 of the (beta/alpha)(8) barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.
  • Young-Min Kim, Ryoko Shimizu, Hiroyuki Nakai, Haruhide Mori, Masayuki Okuyama, Min-Sun Kang, Zui Fujimoto, Kazumi Funane, Doman Kim, Atsuo Kimura
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 91 2 329 - 339 2011年07月 [査読無し][通常論文]
     
    Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCG Delta) or N-VR/C-VR (TM-Delta CG Delta) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-Delta CG Delta did not accept any further protease-degradation during long storage. TM-NCG Delta and TM-Delta CG Delta enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.
  • Young-Min Kim, Ryoko Shimizu, Hiroyuki Nakai, Haruhide Mori, Masayuki Okuyama, Min-Sun Kang, Zui Fujimoto, Kazumi Funane, Doman Kim, Atsuo Kimura
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 91 2 329 - 339 2011年07月 [査読有り][通常論文]
     
    Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCG Delta) or N-VR/C-VR (TM-Delta CG Delta) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-Delta CG Delta did not accept any further protease-degradation during long storage. TM-NCG Delta and TM-Delta CG Delta enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.
  • WAKUTA Shinji, HAMADA Shigeki, ITO Hiroyuki, IMAI Ryozo, MORI Haruhide, MATSUURA Hideyuki, NABETA Kensuke, MATSUI Hirokazu
    Journal of applied glycoscience 58 2 67 - 70 Japanese Society of Applied Glycoscience 2011年04月20日 [査読無し][通常論文]
  • カン ヒゴン, キム ヨンミン, 中井 博之, カン ミンソン, 袴田 航, 奥山 正幸, 森 春英, 西尾 俊幸, 木村 淳夫
    Journal of applied glycoscience 57 4 269 - 272 日本応用糖質科学会 2010年10月20日 [査読無し][通常論文]
     
    Streptococcus mutans ATCC 25175由来のエンド型デキストラナーゼ(SmDex)に対し，3種類のω-エポキシアルキルα-D-グルコピラノシド(3′,4′-エポキシブチルα-D-グルコピラノシド(E4G)，4′,5′-エポキシペンチルα-D-グルコピラノシド(E5G)および5′,6′-エポキシヘキシルα-D-グルコピラノシド(E6G)：アグリコンのアルキル鎖長が異なる)を作用させると，SmDexは擬一次的な活性低下を示した．アルキル鎖長に依存した失活が認められ，失活の度合いはE5G > E6G > E4Gであった．したがってω-エポキシアルキルα-D-グルコピラノシドのグルコース残基とエポキシ基の距離が，SmDexの失活に対し重要であることが判明した．E5Gは可逆的な中間体を形成する失活機構(自殺基質型の失活機構)を与え，不活性化の一次定数(k)と中間体の解離定数(KR)はそれぞれ0.44 min-1および1.45 mMと算出された．SmDexの加水分解反応の生成物であるイソマルトースの存在によりE5Gの失活が防御されたため，E5GはSmDexの触媒部位に結合すると示唆された．本論文は，ω-エポキシアルキルα-D-グルコピラノシドがエンド型デキストラナーゼの自殺基質になることを示す初めての報告である．
  • カンヒゴン, キムヨンミン, 中井博之, カンミンソン, 袴田航, 奥山正幸, 森春英, 西尾俊幸, 木村淳夫
    J Appl Glycosci 57 4 269-272 (J-STAGE) - 272 日本応用糖質科学会 2010年 [査読無し][通常論文]
     
    Streptococcus mutans ATCC 25175由来のエンド型デキストラナーゼ(SmDex)に対し，3種類のω-エポキシアルキルα-D-グルコピラノシド(3′,4′-エポキシブチルα-D-グルコピラノシド(E4G)，4′,5′-エポキシペンチルα-D-グルコピラノシド(E5G)および5′,6′-エポキシヘキシルα-D-グルコピラノシド(E6G)：アグリコンのアルキル鎖長が異なる)を作用させると，SmDexは擬一次的な活性低下を示した．アルキル鎖長に依存した失活が認められ，失活の度合いはE5G > E6G > E4Gであった．したがってω-エポキシアルキルα-D-グルコピラノシドのグルコース残基とエポキシ基の距離が，SmDexの失活に対し重要であることが判明した．E5Gは可逆的な中間体を形成する失活機構(自殺基質型の失活機構)を与え，不活性化の一次定数(k)と中間体の解離定数(KR)はそれぞれ0.44 min-1および1.45 mMと算出された．SmDexの加水分解反応の生成物であるイソマルトースの存在によりE5Gの失活が防御されたため，E5GはSmDexの触媒部位に結合すると示唆された．本論文は，ω-エポキシアルキルα-D-グルコピラノシドがエンド型デキストラナーゼの自殺基質になることを示す初めての報告である．
  • 西村 崇志, 鐘ヶ江 倫世, 本同 宏成, 奥山 正幸, 森 春英, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2010 71 - 71 日本応用糖質科学会 2010年
  • 田上 貴祥, 奥山 正幸, 森 春英, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2010 73 - 73 日本応用糖質科学会 2010年
  • MORI Haruhide, LEE Jin-Ha, OKUYAMA Masayuki, NISHIMOTO Mamoru, OHGUCHI Masao, KIM Doman, KIMURA Atsuo, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 73 11 2466 - 2473 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2009年11月 [査読無し][通常論文]
     
    Trehalase, an anomer-inverting glycosidase, hydrolyzes only α,α-trehalose in natural substrates to release equimolecular β-glucose and α-glucose. Since the hydrolytic reaction is reversible, α,α-[1,1′-<SUP>2</SUP>H]trehalose is capable of synthesis from [1-<SUP>2</SUP>H]glucose through the reverse reaction of trehalase. α-Secondary deuterium kinetic isotope effects (α-SDKIEs) for the hydrolysis of synthesized α,α-[1,1′-<SUP>2</SUP>H]trehalose by honeybee trehalase were measured to examine the catalytic reaction mechanism. Relatively high <I>k</I><SUB>H</SUB>⁄<I>k</I><SUB>D</SUB> value of 1....
  • Min-Sun Kang, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    BIOCHIMIE 91 11-12 1434 - 1442 2009年11月 [査読有り][通常論文]
     
    Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES-GroEL chaperones. Recombinant LJAG31 was an alpha-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of k(cat)/K-m) toward nigerose, maltulose, and kojibiose than other natural substrates having an alpha-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial alpha-1,3-glucosidase to be characterized with a high k(cat)/K-m value for nigerose [alpha-D-Glcp-(1 -> 3)-D-Glcp]. Transglucosylation of 4-nitrophenyl M-D-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl alpha-nigeroside and 4-nitrophenyl alpha-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) alpha-1,3-glucosidase of glycoside hydrolase family 31. (C) 2009 Elsevier Masson SAS. All rights reserved.
  • Masayuki Okuyama, Momoyo Kitamura, Hironori Hondoh, Min-Sun Kang, Haruhide Mori, Atsuo Kimura, Isao Tanaka, Min Yao
    JOURNAL OF MOLECULAR BIOLOGY 392 5 1232 - 1241 2009年10月 [査読有り][通常論文]
     
    Glycoside hydrolase family 97 (GH 97) is a unique glycoside family that contains inverting and retaining glycosidases. Of these, BtGH97a (SusB) and BtGH97b (UniProtKB/TrEMBL entry QM6L0), derived from Bacteroides thetaiotaomicron, have been characterized as an inverting a-glucoside hydrolase and a retaining a-galactosidase, respectively. Previous studies on the three-dimensional structures of BtGH97a and site-directed mutagenesis 508 indicated that Glu532 acts as an acid catalyst and that G function as the catalytic base in the inverting mechanism. However, BtGH97b lacks base catalysts but possesses a putative catalytic nucleophilic residue, Asp415. Here, we report that Asp415 in BtGH97b is the nucleophilic catalyst based on the results of crystal structure analysis and site-directed mutagenesis study. Structural comparison between BtGH97b and BtGH97a indicated that OD1 of Asp415 in BtGH97b is located at a position spatially identical with the catalytic water molecule of BtGH97a, which attacks on the anomeric carbon from the beta-face (i.e., Asp415 is poised for nucleophilic attack on the anomeric carbon). Site-directed mutagenesis of Asp415 leads to inactivation of the enzyme, and the activity is rescued by an external nucleophilic azide ion. That is, Asp415 functions as a nucleophilic catalyst. The multiple amino acid sequence alignment of GH 97 members indicated that almost half of the GH 97 enzymes possess base catalyst residues at the end of beta-strands 3 and 5, while the other half of the fan-Lily show a conserved nucleophilic residue at the end of beta-strand 4. The different positions of functional groups on the beta-face of the substrate, which seem to be due to "hopping of the functional group" during evolution, have led to divergence of catalytic mechanism within the same family. (C) 2009 Elsevier Ltd. All rights reserved.
  • 吉田 拓弥, 奥山 正幸, 本同 宏成, 姚 閔, 森 春英, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2009 38 - 38 日本応用糖質科学会 2009年
  • 田上 貴祥, 奥山 正幸, 森 春英, 田口 和憲, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2009 37 - 37 日本応用糖質科学会 2009年
  • 中塚 大地, 本同 宏成, 大塚 博昭, 佐分利 亘, 森 春英, 奥山 正幸, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2009 34 - 34 日本応用糖質科学会 2009年
  • ギウサラ ルカナ, 森 春英, 奥山 正幸, 千葉 誠哉, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2009 36 - 36 日本応用糖質科学会 2009年
  • 西村 崇志, 鐘ヶ江 倫世, KIM Young-Min, 本同 宏成, 奥山 正幸, 森 春英, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2009 35 - 35 日本応用糖質科学会 2009年
  • Wataru Saburi, Hironori Hondoh, Young-Min Kim, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura
    BIOLOGIA 63 6 1000 - 1005 2008年12月 [査読無し][通常論文]
     
    Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an alpha-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms of substrate chain length specificity. SMDG efficiently hydrolyzes both short- and long-chain substrates, while O16G acts on only short- chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short beta -> alpha loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced by a smaller amino acid, alanine, asparagine or proline; and (ii) short beta -> alpha loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates, as implied by structural analysis.
  • Momoyo Kitamura, Masayuki Okuyama, Fumiko Tanzawa, Haruhide Mori, Yu Kitago, Nobuhisa Watanabe, Atsuo Kimura, Isao Tanaka, Min Yao
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 52 36328 - 36337 2008年12月 [査読有り][通常論文]
     
    SusB, an 84-kDa alpha-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6 angstrom resolution. SusB hydrolyzes the alpha-glucosidic linkage, with inversion of anomeric configuration liberating the beta-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only alpha-1,4-glucosidic linkages but also alpha-1,6-, alpha-1,3-, and alpha-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu(532) is positioned to provide protonic assistance to leaving group departure, with Glu(439) and Glu(508) both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of alpha-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.
  • Masayuki Okuyama, Haruhide Mori, Hironori Hondoh, Hiroyuki Nakai, Wataru Saburi, Min Sung Kang, Young Min Kim, Mamoru Nishimoto, Jintanart Wongchawalit, Takeshi Yamamoto, Mee Son, Jin Ha Lee, San San Mar, Kenji Fukuda, Seiya Chiba, Atsuo Kimura
    Carbohydrate-Active Enzymes: Structure, Function and Applications 64 - 76 2008年09月 [査読有り][通常論文]
     
    α-Glucosidase (EC 3.2.1.20), an exo-glycosylase to hydrolyze α-glucosidic linkage, is characterized by the variety of substrate specificity. Enzyme also catalyzes the transglucosylation, on which industrial interests focus due to the production of valuable glucooligosaccharides. α-Glucosidase is a physiologically important enzyme in most of organisms (microorganisms, insects, plants and animals including human). Therefore, there are many types of α-glucosidases to display unique functions, in which we are interested. This report describes the recently analyzed unique functions of α-glucosidases by mainly focusing on honeybee α-glucosidase isoenzymes, dextran glucosidase, multiple forms of rice α-glucosidases, and Escherichia coli α-xylosidase. © 2008 Woodhead Publishing Limited. All rights reserved.
  • Nakai H, Tanizawa S, Ito T, Kamiya K, Kim YM, Yamamoto T, Matsubara K, Sakai M, Sato H, Imbe T, Okuyama M, Mori H, Chiba S, Sano Y, Kimura A
    Biocatalysis and Biotransformation 26 104 - 110 2008年07月 [査読有り][通常論文]
  • 奥山正幸, カンミンソン, 矢追克郎, 三石安, 森春英, 木村淳夫
    J Appl Glycosci 55 2 111-118 (J-STAGE) - 118 日本応用糖質科学会 2008年 [査読無し][通常論文]
     
    大腸菌YicIはα-xylosidaseは，GH31のなかで最も研究が行われている酵素の一つであり，立体構造解析，生化学的な研究が数多く行われている．YicIの酵素反応の至適pHは7.0，pH安定領域，温度領域はそれぞれ4.7-10.1，47°Cまでの範囲内である．YicIはGH31のα-glucosidaseと30%程度の配列類似性を示すが，YicIは厳密に非還元末端のα-xylosyl基を認識している．二つの変異酵素(TIM-barrelドメインのβ→α loop 1をα-glucosidase様に変化させたL1Chi，loop 2に位置するCys307，Phe308をα-glucosidase様シーケンスに置換したC307I/F308D)では，α-xylosidase活性が低下し，α-glucosidase活性が上昇する．YicIのプラス側サブサイトの特異性を糖転移反応受容体特異性により評価した．YicIはエカトリアルの4-OHを選択して受容体とする．またYicIの糖転移反応は1糖受容体に対しα-1,6結合の1生成物を生成する非常に特異性の狭い反応である．これら糖転移産物のうちα-D-xylopyranosyl-(1→6)-D-mannopyranose，α-D-xylopyranosyl-(1→6)-D-fructofuranose，α-D-xylopyranosyl-(1→3)-D-fructopyranoseは新規糖である．さらにα-D-xylopyranosyl-(1→6)-D-mannopyranoseならびにα-D-xylopyranosyl-(1→6)-D-fructofuranoseラット小腸α-glucosidaseに対して緩い阻害を示す．
  • 鐘ヶ江 倫世, KIM Young-Min, 本同 宏成, 奥山 正幸, 森 春英, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2008 104 - 104 日本応用糖質科学会 2008年
  • 奥山 正幸, 姚 閔, 本同 宏成, 北村 百世, 森 春英, 田中 勲, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2008 139 - 139 日本応用糖質科学会 2008年
  • 田上 貴祥, 奥山 正幸, 森 春英, 田口 和憲, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2008 94 - 94 日本応用糖質科学会 2008年
  • カン ミンソン, 奥山 正幸, 森 春英, 木村 淳夫
    Journal of Applied Glycoscience Supplement 2008 96 - 96 日本応用糖質科学会 2008年
  • M-S. Kang, M. Okuyama, K. Yaoi, Y. Mitsuishi, Y-M. Kim, H. Mori, A. Kimura
    Biocatalysis and Biotransformation 26 1-2 96 - 103 2008年 [査読無し][通常論文]
     
    Escherichia coli YicI is a retaining alpha-xylosidase, which strictly recognizes the alpha-xylosyl moiety at the non-reducing end, belonging to glycoside hydrolase family 31 (GH 31). We have elucidated key residues determining the substrate specificity at both glycone and aglycone sites of Escherichia coli -xylosidase (YicI). Detection of distinguishing features between alpha-xylosidases and alpha-glucosidases of GH 31 in their close evolutionary relationship has been used for the modification of protein function, converting YicI into an alpha-glucosidase. Aglycone specificity has been characterized by its transxylosylation ability. YicI exhibits a preference for aldopyranosyl sugars having equatorial 4-OH as the acceptor substrate with 1,6 regioselectivity, resulting in transfer products. The disaccharide transfer products of YicI, alpha-D-Xylp-(1 -> 6)-D-Manp, alpha-D-Xylp-(1 -> 6)-D-Fruf, and alpha-D-Xylp-(1 -> 3)-D-Frup, are novel oligosaccharides, which have never been reported. The transxylosylation products are moderately inhibitory towards intestinal alpha-glucosidases.
  • Hondoh H, Saburi W, Mori H, Okuyama M, Nakada T, Matsuura Y, Kimura A
    J Mol Biol 378 4 913 - 922 2008年 [査読有り][通常論文]
  • NAKAI Hiroyuki, TANIZAWA Shigeki, ITO Tatsuya, KAMIYA Koutarou, KIM Young-Min, YAMAMOTO Takeshi, MATSUBARA Kazuki, SAKAI Makoto, SATO Hiroyuki, IMBE Tokio, OKUYAMA Masayuki, MORI Haruhide, SANO Yoshio, CHIBA Seiya, KIMURA Atsuo
    The journal of biochemistry 142 4 491 - 500 Japanese Biochemical Society 2007年10月 [査読無し][通常論文]
     
    In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three a-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. alpha-Xylosidase encoded in rice genome was found for the first time.
  • Wataru Saburi, Hironori Hondoh, Hideaki Unno, Masayuki Okuyama, Haruhide Mori, Toshitaka Nakada, Yoshiki Matsuura, Atsuo Kimura
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 63 9 774 - 776 2007年09月 [査読無し][通常論文]
     
    Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal alpha-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 angstrom. A native data set was collected to 2.2 angstrom resolution from a single crystal.
  • Jin-Ha Lee, Saori Saito, Haruhide Mori, Mamoru Nishimoto, Masayuki Okuyama, Doman Kim, Jintanart Wongchawalit, Atsuo Kimura, Seiya Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 9 2256 - 2265 2007年09月 [査読無し][通常論文]
     
    cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5 ' untranslated region (UTR) of 869 bp, and the 3 ' UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M-r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.
  • NISHIMOTO Mamoru, MORI Haruhide, MOTEKI Tsuneharu, TAKAMURA Yukiko, IWAI Gaku, MIYAGUCHI Yu, OKUYAMA Masayuki, WONGCHAWALIT Jintanart, SURARIT Rudee, SVASTI Jisnuson, KIMURA Atsuo, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 71 7 1703 - 1716 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2007年07月 [査読無し][通常論文]
     
    cDNAs encoding three α-glucosidases (HBGases I, II, and III) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 bp in length, and included ORFs of 1,767, 1,743, and 1,704 bp encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases I, II, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. I...
  • Nakai Hiroyuki, Ito Tatsuya, Hayashi Masatoshi, Kamiya Koutarou, Yamamoto Takeshi, Matsubara Kazuki, Kim Young-Min, Jintanart Wongchawalit, Okuyama Masayuki, Mori Haruhide, Chiba Seiya, Sanoa Yoshio, Kimura Atsuo
    Biochimie 89 1 49 - 62 Elsevier Masson SAS 2007年01月 [査読無し][通常論文]
     
    Two isoforms of α-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6 kDa-peptide and 88 kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88 kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88 kDa-peptide of ONG2-I. The peptides between 6 kDa and 88 kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were rem...
  • WONGCHAWALIT Jintanart, YAMAMOTO Takeshi, NAKAI Hiroyuki, KIM Young-Min, SATO Natsuko, NISHIMOTO Mamoru, OKUYAMA Masayuki, MORI Haruhide, SAJI Osamu, CHANCHAO Chanpen, WONGSIRI Siriwat, SURARIT Rudee, SVASTI Jisnuson, CHIBA Seiya, KIMURA Atsuo
    Bioscience, biotechnology, and biochemistry 70 12 2889 - 2898 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2006年12月 [査読無し][通常論文]
     
    α-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 °C and in a...
  • HACHEM Maher Abou, BOZONNET Sophie, WILLEMOES Martin, BONSAGER Birgit C, NIELSEN Morten Munch, FUKUDA Kenji, KRAMHOFT Birte, MAEDA Kenji, SIGURSKJOLD Bent W, HAGGLUND Per, FINNIE Christine, MORI Haruhide, ROBERT Xavier, JENSEN Malene H, TRANIER Samuel, AGHAJARI Nushin, HASER Richard, SVENSSON Birte
    Journal of applied glycoscience 53 2 163 - 169 日本応用糖質科学会 2006年04月20日 [査読無し][通常論文]
     
    Barley α-amylase binds sugars at two sites on the enzyme surface in addition to the active site. Crystallography and site-directed mutagenesis highlight the importance of aromatic residues at these surface sites as demonstrated by Kd values determined for β-cyclodextrin by surface plasmon resonance and for starch granules by adsorption analysis. Activity towards amylopectin and amylose follows two different kinetic models, degradation of amylopectin being composed of a fast and a slow component, perhaps reflecting attack on A and B chains, respectively, whereas amylose hydrolysis follows a simple Michaelian kinetics. β-cyclodextrin binding at surface sites inhibits only the fast reaction in amylopectin degradation. Site-directed mutagenesis and activity analysis, furthermore show that one of the surface binding sites as well as individual subsites in the active site cleft have distinct roles in the multiple attack on amylose. Although the two isozymes AMY1 and AMY2 share ligands for three structural calcium ions, they differ importantly in the effect of calcium on activity and stability, AMY1 having the higher affinity and the lower stability. The role of the individual calcium ions is studied by mutagenesis, crystallography and microcalorimetry. Further improvement of recombinant AMY2 production allows future direct mutational analysis in this isozyme. Specific proteinaceous inhibitors act on α-amylases of different origin. In the complex of barley α-amylase/subtilisin inhibitor (BASI) with AMY2, a fully hydrated calcium ion at the protein interface mediates contact between inhibitor residues and the enzyme catalytic groups in a manner that depends on calcium and which can be suppressed by site-directed mutagenesis of Glu168 in BASI. Finally certain inhibitors and enzymes are targets of the disulphide reductase thioredoxin h that attacks a specific disulphide bond in BASI and, remarkably, reduces two different disulphide bonds in the barley monomeric and dimeric amylase inhibitors that both belong to the CM-proteins and inhibit animal α-amylase.
  • 中井 博之, 伊藤 達也, 谷沢 茂紀, 松原 一樹, 山本 健, 奥山 正幸, 森 春英, 千葉 誠哉, 佐野 芳雄, 木村 淳夫
    Journal of applied glycoscience 53 2 137 - 142 日本応用糖質科学会 2006年04月20日 [査読無し][通常論文]
     
    植物種子が発芽する際に貯蔵物質である澱粉は，各種加水分解酵素により酵素分解され，生長に必要な物質・エネルギーの供給源となる．澱粉は植物種子中に不溶性の澱粉粒として存在するため，現在までα-アミラーゼが唯一澱粉粒に直接作用できる酵素であると考えられてきた．しかし，我々は植物α-グルコシダーゼが澱粉粒への吸着・分解能をもつことを明らかにし，第2の澱粉分解経路 (澱粉粒にα-グルコシダーゼが作用し，直接グルコースを遊離する経路) の存在を示した．さらに，植物α-グルコシダーゼには，触媒ドメインとは独立して機能する澱粉粒吸着ドメインがC末端に存在することを解明した．本ドメインに保存された芳香族アミノ酸に対する部位特異的変異導入により，澱粉粒吸着に関与する残基を推定した．植物種子中には，複数のα-グルコシダーゼが存在する．我々は，イネ種子中に可溶性および不溶性 (界面活性剤により可溶化が可能) の性質を示す2種のアイソザイムを見出した．両酵素の発現様式から，14品種のイネ種子を二つのグループ (グループ1，2) に大別した．グループ1は，不溶性酵素のみが乾燥種子に検出されるイネ品種である．不溶性α-グルコシダーゼが登熟期で合成され，完熟に伴い種子中に保存されるが，発芽の進行につれ消失する．発芽後に可溶性酵素が新たに合成される．グループ2の品種では，可溶性および不溶性の酵素が乾燥種子に検出された．両酵素は登熟期で合成され，発芽期間中の活性は変化せず，一定のレベルで推移する．グループ1からは赤米を，グループ2では日本晴を実験対象に選び，α-グルコシダーゼの機能解析を行った．翻訳後修飾やゲノム遺伝子発現調節によるアイソフォームやアイソザイムの形成機構ならびに精製酵素を用いた性質の解明を行った．多様なα-グルコシダーゼが関与する澱粉代謝の一端が明らかにされた．N末領域に生じる翻訳後限定分解は，植物酵素に対し一般的に観察される現象であった．
  • 森春英
    J Appl Glycosci 53 1 51 - 56 2006年01月20日 [査読無し][通常論文]
  • T Shinano, K Nakajima, J Wasaki, H Mori, T Zheng, M Osaki
    PHOTOSYNTHETICA 44 1 1 - 10 2006年 [査読無し][通常論文]
     
    mRNA expression patterns of genes for metabolic key enzymes sucrose phosphate synthase (SPS), phosphoenolpyruvate carboxylase (PEPC), pyruvate kinase, ribulose 1,5-bisphosphate carboxylase/oxygenase, glutamine synthetase 1, and glutatmine synthetase 2 were investigated in leaves of rice plants grown at two nitrogen (N) supplies (N(0.5), N(3.0)). The relative gene expression patterns were similar in all leaves except for 9(th) leaf, in which mRNA levels were generally depressed. Though increased N supply prolonged the expression period of each mRNA, it did not affect the relative expression intensity of any mRNA in a given leaf. SPS V(max) SPS limiting and PEPC activities, and carbon flow were exartimed. The ratio between PEPC activity and SPS V(max) was higher in leaves developed at the vegetative growth stage (vegetative leaves: 5(th) and 7(th) leaves) than in leaves developed after the ear primordia formation stage (reproductive leaves: 9(th) and flag leaves). PEPC activity and SPS V(max) decreased with declining leaf N content. After using (14)CO(2) the (14)C photosynthate distribution in the amino acid fraction was higher in vegetative than in reproductive leaves when compared for the same leaf N status. Thus, at high PEPC/SPS activities ratio, more (14)C photosynthate was distributed to the amino acid pool, whereas at higher SPS activity more (14)C was channelled into the saccharide fraction. Thus, leaf ontooeny was an important factor controlling photosyntliate distribution to the N- or C-pool, respectively, regardless of the leaf N status.
  • SATO Fumiaki, OKUYAMA Masayuki, NAKAI Hiroyuki, MORI Haruhide, KIMURA Atsuo, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 69 10 1905 - 1913 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2005年10月 [査読無し][通常論文]
     
    A starch-hydrolyzing enzyme from <I>Schwanniomyces occidentalis</I> has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of <I>S. occidentalis</I>. The enzyme showed high catalytic efficiency (<I>k</I><SUB>cat</SUB>⁄<I>K</I><SUB>m</SUB>) for maltooligosaccharides, compared with that for soluble starch. The product anomer was α-glucose, differing from glucoamylase as a β-glucose producing enzyme. These findings are striking characteristics of α-glucosid...
  • Robert, X, R Haser, H Mori, B Svensson, N Aghajari
    JOURNAL OF BIOLOGICAL CHEMISTRY 280 38 32968 - 32978 2005年09月 [査読有り][通常論文]
     
    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1(D180A)) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."
  • Sophie Bozonnet, Birgit C. Boønsager, Birte Kramhøft, Haruhide Mori, Maher Abou Hachem, Martin Willemoës, Morten T. Jensen, Kenji Fukuda, Peter K. Nielsen, Nathalie Juge, Nushin Aghajari, Samuel Tranier, Xavier Robert, Richard Haser, Birte Svensson
    Biologia - Section Cellular and Molecular Biology 60 SUPPL. 16 27 - 36 2005年 [査読有り][通常論文]
     
    This review on barley α-amylases 1 (AMY1) and 2 (AMY2) addresses rational mutations at distal subsites to the catalytic site, polysaccharide hydrolysis, and interactions with proteinaceous inhibitors. Subsite mapping of barley α-amylases revealed 6 glycone and 4 aglycone substrate subsites. Moreover, two maltooligosaccharide surface binding sites have been identified. Engineering of outer subsites -6 and +4 alters action patterns and relative specificities. Thus, compared to wild-type, Y105A AMY1 (subsite -6) shows 140%, 15%, and <1% and T212Y (subsite +4) 32%, 370%, and 90% activity towards starch, maltodextrin, and maltoheptaoside, respectively. The enzyme kinetic properties and modeled maltododecaose complexes suggest binding mode multiplicity. Following an initial hydrolytic cleavage of amylose, an average of 1.9 bonds are cleaved per enzyme-substrate encounter, defining a degree of multiple attack (DMA) of 1.9. DMA increased to 3.3 for Y105A and decreased to 1-1.7 for other subsite mutants. The fusion of a starch-binding domain to AMY1 raised the DMA to 3.0 and increased the amount of higher oligosaccharide products. Remarkably, the subsite mutants had unchanged distribution of released oligosaccharides of DP 5-9, but the profiles differed for the shorter products. A recently identified surface binding site, found exclusively in AMY1, involves the conserved Tyr380 which has no effect on the DMA, but proved critical for β-cyclodextrin binding as shown by mutational and surface plasmon resonance analyses. Accordingly, AMY2 has lower affinity for β-cyclodextrin. Hydrolysis of amylopectin proceeds via a fast and a slow reaction rate, with β-cyclodextrin inhibiting the fast one, implicating a distinct role for Tyr380 in activity on amylopectin. Barley seeds produce different proteinaceous inhibitors acting specifically on insect, animal or plant α-amylases. Rational mutagenesis of barley α-amylase/ subtilisin inhibitor (BASI) identified structural elements responsible for AMY2 inhibition and demonstrated the importance of ionic bonds for inhibitory activity.
  • Nakai H, Okuyama M, Kim YM, Saburi W, Wongchawalit J, Mori H, Chiba S, Kimura A
    Biologia, Bratislava 60 131 - 135 2005年 [査読有り][通常論文]
  • KUBOTA Masaki, TSUJI Masahisa, NISHIMOTO Mamoru, WONGCHAWALIT Jintanart, OKUYAMA Masayuki, MORI Haruhide, MATSUI Hirokazu, SURARIT Rudee, SVASTI Jisnuson, KIMURA Atsuo, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 68 11 2346 - 2352 Japan Society for Bioscience, Biotechnology, and Agrochemistry 2004年11月 [査読無し][通常論文]
     
    Three kinds of α-glucosidases, I, II, and III, were purified from European honeybees, <I>Apis mellifera</I> L. In addition, an α-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey α-glucosidase, were almost the same as those of α-glucosidase III. Specific antisera against the α-glucosidases were prepared to examine the localization of α-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that α-glucosidase I was present in ventriculus, and α-glucosidase II, in ventriculus and haemolymph. α-Glucosid...
  • Fukiya S, Mizoguchi H, Mori H
    FEMS microbiology letters 234 2 325 - 331 2 2004年05月 [査読有り][通常論文]
  • FINNIE Christine, OSTERGAARD Ole, BAK-JENSEN Kristian Sass, NIELSEN Peter K, BONSAGER Birgit C, MORI Haruhide, NOHR Jane, KRAMHOFT Birte, JUGE Nathalie, SVENSSON Birte
    Journal of applied glycoscience 50 2 277 - 282 日本応用糖質科学会 2003年07月14日 [査読無し][通常論文]
     
    Proteomes of barley seeds were described by 2-D gel electrophoresis and spots selected for proteinidentification by mass spectrometry and database searches. Proteins were categorised according to temporalappearance during seed development and maturation. Fragments of β-amylases appeared transiently at midgrain filling and during germination. The α-amylase/trypsin inhibitors increased during grain filling andtypical housekeeping enzymes were present throughout the period. Germination altered the proteome and dissection of micromalted seeds enabled localization of selected proteins to specifi...
  • SVENSSON Birte, MORI Haruhide, BAK-JENSEN Kristian Sass, JENSEN Morten Tovborg
    Journal of applied glycoscience 50 2 143 - 145 日本応用糖質科学会 2003年07月14日 [査読無し][通常論文]
     
    The mutational analysis of the roles of specific side chains at individual subsites have been conducted for barley α-amylase 1(AMY1) across the ten subsites long substrate binding cleft. The present study specifically focuses on such mutants in which the AMY2 structure has been mimicked. Generally the kinetics parameters for mutants at subsites accommodating the substrate glycone part showed decreased affinity for oligosaccharide and amylose DP 17 whereas an aglycon binding subsite +4 AMY2 mimic had increased affinity but reduced activity. Among barley α-amylase/subtilisin inhibitor (BASI) ...
  • MAR San San, MORI Haruhide, LEE Jin-Ha, FUKUDA Kenji, SABURI Wataru, FUKUHARA Arinobu, OKUYAMA Masayuki, CHIBA Seiya, KIMURA Atsuo
    Bioscience, biotechnology, and biochemistry 67 5 1080 - 1093 社団法人日本農芸化学会 2003年05月 [査読無し][通常論文]
     
    Two α-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a β-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl_2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17) : k_<cat>, 71.8 and 55.5 s^<-1>, K_m, 0.113 and 0.097 mg/ml ; for blocked 4-nitr...
  • 孫 美, 森 春英, 奥山 正幸, 木村 淳夫, 千葉 誠哉
    Journal of applied glycoscience 50 1 41 - 44 日本応用糖質科学会 2003年01月20日 [査読無し][通常論文]
     
    炭水化物加水分解酵素の水解反応は,その逆反応(縮合反応)が必然的に伴った反応である.従って,水解され得る基質のみが逆反応によって生成される.ミツバチα-グルコシダーゼI は,イソマルトースを全く水解できないが,他の二糖マルトース,コジビオースを水解できる.わずかではあるが,ニゲロースをも水解する.しかしながら,本酵素は高濃度のグルコースから逆反応によってマルトース,コジビオースおよびニゲロースと共にイソマルトースをも生成した.このような事実は,本酵素がイソマルトースに対して水解作用をもたないという知見と矛盾している.この特異な現象に関する矛盾の解析が試みられた.その結果,本来,グルコースから逆反応によってイソマルトースが直接生成されることはあり得ないことであるが,逆反応によって容易に生成され得るマルトースやコジビオース等の他の二糖からの分子内糖転移反応によってイソマルトースへ変換されイソマルトースが生成されると推定された.炭水化物水解酵素の反応において,通常の糖転移反応と考えられている反応には,上述のような分子内転移反応が同時に起こっていることが示唆された.
  • 孫 美, 森 春英, 奥山 正幸, 木村 淳夫, 千葉 誠哉
    Journal of Applied Glycoscience 50 1 41 - 44 日本応用糖質科学会 2003年 [査読無し][通常論文]
     
    The hydrolytic reaction of carbohydrate-hydrolase is essentially accompanied by a reverse reaction (the condensation reaction), meaning that only the substrate capable of being hydrolyzed is produced by the reverse reaction. Honeybee α-glucosidase I can't hydrolyze isomaltose, but is capable of hydrolyzing maltose, kojibiose and slightly nigerose. Nevertheless, the enzyme catalyzes the formation and accumulation of isomaltose from glucose together with α-glucobioses such as maltose, kojibiose and nigerose. This finding is in conflict with the data that the enzyme has no hydrolytic activity ...
  • FUKUDA Kenji, MORI Haruhide, OKUYAMA Masayuki, KIMURA Atsuo, OZAKI Hachiro, YONEYAMA Michio, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 66 10 2060 - 2067 社団法人日本農芸化学会 2002年10月 [査読無し][通常論文]
     
    Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of β-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of β-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and K_m values on pH, when the substrate concentration was sufficiently lower than K_m, gave the values of 4.1 and 7.2 for the ionization constants, pK_<e1> a...
  • 福田 健二, 白川 康, 森 春英, 奥山 正幸, 木村 淳夫, 尾崎 八郎, 米山 道男, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 49 3 265 - 272 日本応用糖質科学会 2002年07月18日 [査読無し][通常論文]
     
    Streptomyces sp.から精製したβ-D-グルコシダーゼF1(分子量、50,O00;最適pH、5.5)の基質特異性と活性部位について反応速度論的な検討を行った。本酵素は、β-グルコシド結合からなる各種の合成基質や二糖に対し広い基質特異性を示したが、二糖よりも合成基質に対し高い特異性を示した。合成基質ではp-ニトロフェニルβ-グルコシド(Km,0.72mM;k0,63s-1)よりもp-ニトロフェニルβ-フコシド(Km,O.19mM;k0,44s-1)が、二糖ではセロビオースよりもラミナリビオース(Km,1.6mM;k0,70s-1)が加水分解の反応効率からみてより優れた基質と考えられた。P-ニトロフェニルβ-グルコシド(PNPG)、β-フコシド(PNPF)およびβ-ガラクトシド(PNPGal)が共通の活性部位で加水分解されるか否かについて、混合基質を用いた反応系において速度論的解析を行った。PNPGとPNPF(またはPNPGal)の混合基質の加水分解におけるいくつかの速度論的特徴、特にLineweaver-Burkプロットの直線性、あるいはPNPGの他基質に対するモル分率(f),f=[PNPG]/([PNPG]+[PNPFまたはPNPGal])の最大速度とミカエリス定数への依存性の実験値は単一触媒部位機構において理論的に予測される値によく一致した。これらの実験結果は、...
  • 西本完, 森春英, 木村淳夫, 千葉誠哉
    J Appl Glycosci 49 2 191 - 197 The Japanese Society of Applied Glycoscience 2002年04月01日 [査読無し][通常論文]
     
    The genes of three a-glucosidases (HBG I, HBG II and HBG III) were isolated from the cDNA library of honeybee, Apis mellifera L. The nucleotide sequences of HBG I, II and III were consisted of 1974, 1910 and 1916 base pair and encoding 588, 580 and 567 amino acid residues, respectively. The putative primary structures showed high homology ranging from N- to C-terminals, and three enzymes belonged to a-glucosidase family I, in which four conservative regions of aamylase family were observed in their sequences. To obtain the recombinant enzymes, we tried to express the cDNAs in Pichia pastoris of heterologous host cells. Although recombinant HBG I was not produced, the recombinant HBG II and III of 2.4 and 1.2 U/mg were respectively expressed and secreted into culture supernatant. The active recombinant enzymes purified had the same properties as those of native ones except sugar content. To investigate the catalytic residues in HBGs, four mutated enzymes (D206N, E259Q, E269Q and D33 1N) of HBG III were constructed, and their specific activities were found to be 0.0004, 4.9, 0.004 and 0.0002 U/mg, respectively. E259Q remained half activity of wild type and those of the others disappeared, implying that three catalyticresidues of HBGs were D212, E281 and D343 of HBG I, D202, E271 and D333 of HBG II, D206, E269 and D331 of HBG III. The homology modeling showed that three enzymes had Ndomain ((β/α)8 barrel), subdomain, and C-domain(β-sheet structure mainly) like oligo-l, 6-glucosidase from Bacillus cereus.
  • 奥山正幸, 森春英, 木村淳夫, 千葉誠哉
    J Appl Glycosci 49 2 211 - 219 The Japanese Society of Applied Glycoscience 2002年04月01日 [査読無し][通常論文]
     
    cDNA encoding Schizosaccharomyces pombe a-glucosidase was cloned, and expressed in Saccharomyces cerevisiae. The deduced amino acid sequence categorized under the α-glucosidase family II showed a high homology to those of a-glucosidase from molds, plants and mammals. By site direct mutagenesis, Asp481, G1u484, and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COON) of the Asp647 residue was for the first time pointed out to be the candidate of proton donor in the a-glucosidase of family II. The carboxylate group (-COO-) of the Asp481 residue was assumed to be the secondary carboxylate group, which stabilize the oxocarbenium ion through electrostatic interaction, and the Asp481 was considered to be modified by the chemical modification with conduritol B epoxide. The role of the G1u484 residue, which was the third residue, was presumed to be to fix the reaction intermediate of substrates.
  • 奥山 正幸, 森 春英, 木村 淳夫, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 49 2 211 - 219 日本応用糖質科学会 2002年04月01日 [査読無し][通常論文]
     
    cDNA encoding Schizosaccharomyces pombe a-glucosidase was cloned, and expressed in Saccharomyces cerevisiae. The deduced amino acid sequence categorized under the α-glucosidase family II showed a high homology to those of a-glucosidase from molds, plants and mammals. By site direct mutagenesis, Asp481, G1u484, and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COON) of the Asp647 residue was for the first time pointed out to be the candidate of proton donor in the a-glucosidase of family II. The carboxylate group (-COO-) of the Asp481 residue was assumed to be the secondary carboxylate group, which stabilize the oxocarbenium ion through electrostatic interaction, and the Asp481 was considered to be modified by the chemical modification with conduritol B epoxide. The role of the G1u484 residue, which was the third residue, was presumed to be to fix the reaction intermediate of substrates.
  • 西本 完, 森 春英, 木村 淳夫, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 49 2 191 - 197 日本応用糖質科学会 2002年04月01日 [査読無し][通常論文]
     
    The genes of three a-glucosidases (HBG I, HBG II and HBG III) were isolated from the cDNA library of honeybee, Apis mellifera L. The nucleotide sequences of HBG I, II and III were consisted of 1974, 1910 and 1916 base pair and encoding 588, 580 and 567 amino acid residues, respectively. The putative primary structures showed high homology ranging from N- to C-terminals, and three enzymes belonged to a-glucosidase family I, in which four conservative regions of aamylase family were observed in their sequences. To obtain the recombinant enzymes, we tried to express the cDNAs in Pichia pastoris of heterologous host cells. Although recombinant HBG I was not produced, the recombinant HBG II and III of 2.4 and 1.2 U/mg were respectively expressed and secreted into culture supernatant. The active recombinant enzymes purified had the same properties as those of native ones except sugar content. To investigate the catalytic residues in HBGs, four mutated enzymes (D206N, E259Q, E269Q and D33 1N) of HBG III were constructed, and their specific activities were found to be 0.0004, 4.9, 0.004 and 0.0002 U/mg, respectively. E259Q remained half activity of wild type and those of the others disappeared, implying that three catalyticresidues of HBGs were D212, E281 and D343 of HBG I, D202, E271 and D333 of HBG II, D206, E269 and D331 of HBG III. The homology modeling showed that three enzymes had Ndomain ((β/α)8 barrel), subdomain, and C-domain(β-sheet structure mainly) like oligo-l, 6-glucosidase from Bacillus cereus.
  • OKUYAMA Masayuki, MORI Haruhide, WATANABE Kotomi, KIMURA Atsuo, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 66 4 928 - 933 社団法人日本農芸化学会 2002年04月 [査読無し][通常論文]
     
    Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe α-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an α-glucosidic linkage from β-glucosyl fluoride and 4-nitrophenyl (PNP) α-glucoside to produce two kinds of PNP α-diglucosides, α-isomaltoside and α-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP α-isomaltoside and α-maltoside, respectively. PNP monoglycosides, such as α-xyloside, α-mannoside, or β-glucoside, acted as the substrate, but PNP α-galac...
  • Nishimoto M, Mori H, Kimura A & Chiba S, "Study on Three a-Glucosidase Isozymes from Honeybee, Apis mellifera L." J. Appl. Glycosci., 49(2) 191-197, 2002.
    2002年 [査読無し][通常論文]
  • Okuyama M, Mori H, Kimura A & Chiba S, "Catalytic Amino Acid Residue Providing Proton Donar in alpha-Glucosidase Family II" J.Appl.Glycosci., 49(2) 211-219, 2002
    2002年 [査読無し][通常論文]
  • Svensson B, Sauer J, Mori H, Jensen MT, Bak-Jensen KS, Kramhoft B, Juge N, Nohr J, Greffe L, Framdsem TP, Palcic MM, Williamson G, and Driguez H, "(Gluco)amylases, what have we learned so far ?" Carbohydrate Bioengineering, 67-75, 2002.
    2002年 [査読無し][通常論文]
  • 福田 健二, 白川 康, 森 春英, 奥山 正幸, 木村 淳夫, 尾崎 八郎, 米山 道男, 千葉 誠哉
    Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ 49 3 265 - 272 日本応用糖質科学会 2002年 [査読無し][通常論文]
     
    The substrate specificity and the active site of β-D-glucosidase F<SUP>-1</SUP> (Mr, 50, 000; optimum pH, 5.5) purified from a Streptomyces sp. were kinetically investigated. The β-D-glucosidase showed a broad substrate specificity for synthetic glycosides and disaccharides having β-glycosidic linkage, but the former was more favorable substrate than the latter. The enzyme was characterized by the ability to hydrolyze rapidly not only ρ-nitrophenyl β-glucoside (K<SUB>m</SUB>, 0.72 mM; k<SUB>0</SUB>, 63 s<SUP>-1</SUP>) but also ρ-nitrophenyl β-fucoside (K<SUB>m</SUB>, 0.19 mM; k<SUB>0</SUB>,...
  • B Svensson, J Sauer, H Mori, MT Jensen, KS Bak-Jensen, B Kramhoft, N Juge, J Nohr, L Greffe, TP Frandsen, MM Palcic, G Williamson, H Driguez
    CARBOHYDRATE BIOENGINEERING: INTERDISCIPLINARY APPROACHES 275 67 - 75 2002年 [査読有り][通常論文]
  • JH Lee, M Tsuji, M Nakamura, M Nishimoto, M Okuyama, H Mori, A Kimura, H Matsui, S Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 12 2657 - 2665 2001年12月 [査読無し][通常論文]
     
    Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl beta -glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1% carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (K-m) was 0.66 nim, and the molecular activity (k(0)) was 86.2 s(-1). The enzyme was an "inverting" type which produced beta -glucose from alpha, alpha -trehalose. Dependence of the V and K-m values on pH gave values for the ionization constants, pKe(1) and pKe(2), of essential ionizable groups I and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe(1), and pKe(2) were shifted to higher values of + 0.2 and + 0.5 pH unit, respectively. The ionization heat (DeltaH) of ionizable group I was estimated to be + 1.8 kcal/mol, and the DeltaH value of group 2 was + 1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (-COO-, ionizable group 1) and the other a protonated type (-COOH, ionizable group 2), although the pKe(2) value is high.
  • 水野 隆文, 森 春英, 西本 完, 伊藤 浩之, 松井 博和, 木村 淳夫, 本間 守, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 48 3 287 - 291 日本応用糖質科学会 2001年07月01日 [査読無し][通常論文]
     
    Brevibacterium fuscum var.dextranlyticum0407株のゲノミックライブラリーよりα-グルコシダーゼ様遺伝子（dexG）のクローニングを行いその配列を決定した。遺伝子はイソマルトトリオーデキストラナーゼ遺伝子（dexT）の5'末端側上流に隣在し、全長1725bpであった。本遺伝子産物は、そのアミノ酸配列からα-グルコシダーゼファミリーIに分類され、Bacillus属由来オリゴ-1、6-グルコシダーゼおよびStreptococcus mutans由来デキストラングルコシダーゼなどと高い相同性を示した。dexGのさらに上流に、糖質輸送に関わる膜タンパク質をコードすると推定される遺伝子群が存在し、dexG、dexTとともにデキストランの資化に関連する遺伝子オペロンを形成していると推定された。
  • NISHIMOTO Mamoru, KUBOTA Masaki, TSUJI Masahisa, MORI Haruhide, KIMURA Atsuo, MATSUI Hirokazu, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 65 7 1610 - 1616 社団法人日本農芸化学会 2001年07月 [査読無し][通常論文]
     
    α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. Th...
  • 水野 隆文, 森 春英, 西本 完, 伊藤 浩之, 松井 博和, 木村 淳夫, 本間 守, 千葉 誠哉
    Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ 48 3 287 - 291 日本応用糖質科学会 2001年 [査読無し][通常論文]
     
    A putative α-glucosidase gene was isolated from the genomic library of Brevibacterium fuscumvar. dextranlyticum strain 0407. The gene, designated dexG, was located upstream of isomaltotriodextranase gene (dexT). The dexG contained an open reading frame of 1725 bp, and its deduced amino acid sequence (DexG) showed a high homology with the enzymes belonging to α-glucosidase Family I and I-like, especially oligo-l, 6-glucosidase from Bacillus sp. and dextran glucosidase from Streptococcus mutans. The DexG has four conserved regions shared with aamylases. In the cloned genomic fragment there we...
  • T Mizuno, H Mori, H Ito, H Matsui, A Kimura, S Chiba
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 63 9 1582 - 1588 1999年09月 [査読無し][通常論文]
     
    The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M-r, 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.
  • 佐伯 雄史, 奥山 正幸, 森 春英, 木村 淳夫, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 45 3 281 - 283 日本応用糖質科学会 1998年08月31日 [査読無し][通常論文]
  • 森 春英, 小林 哲也, 殿川 隆志, 立松 あゆみ, 松井 博和, 木村 淳夫, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 45 3 261 - 267 日本応用糖質科学会 1998年08月31日 [査読無し][通常論文]
  • 野崎 功一, 松井 博和, 殿川 隆, 森 春英, 伊藤 浩之, 本間 守, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 45 2 117 - 122 日本応用糖質科学会 1998年06月30日 [査読無し][通常論文]
  • KIMURA Atsuo, TAKATA Masuhiro, FUKUSHI Yukiharu, MORI Haruhide, MATSUI Hirokazu, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 61 7 1091 - 1098 社団法人日本農芸化学会 1997年07月 [査読無し][通常論文]
     
    The catalytic amino acid residue of Aspergillus niger α-glucosidase (ANGase) was identified by modification with conduritol B epoxide (CBE), a mechanism-based irreversible inactivator. The inactivation by CBE followed pseudo-first order kinetics. The interaction of CBE and ANGase conformed to a model with a reversible enzyme-inhibitor complex formed before covalent inactivation. A competitive inhibitor, Tris, decreased the inactivation rate. The incorporation of one mole of CBE per mole of ANGase was completely abolished the enzyme activity. A dissociated carboxyl group (-COO-) in the activ...
  • 松井 博和, 岩波 俊介, 伊藤 浩之, 木村 淳夫, 森 春英, 本間 守, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 44 2 245 - 252 日本応用糖質科学会 1997年06月30日 [査読無し][通常論文]
  • 木村 淳夫, 高柳 勉, 森 春英, 松井 博和, 魚住 武司, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 44 2 233 - 243 日本応用糖質科学会 1997年06月30日 [査読無し][通常論文]
  • 松井博和, 岩波俊介, 伊藤浩之, 木村淳夫, 森春英, 本間守, 千葉誠哉
    応用糖質科学 44 2 245-252  1997年06月 [査読無し][通常論文]
  • 木村淳夫, 高柳勉, 森春英, 魚住武司, 千葉誠哉, 松井博和
    応用糖質科学 44 2 233-243  1997年06月 [査読無し][通常論文]
  • MATSUI Hirokazu, IWANAMI Shunsuke, ITO Hiroyuki, MORI Haruhide, HONMA Mamoru, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 61 5 875 - 880 社団法人日本農芸化学会 1997年05月 [査読無し][通常論文]
     
    A cDNA encoding sugar beet α-glucosidase was cloned from a library constructed from mRNA of suspension-cultured cells. The cDNA, 3056bp in length, had an open reading frame encoding a polypeptide of 913 amino acid residues with a molecular mass of 102,078 Da, included only one of four regions which were conserved in the α-amylase family of enzymes. The deduced amino acid sequence from the analysis of the cDNA contained the sequences of the proteolysis peptides and the active site region peptide of sugar beet α-glucosidase. The primary structure indicated relatively high homology in the rang...
  • KIMURA Atsuo, SOMOTO Akishige, MORI Haruhide, SAKAI Osamu, MATSUI Hirokazu, CHIBA Seiya
    Bioscience, biotechnology, and biochemistry 61 3 475 - 479 社団法人日本農芸化学会 1997年03月 [査読無し][通常論文]
     
    A kinetic study was done to identify the ionizable groups in the active site of Aspergillus niger α-glucosidase (ANGase). From dependence of V and K_m values on pH, we obtained the ionization constants of essential ionizable groups 1 and 2 of free enzyme; pKe_1 = 3.2 and pKe_2 = 6.4. When the dielectric constant of the reaction mixture was decreased, the pKe_1 and pKe_2 were shifted to higher values. The ionization heats (ΔH's) of ionizable groups 1 and 2 were measured to be - 0.4 kcal/mol and 0 kcal/mol, respectively. The water-soluble carbodiimide (WSC), a specific reagent for carboxyl gr...
  • S Onodera, T Murakami, H Ito, H Mori, H Matsui, M Honma, S Chiba, N Shiomi
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 60 11 1780 - 1785 1996年11月 [査読無し][通常論文]
     
    A cDNA and a gene encoding endo-inulinase from Penicillium purpurogenum were isolated, and were cloned for the first time. Two oligonucleotide probes, which were synthesized based on the partial amino acid sequences of the purified endo-inulinase, were used to screen a cDNA library. A 1.7-kb DNA fragment encoding endo-inulinase was isolated and analyzed. A single open reading frame, consisting of 1548-bp, was found to encode a polypeptide that comprised a 25-amino acid signal peptide and 490-amino acid mature protein. All the partial amino acid sequences of the purified enzyme were discovered in the deduced ones. The deduced amino acid sequences of endo-inulinase had similar sequences to those of fructan hydrolases. A 3.5-kb chromosomal DNA fragment encoding endo-inulinase was also isolated and analyzed. The same ORF with the cDNA clone was identified. There were no introns in the endo-inulinase gene.
  • 水野 隆文, 松井 博和, 伊藤 浩之, 森 春英, 木村 淳夫, 本間 守, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 43 3 347 - 353 日本応用糖質科学会 1996年08月31日 [査読無し][通常論文]
  • 森 春英, 立松 あゆみ, 斎藤 明子, 松井 博和, 木村 淳夫, 千葉 誠哉
    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience 42 4 387 - 394 日本応用糖質科学会 1995年12月01日 [査読無し][通常論文]
  • Iwanami Shunsuke, Matsui Hirokazu, Kimura Atsuo, Ito Hiroyuki, Mori Haruhide, Honma Mamoru, Chiba Seiya
    Bioscience, biotechnology, and biochemistry 59 3 459 - 463 社団法人日本農芸化学会 1995年03月 [査読無し][通常論文]
     
    The modification of amino acid residues in sugar beet α-glucosidase with conduritol B epoxide (CBE), an affinity labeling reagent, inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The enzyme was protected from inactivation by a competitive inhibitor, Tris, and the partially inactivated enzymes showed only the decrease of V values and no change in Km value. An ^3H-CBE labeled peptide isolated from the digest of the inactivated enzyme with Lys-C protease was sequenced. The -COO - group of Asp was found to be specifically labeled, implicating that it is a catalyti...
  • Kawagishi H, Mori H, Uno A, Kimura A, Chiba S
    FEBS Lett 340 1 56 - 58 1994年 [査読有り][通常論文]
  • H MORI, A TATEMATSU, H MATSUI, T TAKAYANAGI, M HONMA, S CHIBA
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 56 9 1499 - 1500 1992年09月 [査読無し][通常論文]
  • H MORI, K HIGO, H HIGO, Y MINOBE, H MATSUI, S CHIBA
    PLANT MOLECULAR BIOLOGY 18 5 973 - 976 1992年03月 [査読無し][通常論文]

MISC

  • 太田 智也, 佐分利 亘, 森 春英 化学と生物 62 (8) 362 -364 2024年08月 [査読無し][通常論文]
  • 内山昌典, 佐分利亘, 武井梓穂, 尾瀬農之, 森春英 日本農芸化学会北海道支部学術講演会講演要旨集(Web) 2023 2023年
  • 太田智也, 佐分利亘, 山下恵太郎, 田上貴祥, 于健, 今場司朗, ジュウェルリンダ, シャントム, 今井亮三, 姚閔, 森春英 応用糖質科学 13 (1) 2023年 [査読無し][招待有り]
  • 太田智也, 佐分利亘, 今場司朗, 森春英 日本農芸化学会大会講演要旨集(Web) 2023 2023年
  • 太田智也, 佐分利亘, JEWELL Linda, HSIANG Tom, 今井亮三, 森春英 日本栄養・食糧学会北海道支部大会講演要旨集 53rd (CD-ROM) 2023年
  • 太田智也, 佐分利亘, JEWELL Linda, HSIANG Tom, 今井亮三, 森春英 応用糖質科学 13 (3) 2023年
  • 太田智也, 佐分利亘, 今場司朗, JEWELL Linda, HSIANG Tom, 今井亮三, 森春英 日本農芸化学会大会講演要旨集(Web) 2022 2022年
  • 太田智也, 佐分利亘, 山下恵太郎, 田上貴祥, 于健, 今場司朗, JEWELL Linda, HSIANG Tom, 今井亮三, 姚閔, 森春英 応用糖質科学 12 (3) 2022年
  • 太田智也, 佐分利亘, JEWELL Linda, HSIANG Tom, 今井亮三, 森春英 応用糖質科学 10 (4) 2020年
  • 太田智也, 佐分利亘, JEWELL Linda, HSIANG Tom, 今井亮三, 森春英 日本農芸化学会大会講演要旨集(Web) 2020 2020年
  • 太田智也, 佐分利亘, 今井亮三, 森春英 応用糖質科学 9 (3) 2019年
  • 阪中幹祥, 阪中幹祥, 阪中幹祥, 中川路伸吾, 中島森, 阿部光紗, 佐分利亘, 森春英, 横田篤, 吹谷智 日本農芸化学会大会講演要旨集(Web) 2019 2019年
  • 佐分利亘, 加藤公児, 于健, 姚閔, 森春英 応用糖質科学 8 (3) (40) 2018年08月20日 [査読無し][通常論文]
  • 手塚大介, 手塚大介, 佐分利亘, 森春英, 松浦英幸, 今井亮三 植物の生長調節 53 (Supplement) 2018年
  • 手塚大介, 手塚大介, 川又彩, 加藤英樹, 佐分利亘, 森春英, 今井亮三, 今井亮三 日本植物細胞分子生物学会大会・シンポジウム講演要旨集 36th 2018年
  • Wade Abbott, Orly Alber, Ed Bayer, Jean-Guy Berrin, Alisdair Boraston, Harry Brumer, Ryszard Brzezinski, Anthony Clarke, Beatrice Cobucci-Ponzano, Darrell Cockburn, Pedro Coutinho, Mirjam Czjzek, Bareket Dassa, Gideon John Davies, Vincent Eijsink, Jens Eklof, Alfons Felice, Elizabeth Ficko-Blean, Geoff Pincher, Thierry Fontaine, Zui Fujimoto, Kiyotaka Fujita, Shinya Fushinobu, Harry Gilbert, Tracey Gloster, Ethan Goddard-Borger, Ian Greig, Jan-Hendrik Hehemann, Glyn Hemsworth, Bernard Henrissat, Masafumi Hidaka, Ramon Hurtado-Guerrero, Kiyohiko Igarashi, Takuya Ishida, Stefan Janecek, Seino Jongkees, Nathalie Juge, Satoshi Kaneko, Takane Katayama, Motomitsu Kitaoka, Naotake Konno, Daniel Kracher, Anna Kulminskaya, Alicia Lammerts van Bueren, Sine Larsen, Junho Lee, Markus Linder, Leila LoLeggio, Roland Ludwig, Ana Luis, Mirko Maksimainen, Brian Mark, Richard McLean, Gurvan Michel, Gurvan Michel, Cedric Montanier, Marco Moracci, Haruhide Mori, Hiroyuki Nakai, Wim Nerinckx, Takayuki Ohnuma, Richard Pickersgill, Kathleen Piens, Tirso Pons, Etienne Rebuffet, Peter Reilly, Magali Remaud-Simeon, Brian Rempel, Kyle Robinson, David Rose, Juha Rouvinen, Wataru Saburi, Yuichi Sakamoto, Mats Sandgren, Fathima Shaikh, Yuval Shoham, Franz St John, Jerry Stahlberg, Michael Suits, Gerlind Sulzenbacher, Tomomi Sumida, Ryuichiro Suzuki, Birte Svensson, Toki Taira, Ed Taylor, Takashi Tonozuka, Breeanna Urbanowicz, Gustav Vaaje-Kolstad, Wim Van den Ende, Annabelle Varrot, Maxime Versluys, Florence Vincent, David Vocadlo, Warren Wakarchuk, Tom Wennekes, Rohan Williams, Spencer Williams, David Wilson, Stephen Withers, Katsuro Yaoi, Vivian Yip, Ran Zhang GLYCOBIOLOGY 28 (1) 3 -8 2018年01月 
    CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.
  • 武藤洋彦, 佐分利亘, 藤原孝彰, 加藤公児, YAO Min, 森春英 日本農芸化学会大会講演要旨集(Web) 2017 ROMBUNNO.3J33p07 (WEB ONLY) 2017年03月05日 [査読無し][通常論文]
  • 手塚大介, 手塚大介, 川又彩, 加藤英樹, 佐分利亘, 森春英, 今井亮三 植物の生長調節 51 (Supplement) 59 2016年10月07日 [査読無し][通常論文]
  • 田口陽大, 佐分利亘, 今井亮三, 森春英 応用糖質科学 6 (3) 46 2016年08月20日 [査読無し][通常論文]
  • 佐分利亘, 加藤公児, 姚閔, 松井博和, 森春英 応用糖質科学 6 (3) 65 -65 2016年08月20日 [査読無し][通常論文]
  • 田口陽大, 佐分利亘, 今井亮三, 今井亮三, 森春英 日本農芸化学会大会講演要旨集(Web) 2016 4D028 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  • 武藤洋彦, 佐分利亘, 森春英 日本農芸化学会大会講演要旨集(Web) 2016 4D008 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  • 貞廣樹里, 森春英, 佐分利亘, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集(Web) 2016 4D013 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  • 坂井未悠, 佐分利亘, 森春英 日本農芸化学会大会講演要旨集(Web) 2016 2E063 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  • 塩田咲耶子, 尾高伶, 佐分利亘, YE Yuxin, 薦田圭介, 加藤公児, 西本完, 北岡本光, YAO Min, 森春英 日本農芸化学会大会講演要旨集(Web) 2016 4D009 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  • 手塚 大介, 和久田 真司, 加藤 英樹, 松浦 英幸, 佐分 利亘, 森 春英, 松井 博和, 今井 亮三 植物化学調節学会研究発表記録集 50 (0) 73 -73 2015年10月01日 [査読無し][通常論文]
  • 手塚大介, 手塚大介, 和久田真司, 加藤英樹, 松浦英幸, 佐分利亘, 森春英, 松井博和, 今井亮三, 今井亮三 植物の生長調節 50 (Supplement) 73 2015年10月01日 [査読無し][通常論文]
  • 田上 貴祥, 山下 恵太郎, 奥山 正幸, 森 春英, 姚 閔, 木村 淳夫 応用糖質科学 : 日本応用糖質科学会誌 5 (3) B59 2015年08月20日
  • 川田 恭平, 藤本 瑞, 鐘ケ江 倫世, 西村 崇志, 奥山 正幸, 森 春英, 木村 淳夫 応用糖質科学 : 日本応用糖質科学会誌 5 (3) B41 2015年08月20日
  • 尾高 伶, 佐分 利亘, Ye Yuxin, 薦田 圭介, 加藤 公児, 西本 完, 北岡 本光, 姚 閔, 森 春英 応用糖質科学 : 日本応用糖質科学会誌 5 (3) B42 2015年08月20日 [査読無し][通常論文]
  • 佐分利亘, 奥山正幸, 熊谷祐也, 木村淳夫, 森春英 日本農芸化学会大会講演要旨集(Web) 2015 3E32P02 (WEB ONLY) 2015年03月05日 [査読無し][通常論文]
  • 武藤洋彦, 佐分利亘, 藤原孝彰, YAO Min, 森春英 日本農芸化学会大会講演要旨集(Web) 2015 2E32A08 (WEB ONLY) 2015年03月05日 [査読無し][通常論文]
  • 菅原好美, 佐分利亘, 谷口沙希, 今井亮三, 森春英 日本農芸化学会大会講演要旨集(Web) 2015 2E32A11 (WEB ONLY) 2015年03月05日 [査読無し][通常論文]
  • 田上貴祥, 山下恵太郎, 奥山正幸, 森春英, YAO Min, YAO Min, 木村淳夫 日本農芸化学会大会講演要旨集(Web) 2015 2015年
  • 高橋 直希, 川又 彩, 手塚 大介, 佐分利 亘, 松浦 英幸, 森 春英, 今井 亮三 植物化学調節学会研究発表記録集 49 (0) 37 -37 2014年10月01日 [査読無し][通常論文]
  • 竹松 知紀, 瀬戸 義哉, 宮澤 吉郎, 和久田 真司, 佐分利 亘, 森 春英, 高橋 公咲, 松浦 英幸 植物化学調節学会研究発表記録集 49 (0) 61 -61 2014年10月01日 [査読無し][通常論文]
  • 手塚 大介, 坂井 志帆, 和久田 真司, 加藤 英樹, 松浦 英幸, 佐分利 亘, 森 春英, 松井 博和, 今井 亮三 植物化学調節学会研究発表記録集 49 (0) 70 -70 2014年10月01日 [査読無し][通常論文]
  • 武田 遼介, 佐分利 亘, 姫野 奈美, 和久田 真司, 松浦 英幸, 今井 亮三, 松井 博和, 森 春英 植物化学調節学会研究発表記録集 49 (0) 80 -80 2014年10月01日 [査読無し][通常論文]
  • 武田遼介, 佐分利亘, 姫野奈美, 和久田真司, 松浦英幸, 今井亮三, 松井博和, 森春英 植物の生長調節 49 (Supplement) 80 2014年10月01日 [査読無し][通常論文]
  • 手塚大介, 坂井志帆, 和久田真司, 加藤英樹, 松浦英幸, 佐分利亘, 森春英, 松井博和, 今井亮三 植物の生長調節 49 (Supplement) 70 2014年10月01日 [査読無し][通常論文]
  • 竹松知紀, 瀬戸義哉, 宮澤吉郎, 和久田真司, 佐分利亘, 森春英, 高橋公咲, 松浦英幸 植物の生長調節 49 (Supplement) 61 2014年10月01日 [査読無し][通常論文]
  • 高橋直希, 川又彩, 手塚大介, 佐分利亘, 松浦英幸, 森春英, 今井亮三 植物の生長調節 49 (Supplement) 37 2014年10月01日 [査読無し][通常論文]
  • 城戸悠輔, 佐分利亘, 小島晃代, SHEN Xing, 薦田圭介, 姚閔, 松井博和, 森春英 日本農芸化学会北海道支部講演会講演要旨 2014 32 2014年09月22日 [査読無し][通常論文]
  • Lang Weeranuch, Kumagai Yuya, Sadahiro Juri, Okuyama Masayuki, Mori Haruhide, Sakairi Nobuo, Kimura Atsuo 応用糖質科学 : 日本応用糖質科学会誌 4 (3) B42 2014年08月20日
  • 岩藤伸治, 佐分利亘, 松井博和, 今井亮三, 森春英 応用糖質科学 4 (3) (40) 2014年08月20日 [査読無し][通常論文]
  • 村上祐紀, 佐分利亘, 森春英, 松井博和, 田辺創一, 鈴木卓弥 日本栄養・食糧学会大会講演要旨集 68th 257 2014年04月30日 [査読無し][通常論文]
  • 城戸悠輔, 佐分利亘, 小島晃代, 松井博和, 森春英 日本農芸化学会大会講演要旨集(Web) 2014 2D02A05 (WEB ONLY) 2014年03月05日 [査読無し][通常論文]
  • 尾高伶, 佐分利亘, 福士江里, 西本完, 北岡本光, 松井博和, 森春英 日本農芸化学会大会講演要旨集(Web) 2014 3D02P09 (WEB ONLY) 2014年03月05日 [査読無し][通常論文]
  • 坂谷 敦, 熊谷 祐也, 貞廣 樹里, Weeranuch Lang, 奥山 正幸, 森 春英, 木村 淳夫 応用糖質科学：日本応用糖質科学会誌 4 (3) B39 2014年 [査読無し][通常論文]
  • Saburi Wataru, M. Ueno Hiroshi, Matsui Hirokazu, Mori Haruhide Journal of Applied Glycoscience 61 (2) 53 -57 2014年 [査読無し][通常論文]
     
    Acidophilic β-galactosidase is a useful enzyme as digestive supplement used to alleviate symptoms of lactose intolerance. Aspergilli are the source of several acidophilic β-galactosidases that retain enzymatic activity under gastric conditions. In this study, we investigated the extracellular acidophilic β-galactosidase activity of six Aspergillus niger strains, AHU7104, AHU7120, AHU7217, AHU7294, AHU7295 and AHU7296; A. niger AHU7120 was selected as an enzyme source. β-Galactosidase from A. niger AHU7120 (AnBGal) was purified from culture supernatant. Its N-terminal sequence was identical to that of An01g12150, which belongs to the glycoside hydrolase family 35, from A. niger CBS 513.88. The DNA sequence of AnBGal was identical to An01g12150. Recombinant AnBGal (rAnBGal) harboring yeast α-factor signal sequence was expressed in Pichia pastoris, and 21.9 mg of purified rAnBGal with 129 U/mg of enzyme activity was isolated from 200 mL of culture supernatant. Native and recombinant AnBGal enzymes showed similar pH optima, pH stability, and kinetics for p-nitrophenyl β-D-galactopyranoside and lactose; rAnBGal showed slightly lower thermal stability than the native enzyme. Lactose in milk was rapidly degraded by rAnBGal at higher pH values (range, 2.0‒3.5), consistent with the pH optimum of AnBGal. We estimated that 3.5 μM AnBGal may degrade ≥ 66% of lactose before gastric half-emptying of ingested milk. These data indicate that AnBGal may help alleviate symptoms of lactose intolerance.
  • Jaito Nongluck, Saburi Wataru, Muto Hirohiko, Matsui Hirokazu, Mori Haruhide Journal of Applied Glycoscience 61 (4) 117 -119 2014年 [査読無し][通常論文]
     
    Spectrophotometric quantification method of carbohydrates is useful for processing multiple samples. In this study, we established colorimetric quantification for 4-O-β-D-mannosyl-D-glucose (Man-Glc) and β-(1→4)-mannobiose (Man2). For quantification of Man-Glc, phosphorolysis of Man-Glc catalyzed by 4-O-β-D-mannosyl-D-glucose phosphorylase (MGP) was coupled with quantification of D-glucose by the glucose oxidase-peroxidase method. In addition to MGP, cellobiose 2-epimerase (CE) was added for quantification of Man2. In both quantifications, a good linear relationship was obtained between A505 and the sample concentration (0-0.5 mM). The A505 values obtained at various concentrations of Man2 and Man-Glc were almost identical to those with equivalent D-glucose concentrations. Kinetic parameters of Ruminococcus albus and Rhodothermus marinus CEs for the epimerization of Man2 were determined using the quantification method for Man-Glc. Both enzymes showed 5-15-fold higher kcat/Km values than those for cellobiose and lactose, which supports the prediction that these enzymes utilize Man2 as a substrate in the β-mannan metabolic pathway.
  • 谷口沙希, 佐分利亘, 松浦英幸, 今井亮三, 松井博和, 森春英 日本農芸化学会北海道支部講演会講演要旨 2013 36 2013年11月27日 [査読無し][通常論文]
  • 貞廣樹里, 森春英, 佐分利亘, 奥山正幸, 木村淳夫 日本農芸化学会北海道支部講演会講演要旨 2013 17 2013年11月27日 [査読無し][通常論文]
  • 石塚佐都子, 和久田真司, 佐分利亘, 今井亮三, 森春英 日本農芸化学会北海道支部講演会講演要旨 2013 23 2013年11月27日 [査読無し][通常論文]
  • 谷口沙希, 佐分利亘, 松浦英幸, 今井亮三, 松井博和, 森春英 日本農芸化学会北海道支部講演会講演要旨 2013 23 2013年11月27日 [査読無し][通常論文]
  • 田中佑果, 佐分利亘, 森春英 日本農芸化学会北海道支部講演会講演要旨 2013 25 2013年11月27日 [査読無し][通常論文]
  • 宮本 大司, 奥山 正幸, 松尾 一郎, 森 春英, 木村 淳夫 応用糖質科学 : 日本応用糖質科学会誌 3 (3) B37 2013年08月20日
  • 西堀 貴哉, 鈴木 志保, 森 春英, 木村 淳夫, 秋吉 一成, 北村 進一 応用糖質科学 : 日本応用糖質科学会誌 3 (3) B43 2013年08月20日
  • 小林 桃子, 森 春英, 奥山 正幸, 木村 淳夫 応用糖質科学 : 日本応用糖質科学会誌 3 (3) B38 2013年08月20日
  • 城戸悠輔, 佐分利亘, 小島晃代, 松井博和, 森春英 応用糖質科学 3 (3) 37 2013年08月20日 [査読無し][通常論文]
  • 佐分利亘, 森本奈保喜, 向井惇, KIM Dae Hoon, 竹花稔彦, 小池誠治, 松井博和, 森春英 応用糖質科学 3 (3) 38 2013年08月20日 [査読無し][通常論文]
  • 藤原孝彰, 佐分利亘, 松井博和, 森春英, 田中勲, 姚閔 応用糖質科学 3 (3) 30 2013年08月20日 [査読無し][通常論文]
  • 佐分利亘, 小島晃代, 佐藤央基, 田口秀典, 森春英, 松井博和 応用糖質科学 3 (2) 137 -142 2013年05月20日 [査読無し][通常論文]
     
    セロビオース2-エピメラーゼ(CE)は,セロビオースやラクトースなどβ-1,4結合からなるオリゴ糖の還元末端のグルコース残基をマンノース残基に可逆的に異性化する。CEをラクトースに作用させて得られるエピラクトース(4-O-β-D-ガラクトシル-D-マンノース)は優れた腸内細菌叢改善効果を有し,新たな食品素材として有望である。本研究では,CEを用いたエピラクトースの実用的合成法の確立を目的とした。Ruminococcus albus由来CEのアミノ酸配列を基に配列類似性検索を行い,大規模培養が可能な好気性細菌よりCEホモログを探索した。見出されたCEのうちRhodothermus marinus由来酵素(RmCE)は耐熱性に優れ,エピラクトースの工業的製造に適した特性を備えていた。固定化RmCEによる連続反応では,遊離酵素よりも少ない酵素量でエピラクトースを合成可能なことが示された。反応液を濃縮してラクトースを結晶化,除去した後,樹脂分画もしくは結晶化を行うことでCE反応液からエピラクトースを約90%の純度に精製した。
  • Aki Shinoki, Weeranuch Lang, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka, Hiroshi Hara FASEB JOURNAL 27 2013年04月 [査読無し][通常論文]
  • 姫野奈美, 佐分利亘, 武田遼介, 和久田真司, 松浦英幸, 鍋田憲助, 森春英, 今井亮三, 松井博和 日本農芸化学会大会講演要旨集(Web) 2013 3C16A13 (WEB ONLY) 2013年03月05日 [査読無し][通常論文]
  • 貞廣樹里, 森春英, 佐分利亘, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集(Web) 2013 2C16A12 (WEB ONLY) 2013年03月05日 [査読無し][通常論文]
  • 武田遼介, 姫野奈美, 佐分利亘, 和久田真司, 森春英, 松浦英幸, 鍋田憲助, 今井亮三, 松井博和 日本農芸化学会大会講演要旨集(Web) 2013 3C16A12 (WEB ONLY) 2013年03月05日 [査読無し][通常論文]
  • 城戸悠輔, 佐分利亘, 小島晃代, 森春英, 松井博和 日本農芸化学会大会講演要旨集(Web) 2013 3C16A01 (WEB ONLY) 2013年03月05日 [査読無し][通常論文]
  • 熊谷 祐也, Lang Weeranuch, 貞廣 樹里, 奥山 正幸, 森 春英, 木村 淳夫 応用糖質科学：日本応用糖質科学会誌 3 (3) B46 2013年 [査読無し][通常論文]
  • 玉村尚也, 向井惇, 森本奈保喜, 竹花稔彦, 佐分利亘, 森春英, 小池誠治, 松井博和 日本農芸化学会北海道支部講演会講演要旨 2012 24 2012年11月01日 [査読無し][通常論文]
  • 姫野奈美, 和久田真司, 武田遼介, 佐分利亘, 森春英, 松浦英幸, 鍋田憲助, 今井亮三, 松井博和 日本農芸化学会北海道支部講演会講演要旨 2012 24 2012年11月01日 [査読無し][通常論文]
  • 武田遼介, 姫野奈美, 佐分利亘, 和久田真司, 森春英, 松浦英幸, 鍋田憲助, 今井亮三, 松井博和 日本農芸化学会北海道支部講演会講演要旨 2012 23 2012年11月01日 [査読無し][通常論文]
  • 羽村 健, 佐分 利亘, 森 春英, 松井 博和 応用糖質科学 : 日本応用糖質科学会誌 2 (3) B37 2012年08月20日 [査読無し][通常論文]
  • 佐分 利亘, 小島 晃代, 佐藤 央基, 田口 秀典, 森 春英, 松井 博和 応用糖質科学 : 日本応用糖質科学会誌 2 (3) B58 2012年08月20日 [査読無し][通常論文]
  • 藤原孝彰, 佐分利亘, 井上聡太, 森春英, 松井博和, 姚閔, 田中勲 応用糖質科学 2 (3) (53) 2012年08月20日 [査読無し][通常論文]
  • 田上貴祥, 山下恵太郎, 田中良幸, 奥山正幸, 森春英, 姚閔, 木村淳夫 応用糖質科学 2 (3) (34) 2012年08月20日 [査読無し][通常論文]
  • 羽村健, 佐分利亘, 森春英, 松井博和 応用糖質科学 2 (3) (37) 2012年08月20日 [査読無し][通常論文]
  • 佐分利亘, 小島晃代, 佐藤央基, 田口秀典, 森春英, 松井博和 応用糖質科学 2 (3) (58) 2012年08月20日 [査読無し][通常論文]
  • 澤野達也, 佐分利亘, 森春英, 松井博和 応用糖質科学 2 (3) (38) 2012年08月20日 [査読無し][通常論文]
  • 尾高伶, 佐分利亘, 川原良介, 森春英, 松井博和 応用糖質科学 2 (3) (38) 2012年08月20日 [査読無し][通常論文]
  • 佐藤央基, 佐分利亘, 小島晃代, 田口秀典, 森春英, 松井博和 日本農芸化学会大会講演要旨集(Web) 2012 4C10A04 (WEB ONLY) 2012年03月05日 [査読無し][通常論文]
  • 羽村健, 佐分利亘, 森本奈保喜, 森春英, 松井博和 日本農芸化学会大会講演要旨集(Web) 2012 2C10P21 (WEB ONLY) 2012年03月05日 [査読無し][通常論文]
  • 鈴木喜大, 金泳みん, 金泳みん, 藤本瑞, 門間充, 奥山正幸, 森春英, 舟根和美, 木村淳夫 PFシンポジウム要旨集 29th 28 2012年 [査読無し][通常論文]
  • Teruyo Ojima, Wataru Saburi, Hiroki Sato, Takeshi Yamamoto, Haruhide Mori, Hirokazu Matsui BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (11) 2162 -2168 2011年11月 [査読無し][通常論文]
     
    Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of beta-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinas DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 degrees C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 degrees C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.
  • 森春英 飯島記念食品科学振興財団年報 2009 348 2011年08月 [査読無し][通常論文]
  • Momoko Kobayashi, Hironori Hondoh, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (8) 1557 -1563 2011年08月 [査読無し][通常論文]
     
    Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl2 increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl2 also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca2+, were considered to stabilize SmDG. On the other hand, CaCl2 had no significant effect on catalytic reaction. The enhanced stability by Ca2+ was probably related to calcium binding in the beta -> alpha loop 1 of the (beta/alpha)(8) barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.
  • 阪本安希, 中島碧, 田口秀典, 佐分利亘, 森春英, 松井博和 応用糖質科学 1 (3) (50) 2011年07月20日 [査読無し][通常論文]
  • 羽村健, 阿部正太郎, 河内慎平, 森本奈保喜, 佐分利亘, 森春英, 松井博和 応用糖質科学 1 (3) (51) 2011年07月20日 [査読無し][通常論文]
  • 青山泰, 西村崇志, 鐘ケ江倫世, 本同宏成, 奥山正幸, 森春英, 木村淳夫 応用糖質科学 1 (3) (36) 2011年07月20日 [査読無し][通常論文]
  • 姫野奈美, 和久田真司, 佐分利亘, 森春英, 松浦英幸, 鍋田憲助, 今井亮三, 松井博和 応用糖質科学 1 (3) (47) 2011年07月20日 [査読無し][通常論文]
  • 小島晃代, 佐分利亘, 佐分利亘, 山本健, 佐藤央基, 森春英, 松井博和 応用糖質科学 1 (3) (51) 2011年07月20日 [査読無し][通常論文]
  • 小林桃子, 山下恵太郎, 田上貴祥, 本同宏成, 森春英, 奥山正幸, 姚閔, 木村淳夫 応用糖質科学 1 (3) (37) 2011年07月20日 [査読無し][通常論文]
  • 田上貴祥, 奥山正幸, 森春英, 木村淳夫 応用糖質科学 1 (3) (36) 2011年07月20日 [査読無し][通常論文]
  • 川原良介, 伊藤重陽, 田口秀典, 佐分利亘, 森春英, 松井博和 応用糖質科学 1 (3) (51) 2011年07月20日 [査読無し][通常論文]
  • アンストーン ワスサン, 佐分利亘, 和久田真司, 濱田茂樹, 伊藤浩之, 森春英, 今井亮三, 松井博和 応用糖質科学 1 (3) (34) 2011年07月20日 [査読無し][通常論文]
  • 平内亨, 牧孝多朗, 森春英, 奥山正幸, 木村淳夫 応用糖質科学 1 (3) (36) 2011年07月20日 [査読無し][通常論文]
  • 宮崎剛亜, 松田佳奈, 森春英, 北岡本光, 北野克和, 西河淳, 殿塚隆史 応用糖質科学 1 (3) (35) 2011年07月20日 [査読無し][通常論文]
  • 齊藤みどり, KANG Hee‐Kwon, 森春英, 奥山正幸, 藤本瑞, 舟根和美, 小林幹彦, 木村淳夫 応用糖質科学 1 (3) (39) 2011年07月20日 [査読無し][通常論文]
  • Young-Min Kim, Ryoko Shimizu, Hiroyuki Nakai, Haruhide Mori, Masayuki Okuyama, Min-Sun Kang, Zui Fujimoto, Kazumi Funane, Doman Kim, Atsuo Kimura APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 91 (2) 329 -339 2011年07月 [査読無し][通常論文]
     
    Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCG Delta) or N-VR/C-VR (TM-Delta CG Delta) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-Delta CG Delta did not accept any further protease-degradation during long storage. TM-NCG Delta and TM-Delta CG Delta enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.
  • 中島碧, 伊藤重陽, 田口秀典, 佐分利亘, 森春英, 松井博和 日本農芸化学会大会講演要旨集 2011 106 2011年03月05日 [査読無し][通常論文]
  • 阿部正太郎, 羽村健, 河内慎平, 伊藤重陽, 森本奈保喜, 佐分利亘, 森春英, 松井博和 日本農芸化学会大会講演要旨集 2011 44 2011年03月05日 [査読無し][通常論文]
  • 桝田安志, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2011 43 2011年03月05日 [査読無し][通常論文]
  • 小林桃子, 本同宏成, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2011 43 2011年03月05日 [査読無し][通常論文]
  • 平内亨, 牧孝多朗, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2011 44 2011年03月05日 [査読無し][通常論文]
  • 齋藤みどり, KANG Hee‐Kwon, 森春英, 奥山正幸, 藤本瑞, 舟根和美, 小林幹彦, 木村淳夫 日本農芸化学会大会講演要旨集 2011 42 2011年03月05日 [査読無し][通常論文]
  • 澤野達也, 佐分利亘, 森本奈保喜, 森春英, 松井博和 日本農芸化学会北海道支部講演会講演要旨 2011 14 2011年 [査読無し][通常論文]
  • 向井惇, 金大勳, 森本奈保喜, 竹花稔彦, 佐分利亘, 森春英, 小池誠治, 松井博和 日本農芸化学会北海道支部講演会講演要旨 2011 14 2011年 [査読無し][通常論文]
  • 佐藤央基, 佐分利亘, 小島晃代, 田口秀典, 森春英, 松井博和 日本農芸化学会北海道支部講演会講演要旨 2011 7 2011年 [査読無し][通常論文]
  • Wakuta Shinji, Hamada Shigeki, Ito Hiroyuki, IMAI Ryozo, MORI Haruhide, MATSUURA Hideyuki, NABETA Kensuke, MATSUI Hirokazu J. Appl. Glycosci. 58 (2) 67 -70 2011年 [査読無し][通常論文]
  • 寺田智明, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会東北支部大会プログラム・講演要旨集 145th 49 2010年09月27日 [査読無し][通常論文]
  • 砂守このみ, 森春英, 奥山正幸, 森本奈保喜, 松井博和, 木村淳夫 日本農芸化学会東北支部大会プログラム・講演要旨集 145th 49 2010年09月27日 [査読無し][通常論文]
  • 西村崇志, 鐘ケ江倫世, 本同宏成, 奥山正幸, 森春英, 木村淳夫 J Appl Glycosci 57 (Suppl.) 43 2010年07月20日 [査読無し][通常論文]
  • 田上貴祥, 奥山正幸, 森春英, 木村淳夫 J Appl Glycosci 57 (Suppl.) 44 2010年07月20日 [査読無し][通常論文]
  • 寺田智明, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2010 22 2010年03月05日 [査読無し][通常論文]
  • 貞廣樹里, 佐分利亘, 森春英, 奥山正幸, 岡田嚴太郎, 木村淳夫 日本農芸化学会大会講演要旨集 2010 22 2010年03月05日 [査読無し][通常論文]
  • 田上貴祥, 西村崇志, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2010 22 2010年03月05日 [査読無し][通常論文]
  • カン ヒゴン, キム ヨンミン, 中井 博之, カン ミンソン, 袴田 航, 奥山 正幸, 森 春英, 西尾 俊幸, 木村 淳夫 J. Appl. Glycosci. 57 (4) 269 -277 2010年 [査読無し][通常論文]
     
    Streptococcus mutans ATCC 25175由来のエンド型デキストラナーゼ(SmDex)に対し，3種類のω-エポキシアルキルα-D-グルコピラノシド(3′,4′-エポキシブチルα-D-グルコピラノシド(E4G)，4′,5′-エポキシペンチルα-D-グルコピラノシド(E5G)および5′,6′-エポキシヘキシルα-D-グルコピラノシド(E6G)：アグリコンのアルキル鎖長が異なる)を作用させると，SmDexは擬一次的な活性低下を示した．アルキル鎖長に依存した失活が認められ，失活の度合いはE5G > E6G > E4Gであった．したがってω-エポキシアルキルα-D-グルコピラノシドのグルコース残基とエポキシ基の距離が，SmDexの失活に対し重要であることが判明した．E5Gは可逆的な中間体を形成する失活機構(自殺基質型の失活機構)を与え，不活性化の一次定数(k)と中間体の解離定数(KR)はそれぞれ0.44 min-1および1.45 mMと算出された．SmDexの加水分解反応の生成物であるイソマルトースの存在によりE5Gの失活が防御されたため，E5GはSmDexの触媒部位に結合すると示唆された．本論文は，ω-エポキシアルキルα-D-グルコピラノシドがエンド型デキストラナーゼの自殺基質になることを示す初めての報告である．
  • KANG Min-sun, OKUYAMA Masayuki, MORI Haruhide, KIMURA Atsuo Biochimie 91 (11-12) 1434 -1442 2009年11月 [査読無し][通常論文]
     
    Genome analysis of Lactobacillus johnsonii NCC533 has been recently completed. One of its annotated genes, lj0569, encodes the protein having the conserved domain of glycoside hydrolase family 31. Its homolog gene (ljag31) in L. johnsonii NBRC13952 was cloned and expressed using an Escherichia coli expression system, resulting in poor production of recombinant LJAG31 protein due to inclusion body formation. Production of soluble recombinant LJAG31 was improved with high concentration of NaCl in medium, possible endogenous chaperone induction by benzyl alcohol, and over-expression of GroES-GroEL chaperones. Recombinant LJAG31 was an alpha-glucosidase with broad substrate specificity toward both homogeneous and heterogeneous substrates. This enzyme displayed higher specificity (in terms of k(cat)/K-m) toward nigerose, maltulose, and kojibiose than other natural substrates having an alpha-glucosidic linkage at the non-reducing end, which suggests that these sugars are candidates for prebiotics contributing to the growth of L. johnsonii. To our knowledge, LJAG31 is the first bacterial alpha-1,3-glucosidase to be characterized with a high k(cat)/K-m value for nigerose [alpha-D-Glcp-(1 -> 3)-D-Glcp]. Transglucosylation of 4-nitrophenyl M-D-glucopyranoside produced two 4-nitrophenyl disaccharides (4-nitrophenyl alpha-nigeroside and 4-nitrophenyl alpha-isomaltoside). These hydrolysis and transglucosylation properties of LJAG31 are different from those of mold (Acremonium implicatum) alpha-1,3-glucosidase of glycoside hydrolase family 31. (C) 2009 Elsevier Masson SAS. All rights reserved.
  • MORI Haruhide, LEE Jin-ha, LEE Jin-ha, OKUYAMA Masayuki, NISHIMOTO Mamoru, OHGUCHI Masao, KIM Doman, KIMURA Atsuo, CHIBA Seiya Biosci Biotechnol Biochem 73 (11) 2466 -2473 2009年11月 [査読無し][通常論文]
     
    Trehalase, an anomer-inverting glycosidase, hydrolyzes only alpha,alpha-trehalose in natural substrates to release equimolecular beta-glucose and alpha-glucose. Since the hydrolytic reaction is reversible, alpha,alpha-[1,1'-H-2]trehalose is capable of synthesis from [1-H-2]glucose through the reverse reaction of trehalase. alpha-Secondary deuterium kinetic isotope effects (alpha-SDKIEs) for the hydrolysis of synthesized alpha,alpha-[1,1'-H-2]trehalose by honeybee trehalase were measured to examine the catalytic reaction mechanism. Relatively high k(H)/k(D) value of 1.53 for alpha-SDKIEs was observed. The data imply that the catalytic reaction of the trehalase occurs by the oxocarbenium ion intermediate mechanism. In addition, the hydrolytic reaction of glycosidase is discussed from the viewpoint of chemical reactivity for the hydrolysis of acetal in organic chemistry. As to the hydrolytic reaction mechanism of glycosidases, oxocarbenium ion intermediate and nucleophilic displacement mechanisms have been widely recognized, but it is pointed out for the first time that the former mechanism is rational and valid and generally the latter mechanism is unlikely to occur in the hydrolytic reaction of glycosidases.
  • 中塚大地, 本同宏成, 大塚博昭, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 J Appl Glycosci 56 (Suppl.) 37 2009年07月20日 [査読無し][通常論文]
  • 西村崇志, 鐘ケ江倫世, KLM Young‐Min, 本同宏成, 奥山正幸, 森春英, 木村淳夫 J Appl Glycosci 56 (Suppl.) 37 2009年07月20日 [査読無し][通常論文]
  • 田上貴祥, 奥山正幸, 森春英, 田口和憲, 木村淳夫 J Appl Glycosci 56 (Suppl.) 38 2009年07月20日 [査読無し][通常論文]
  • 吉田拓弥, 奥山正幸, 本同宏成, 銚閔, 森春英, 木村淳夫 J Appl Glycosci 56 (Suppl.) 38 2009年07月20日 [査読無し][通常論文]
  • 本同宏成, 大塚博昭, 中塚大地, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 J Appl Glycosci 56 (Suppl.) 37 -33 2009年07月20日 [査読無し][通常論文]
  • 本同 宏成, 大塚 博昭, 佐分利 亘, 森 春英, 奥山 正幸, 木村 淳夫 Journal of applied glycoscience 56 (2) 111 -117 2009年04月20日 [査読無し][通常論文]
     
    Streptococcus mutans由来のdextran glucosidase(DGase)は，デキストランやイソマルトオリゴ糖の非還元末端のα-1,6結合を加水分解し，α-glucoseを遊離する保持型酵素である．またDGaseは，一次構造に基づきGH family 13に分類されることや，高い糖転移活性を示すことも明らかとなっている．本研究では，α-1,6結合に特異的に作用するDGaseの基質認識機構をX線結晶構造解析により解明した．X線結晶構造解析の結果，DGaseは(β/α)8バレル構造を持ち，その活性部位はポケット状になっていることが明らかとなった．イソマルトトリオース複合体の構造解析より，三つのサブサイト(-1～+2)が確認された．サブサイト-1において基質は多くの水素結合により認識されており，その様式はGH family 13の他の酵素と類似していた．加えてAsp60とArg398が基質のグルコース残基のO4原子と水素結合を形成しており，これらの結合が基質の非還元末端の認識に重要であると考えられる．サブサイト+1では，Lys275とGlu371が基質と水素結合しており，基質のコンフォメーションを制御していた．加えて切断されるα-1,6結合の6位のメチレン基とVal195の間に疎水的相互作用が確認され，これらの残基が基質認識に重要であることが示唆された．
  • 西村崇志, 鐘ケ江倫世, KIM Young‐Min, 本同宏成, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2009 41 2009年03月05日 [査読無し][通常論文]
  • 田中良幸, 森春英, 河合正悟, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2009 44 2009年03月05日 [査読無し][通常論文]
  • 牧孝多朗, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2009 41 2009年03月05日 [査読無し][通常論文]
  • ウォンチャワリット ジンタナート, 橋床 泰之, 奥山 正幸, 森 春英, 千葉 誠哉, 木村 淳夫 Journal of Applied Glycoscience Supplement 2009 (0) 39 -39 2009年 [査読無し][通常論文]
  • 本同宏成, 大塚博昭, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 J Appl Glycosci 56 (2) 111-117 (J-STAGE) -117 2009年 [査読無し][通常論文]
     
    Streptococcus mutans由来のdextran glucosidase(DGase)は，デキストランやイソマルトオリゴ糖の非還元末端のα-1,6結合を加水分解し，α-glucoseを遊離する保持型酵素である．またDGaseは，一次構造に基づきGH family 13に分類されることや，高い糖転移活性を示すことも明らかとなっている．本研究では，α-1,6結合に特異的に作用するDGaseの基質認識機構をX線結晶構造解析により解明した．X線結晶構造解析の結果，DGaseは(β/α)8バレル構造を持ち，その活性部位はポケット状になっていることが明らかとなった．イソマルトトリオース複合体の構造解析より，三つのサブサイト(-1～+2)が確認された．サブサイト-1において基質は多くの水素結合により認識されており，その様式はGH family 13の他の酵素と類似していた．加えてAsp60とArg398が基質のグルコース残基のO4原子と水素結合を形成しており，これらの結合が基質の非還元末端の認識に重要であると考えられる．サブサイト+1では，Lys275とGlu371が基質と水素結合しており，基質のコンフォメーションを制御していた．加えて切断されるα-1,6結合の6位のメチレン基とVal195の間に疎水的相互作用が確認され，これらの残基が基質認識に重要であることが示唆された．
  • Momoyo Kitamura, Masayuki Okuyama, Fumiko Tanzawa, Haruhide Mori, Yu Kitago, Nobuhisa Watanabe, Atsuo Kimura, Isao Tanaka, Min Yao JOURNAL OF BIOLOGICAL CHEMISTRY 283 (52) 36328 -36337 2008年12月 [査読無し][通常論文]
     
    SusB, an 84-kDa alpha-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6 angstrom resolution. SusB hydrolyzes the alpha-glucosidic linkage, with inversion of anomeric configuration liberating the beta-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only alpha-1,4-glucosidic linkages but also alpha-1,6-, alpha-1,3-, and alpha-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu(532) is positioned to provide protonic assistance to leaving group departure, with Glu(439) and Glu(508) both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of alpha-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.
  • 本同宏成, 大塚博昭, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 J Appl Glycosci 55 (Suppl.) 62 -148 2008年07月20日 [査読無し][通常論文]
  • 森春英, 西塔沙織, 尾川陽, 牧孝多朗, 奥山正幸, 木村淳夫 J Appl Glycosci 55 (Suppl.) 50 -103 2008年07月20日 [査読無し][通常論文]
  • 本同宏成, 大塚博昭, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 J Appl Glycosci 55 (Suppl.) 49 -99 2008年07月20日 [査読無し][通常論文]
  • 奥山正幸, 姚閔, 本同宏成, 北村百世, 森春英, 田中勲, 木村淳夫 J Appl Glycosci 55 (Suppl.) 59 2008年07月20日 [査読無し][通常論文]
  • 貞廣樹里, 佐分利亘, 森春英, 奥山正幸, 岡田嚴太郎, 木村淳夫 J Appl Glycosci 55 (Suppl.) 49 -100 2008年07月20日 [査読無し][通常論文]
  • 鐘ケ江倫世, KIM Young‐Min, 本同宏成, 奥山正幸, 森春英, 木村淳夫 J Appl Glycosci 55 (Suppl.) 50 2008年07月20日 [査読無し][通常論文]
  • 田上貴祥, 奥山正幸, 森春英, 田口和憲, 木村淳夫 J Appl Glycosci 55 (Suppl.) 48 2008年07月20日 [査読無し][通常論文]
  • Hironori Hondoh, Wataru Saburi, Haruhide Mori, Masayuki Okuyama, Toshitaka Nakada, Yoshiki Matsuura, Atsuo Kimura JOURNAL OF MOLECULAR BIOLOGY 378 (4) 913 -922 2008年05月 [査読無し][通常論文]
     
    We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the alpha-1,6-glucosidic linkage of isomaltooli-gosaccharides from their non-reducing ends to produce alpha-glucose. By using the mutant of catalytic acid Glu236 -> Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 angstrom resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (beta/alpha)(8)-barrel in domain A, which is common to the alpha-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites -1 to +2. The environment of the glucose residue at subsite -1 is similar to the environment of this residue in the alpha-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite -1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile alpha-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile alpha-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the alpha-amylase structure from Bacillus subtilis. The comparison with the alpha-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of alpha-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes. (c) 2008 Elsevier Ltd. All rights reserved.
  • 奥山 正幸, カン ミンソン, 矢追 克郎, 三石 安, 森 春英, 木村 淳夫 Journal of applied glycoscience 55 (2) 111 -118 2008年04月20日 [査読無し][通常論文]
     
    大腸菌YicIはα-xylosidaseは，GH31のなかで最も研究が行われている酵素の一つであり，立体構造解析，生化学的な研究が数多く行われている．YicIの酵素反応の至適pHは7.0，pH安定領域，温度領域はそれぞれ4.7-10.1，47°Cまでの範囲内である．YicIはGH31のα-glucosidaseと30%程度の配列類似性を示すが，YicIは厳密に非還元末端のα-xylosyl基を認識している．二つの変異酵素(TIM-barrelドメインのβ→α loop 1をα-glucosidase様に変化させたL1Chi，loop 2に位置するCys307，Phe308をα-glucosidase様シーケンスに置換したC307I/F308D)では，α-xylosidase活性が低下し，α-glucosidase活性が上昇する．YicIのプラス側サブサイトの特異性を糖転移反応受容体特異性により評価した．YicIはエカトリアルの4-OHを選択して受容体とする．またYicIの糖転移反応は1糖受容体に対しα-1,6結合の1生成物を生成する非常に特異性の狭い反応である．これら糖転移産物のうちα-D-xylopyranosyl-(1→6)-D-mannopyranose，α-D-xylopyranosyl-(1→6)-D-fructofuranose，α-D-xylopyranosyl-(1→3)-D-fructopyranoseは新規糖である．さらにα-D-xylopyranosyl-(1→6)-D-mannopyranoseならびにα-D-xylopyranosyl-(1→6)-D-fructofuranoseラット小腸α-glucosidaseに対して緩い阻害を示す．
  • 鐘ケ江倫世, KIM Young‐Min, 中井博之, 本同宏成, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2008 191 2008年03月05日 [査読無し][通常論文]
  • 本同宏成, 大塚博昭, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2008 191 2008年03月05日 [査読無し][通常論文]
  • 西村茉利子, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2008 33 2008年03月05日 [査読無し][通常論文]
  • 貞廣樹里, 佐分利亘, 森春英, 奥山正幸, 岡田嚴太郎, 木村淳夫 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 2008 25 2008年 [査読無し][通常論文]
  • 鐘ケ江倫世, KIM Young‐Min, 本同宏成, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 2008 25 2008年 [査読無し][通常論文]
  • Min-Sun Kang, Masayuki Okuyama, Katsuro Yaoi, Yasushi Mitsuishi, Young-Min Kim, Haruhide Mori, Doman Kim, Atsuo Kimura FEBS JOURNAL 274 (23) 6074 -6084 2007年12月 [査読無し][通常論文]
     
    The specificity of the aglycone-binding site of Escherichia coli alpha-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and alpha-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl-enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, alpha-D-Xylp-(1 -> 6)-D-Manp, alpha-D-Xylp-(1 -> 6)-D-Fruf, and alpha-D-Xylp-(1 -> 3)-D-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by alpha-1,6-xylosidic linkages. Of the transxylosylation products, alpha-D-Xylp-(1 -> 6)-D-Manp and alpha-D-Xylp(1 -> 6)-D-Fruf inhibited intestinal alpha-glucosidases.
  • Min-Sun Kang, Masayuki Okuyama, Katsuro Yaoi, Yasushi Mitsuishi, Young-Min Kim, Haruhide Mori, Doman Kim, Atsuo Kimura FEBS JOURNAL 274 (23) 6074 -6084 2007年12月 [査読無し][通常論文]
     
    The specificity of the aglycone-binding site of Escherichia coli alpha-xylosidase (YicI), which belongs to glycoside hydrolase family 31, was characterized by examining the enzyme's transxylosylation-catalyzing property. Acceptor specificity and regioselectivity were investigated using various sugars as acceptor substrates and alpha-xylosyl fluoride as the donor substrate. Comparison of the rate of formation of the glycosyl-enzyme intermediate and the transfer product yield using various acceptor substrates showed that glucose is the best complementary acceptor at the aglycone-binding site. YicI preferred aldopyranosyl sugars with an equatorial 4-OH as the acceptor substrate, such as glucose, mannose, and allose, resulting in transfer products. This observation suggests that 4-OH in the acceptor sugar ring made an essential contribution to transxylosylation catalysis. Fructose was also acceptable in the aglycone-binding site, producing two regioisomer transfer products. The percentage yields of transxylosylation products from glucose, mannose, fructose, and allose were 57, 44, 27, and 21%, respectively. The disaccharide transfer products formed by YicI, alpha-D-Xylp-(1 -> 6)-D-Manp, alpha-D-Xylp-(1 -> 6)-D-Fruf, and alpha-D-Xylp-(1 -> 3)-D-Frup, are novel oligosaccharides that have not been reported previously. In the transxylosylation to cello-oligosaccharides, YicI transferred a xylosyl moiety exclusively to a nonreducing terminal glucose residue by alpha-1,6-xylosidic linkages. Of the transxylosylation products, alpha-D-Xylp-(1 -> 6)-D-Manp and alpha-D-Xylp(1 -> 6)-D-Fruf inhibited intestinal alpha-glucosidases.
  • Hiroyuki Nakai, Shigeki Tanizawa, Tatsuya Ito, Koutarou Kamiya, Young-Min Kim, Takeshi Yamamoto, Kazuki Matsubara, Makoto Sakai, Hiroyuki Sato, Tokio Imbe, Masayuki Okuyama, Haruhide Mori, Yoshio Sano, Seiya Chiba, Atsuo Kimura JOURNAL OF BIOCHEMISTRY 142 (4) 491 -500 2007年10月 [査読無し][通常論文]
     
    In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three a-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. alpha-Xylosidase encoded in rice genome was found for the first time.
  • Jin-Ha Lee, Saori Saito, Haruhide Mori, Mamoru Nishimoto, Masayuki Okuyama, Doman Kim, Jintanart Wongchawalit, Atsuo Kimura, Seiya Chiba BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 71 (9) 2256 -2265 2007年09月 [査読無し][通常論文]
     
    cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5 ' untranslated region (UTR) of 869 bp, and the 3 ' UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The M-r of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.
  • Wataru Saburi, Hironori Hondoh, Hideaki Unno, Masayuki Okuyama, Haruhide Mori, Toshitaka Nakada, Yoshiki Matsuura, Atsuo Kimura ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 63 (Pt9) 774 -776 2007年09月 [査読無し][通常論文]
     
    Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal alpha-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 angstrom. A native data set was collected to 2.2 angstrom resolution from a single crystal.
  • 鐘ケ江倫世, KIM Young‐Min, 中井博之, 奥山正幸, 森春英, 木村淳夫 J Appl Glycosci 54 (Suppl.) 43 2007年07月20日 [査読無し][通常論文]
  • 大塚博昭, 本同宏成, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 J Appl Glycosci 54 (Suppl.) 33 2007年07月20日 [査読無し][通常論文]
  • 奥山正幸, KANG Min‐Sun, 矢追克郎, 矢追克郎, 三石安, 森春英, 木村淳夫 J Appl Glycosci 54 (Suppl.) 55 2007年07月20日 [査読無し][通常論文]
  • 飯塚貴久, 小林和之, 中井博之, 奥山正幸, 森春英, 奈良岡哲志, 千葉誠哉, 木村淳夫 J Appl Glycosci 54 (Suppl.) 32 2007年07月20日 [査読無し][通常論文]
  • Mamoru Nishimoto, Haruhide Mori, Tsuneharu Moteki, Yukiko Takamura, Gaku Iwai, Yu Miyaguchi, Masayuki Okuyama, Jintanart Wongchawalit, Rudee Surarit, Jisnuson Svasti, Atsuo Kimura, Seiya Chiba Biosci. Biotechnol. Biochem. 71 (7) 1703 -1716 2007年07月 [査読無し][通常論文]
     
    cDNAs encoding three alpha-glucosidases (HBGases I, II, and 111) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 by in length, and included ORFs of 1,767, 1,743, and 1,704 by encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases 1, 11, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. In spite of remarkable differences in the substrate specificities of the three HBGases, high homologies (3844% identity) were found in the deduced amino acid sequences. In addition, three genomic DNAs, of 13,325, 2,759, and 27,643 bp, encoding HBGases I, II, and III, respectively, were isolated from honeybees, and the sequences were analyzed. The gene of HBGase I was found to be composed of 8 exons and 7 introns. The gene of HBGase II was not divided by intron. The gene of HBGase III was confirmed to be made up of 9 exons and 8 introns, and to be located in the region upstream the gene of HBGase I.
  • 大塚博昭, 佐分利亘, 本同宏成, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2007 206 2007年03月05日 [査読無し][通常論文]
  • 中井博之, 金泳民, 原口慶子, 奥山正幸, 森春英, 舟根和美, 小林幹彦, 木村淳夫 日本農芸化学会大会講演要旨集 2007 65 2007年03月05日 [査読無し][通常論文]
  • 奥山正幸, KANG Minsun, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2007 206 2007年03月05日 [査読無し][通常論文]
  • 飯塚貴久, 中井博之, 奥山正幸, 森春英, 奈良岡哲志, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2007 206 2007年03月05日 [査読無し][通常論文]
  • 本同宏成, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 2007 36 2007年 [査読無し][通常論文]
  • 佐藤なつ子, 鳥羽瀬輝, 中井博之, 西本完, 森春英, 奥山正幸, 木村淳夫 生化学 3P-0212 2007年 [査読無し][通常論文]
  • Hiroyuki Nakai, Tatsuya Ito, Masatoshi Hayashi, Koutarou Kamiya, Takeshi Yamamoto, Kazuki Matsubara, Young-Min Kim, Wongchawalit Jintanart, Masayuki Okuyama, Haruhide Mori, Seiya Chiba, Yoshio Sano, Atsuo Kimura BIOCHIMIE 89 (1) 49 -62 2007年01月 [査読無し][通常論文]
     
    Two isoforms of alpha-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6 kDa-peptide and 88 kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88 kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88 kDa-peptide of ONG2-I. The peptides between 6 kDa and 88 kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three alpha-glucosidase cDNAs (ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of alpha-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice. (c) 2006 Elsevier Masson SAS. All rights reserved.
  • Jintanart Wongchawalit, Takeshi Yamamoto, Hiroyuki Nakai, Young-Min Kim, Natsuko Sato, Mamoru Nishimoto, Masayuki Okuyama, Haruhide Mori, Osamu Saji, Chanpen Chanchao, Siriwat Wongsiri, Rudee Surarit, Jisnuson Svasti, Seiya Chiba, Atsuo Kimura BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 70 (12) 2889 -2898 2006年12月 [査読無し][通常論文]
     
    a-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.
  • 飯塚貴久, 中井博之, 奥山正幸, 森春英, 奈良岡哲志, 千葉誠哉, 木村淳夫 J Appl Glycosci 53 (Suppl.) 30 2006年08月30日 [査読無し][通常論文]
  • 大塚博昭, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 J Appl Glycosci 53 (Suppl.) 30 2006年08月30日 [査読無し][通常論文]
  • 丹澤史子, 奥山正幸, 北村百世, 森春英, 田中勲, 木村淳夫 J Appl Glycosci 53 (Suppl.) 33 2006年08月30日 [査読無し][通常論文]
  • 本同宏成, 佐分利亘, 奥山正幸, 森春英, 松浦良樹, 木村淳夫 J Appl Glycosci 53 (Suppl.) 34 2006年08月30日 [査読無し][通常論文]
  • 佐藤なつ子, 中井博之, 森春英, 奥山正幸, 千葉誠哉, 木村淳夫 J Appl Glycosci 53 (Suppl.) 31 2006年08月30日 [査読無し][通常論文]
  • 谷沢茂紀, 中井博之, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 J Appl Glycosci 53 (Suppl.) 43 2006年08月30日 [査読無し][通常論文]
  • M Okuyama, A Kaneko, H Mori, S Chiba, A Kimura FEBS LETTERS 580 (11) 2707 -2711 2006年05月 [査読無し][通常論文]
     
    Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta -> alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C3071/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Wataru Saburi, Haruhide Mori, Saori Saito, Masayuki Okuyama, Atsuo Kimura BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1764 (4) 688 -698 2006年04月 [査読無し][通常論文]
     
    Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta -> alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar k(cat)/k(m) value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta -> alpha loop 4 of B. subtilis oligo- 1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta ->alpha loop 4 were important for high activity to long-chain substrates. (c) 2006 Elsevier B.V. All rights reserved.
  • 奥山正幸, 北村百世, 森春英, 田中勲, 木村淳夫 日本農芸化学会大会講演要旨集 2006 307 2006年03月05日 [査読無し][通常論文]
  • 大塚博昭, 佐分利亘, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2006 154 2006年03月05日 [査読無し][通常論文]
  • 佐藤なつ子, 中井博之, 森春英, 奥山正幸, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2006 154 2006年03月05日 [査読無し][通常論文]
  • 河合正悟, 浜井英礼, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2006 154 2006年03月05日 [査読無し][通常論文]
  • 須賀原千佳, 佐分利亘, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2006 306 2006年03月05日 [査読無し][通常論文]
  • 森 春英 Journal of applied glycoscience 53 (1) 51 -56 2006年01月20日 [査読無し][通常論文]
     
    In germinating plant seeds, α-amylases degrade starch accumulated in seeds, and that requires two functions: catalysis itself and starch granule binding ability. All plant α-amylases belong to the α-amylase family and share the same catalytic machinery as other members, but are different in extended subsite structure accommodating the non-reducing end side of substrate even with high affinity, particularly in subsite -6, shown in α-amylases of kidney bean as well as barley. Barley α-amylase isozyme 1 (AMY1) mutants introduced site-directed mutagenesis along the predicted substrate binding site and the recent crystal structure solved in complex with a substrate occupying subsite -1 to -7 revealed that amino acid residues situated in a shallow cleft extending between domain A and B were involved in the subsite formation. Although plant α-amylases possess no additional starch-binding domain as seen in several α-amylases from microorganisms, plant α-amylases examined acted on starch granules. The residue corresponding to "sugar tongs" Tyr380AMY1 was proven to be involved in starch granule binding in adzuki bean α-amylase.
  • M Okuyama, Y Tanimoto, T Ito, A Anzai, H Mori, A Kimura, H Matsui, S Chiba ENZYME AND MICROBIAL TECHNOLOGY 37 (5) 472 -480 2005年10月 [査読無し][通常論文]
     
    alpha-Glucosidase secreted from Schizosaccharomyces pombe cell has been purified as a homogeneous protein from culture supernatant. The alpha-glucosidase is hyper-glycosylated form, which included 88% of sugar components, and the relative molecular mass is calculated in 1120 kDa. Heat stability and proteolysis susceptibility of the alpha-glucosidase is descended by enzymatical deglycosylation. By MALDI-TOF NIS analysis, seven Asn residues (Asn185, Asn221, Asn496, Asn499, Asn572, Asn777 and Asn787; numbering from N-terminal of matured form) out of 27 potential N-glycosylation sites of the enzyme are presumed to be modified. The native form of S. pombe a-glucosidase have three subsites in the catalytic site and so prefer alpha-1,4-glucosidic linkage in short substrates, such as maltose and maltotriose, to longer substrate. The enzyme also acts on alpha- 1,2, alpha- 1,3, and alpha-1,6-glucosidic linkage. (c) 2005 Elsevier Inc. All rights reserved.
  • F Sato, M Okuyama, H Nakai, H Mori, A Kimura, S Chiba BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 69 (10) 1905 -1913 2005年10月 [査読無し][通常論文]
     
    A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K-m) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.
  • Robert, X, R Haser, H Mori, B Svensson, N Aghajari JOURNAL OF BIOLOGICAL CHEMISTRY 280 (38) 32968 -32978 2005年09月 [査読無し][通常論文]
     
    Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1(D180A)) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."
  • 森春英 飯島記念食品科学振興財団年報 2003 90-95 2005年08月 [査読無し][通常論文]
  • 佐藤なつ子, 中井博之, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 J Appl Glycosci 52 (Suppl.) 24 2005年07月20日 [査読無し][通常論文]
  • 佐分利亘, 奥山正幸, 森春英, 岡田厳太郎, 木村淳夫 J Appl Glycosci 52 (Suppl.) 26 2005年07月20日 [査読無し][通常論文]
  • 飯塚貴久, 福川太郎, 西岡謙吾, 中井博之, 奥山正幸, 森春英, 吉田孝, 千葉誠哉, 木村淳夫 J Appl Glycosci 52 (Suppl.) 26 2005年07月20日 [査読無し][通常論文]
  • 谷沢茂紀, 中井博之, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 J Appl Glycosci 52 (Suppl.) 25 2005年07月20日 [査読無し][通常論文]
  • 中井博之, 谷沢茂紀, 松原一樹, 奥山正幸, 森春英, 千葉誠哉, 佐野芳雄, 木村淳夫 J Appl Glycosci 52 (Suppl.) 52 2005年07月20日 [査読無し][通常論文]
  • 奥山正幸, 北村百世, 丹沢史子, 北郷悠, 森春英, 田中勲, 木村淳夫 J Appl Glycosci 52 (Suppl.) 25 2005年07月20日 [査読無し][通常論文]
  • J Wongchawalit, T Yamamoto, M Okuyama, H Mori, R Surarit, J Svasti, S Chiba, AK Kimura FEBS JOURNAL 272 101 -101 2005年07月 [査読無し][通常論文]
  • 山本英治, 金泳民, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2005 196 2005年03月05日 [査読無し][通常論文]
  • 河合正悟, 浜井英礼, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2005 195 2005年03月05日 [査読無し][通常論文]
  • 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2005 30 2005年03月05日 [査読無し][通常論文]
  • 中井博之, 伊藤真吾, 奥山正幸, 森春英, 千葉誠哉, 佐藤芳雄, 木村淳夫 日本農芸化学会大会講演要旨集 2005 30 2005年03月05日 [査読無し][通常論文]
  • 佐藤なつ子, 高橋有志, 中井博之, 光畑雅宏, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2005 30 2005年03月05日 [査読無し][通常論文]
  • 谷沢茂紀, 中井博之, 奥山正幸, 森春英, 千葉誠哉, 佐野芳雄, 木村淳夫 日本農芸化学会大会講演要旨集 2005 31 2005年03月05日 [査読無し][通常論文]
  • 西塔沙織, LEE Jin‐Ha, 西本完, 森春英, 奥山正幸, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2005 196 2005年03月05日 [査読無し][通常論文]
  • 須賀原千佳, 佐分利亘, 奥山正幸, 森春英, 木村淳夫 日本農芸化学会大会講演要旨集 2005 196 2005年03月05日 [査読無し][通常論文]
  • 佐分利亘, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2005 26 2005年03月05日 [査読無し][通常論文]
  • 岩井岳, 森春英, 佐分利亘, 奥山正幸, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2005 30 2005年03月05日 [査読無し][通常論文]
  • B Kramhoft, KS Bak-Jensen, H Mori, N Juge, J Nohr, B Svensson BIOCHEMISTRY 44 (6) 1824 -1832 2005年02月 [査読無し][通常論文]
     
    Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite-6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite-5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and GIO at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and GIO at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites.
  • M Kitamura, T Ose, M Okuyama, H Watanabe, M Yao, H Mori, A Kimura, Tanaka, I ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 (Pt 2) 178 -179 2005年02月 [査読無し][通常論文]
  • 西塔沙織, 森春英, 奥山正幸, 千葉誠哉, 木村淳夫 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 2005 16 2005年 [査読無し][通常論文]
  • 佐分利亘, 森春英, 大塚博昭, 岩井岳, 奥山正幸, 木村淳夫 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 2005 15 2005年 [査読無し][通常論文]
  • 汐川由希子, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 2005 16 2005年 [査読無し][通常論文]
  • YM Kim, M Okuyama, H Mori, H Nakai, W Saburi, S Chiba, A Kimura TETRAHEDRON-ASYMMETRY 16 (2) 403 -409 2005年01月 [査読無し][通常論文]
     
    Aspergillus niger alpha-glucosidase (ANGase) was used for an efficient syntheses of alkyl alpha-D-2-deoxyglucosides (A2DGs) and for regioselectivity studies of alkoxy-hydro additions Of D-glucal in the presence of alkyl alcohols. ANGase showed a high stability with respect to the high concentration of alkyl alcohols. The reaction conditions were optimized for pH, temperature, alkyl alcohol concentration, and D-glucal concentration. On the basis of MS and NMR analyses, A2DGs were confirmed to have only an alpha-2deoxyglucosidic bond and the two-dimensional NMR (HMBC) spectra showed to be made up of 2-deoxyglucosyl and alkyl moieties. (C) 2004 Elsevier Ltd. All rights reserved.
  • M Kubota, M Tsuji, M Nishimoto, J Wongchawalit, M Okuyama, H Mori, H Matsui, R Surarit, J Svasti, A Kimura, S Chiba BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 68 (11) 2346 -2352 2004年11月 [査読無し][通常論文]
     
    Three kinds of alpha-glucosidases, I, II, and III, were purified from European honeybees, Apis mellifera L. In addition, an et-glucosidase was also purified from honey. Some properties, including the substrate specificity of honey a-glucosidase, were almost the same as those of alpha-glucosidase III. Specific antisera against the alpha-glucosidases were prepared to examine the localization of alpha-glucosidases in the organs of honeybees. It was immunologically confirmed for the first time that alpha-glucosidase I was present in ventriculus, and alpha-glucosidase II, in ventriculus and haemolymph. alpha-Glucosidase III, which became apparent to be honey alpha-glucosidase, was present in the hypopharyngeal gland, from which the enzyme may be secreted into nectar gathered by honeybees. Honey may be finally made up through the process whereby sucrose in nectar, in which glucose and fructose also are naturally contained, is hydrolyzed by secreted alpha-glucosidase III.
  • M Okuyama, H Mori, S Chiba, A Kimura PROTEIN EXPRESSION AND PURIFICATION 37 (1) 170 -179 2004年09月 [査読無し][通常論文]
     
    The proteins encoded in the yicI and yihQ gene of Escherichia coli have similarities in the amino acid sequences to glycoside hydrolase family 31 enzymes, but they have not been detected as the active enzymes. The functions of the two proteins have been first clarified in this study. Recombinant YicI and YihQ produced in E coli were purified and characterized. YicI has the activity of Otxylosidase. YicI existing as a hexamer shows optimal pH at 7.0 and is stable in the pH range of 4.7-10.1 with incubation for 24 h at 4 degreesC and also is stable up to 47 degreesC with incubation for 15 min. The enzyme shows higher activity against alpha-xylosyl fluoride, isoprimeverose (6-O-alpha-xylopyranosyl-glucopyranose), and alpha-xyloside in xyloglucan oligosaccharides. The alpha-xylosidase catalyzes the transfer of alpha-xylosyl residue from alpha-xyloside to xylose, glucose, mannose, fructose, maltose, isomaltose, nigerose, kojibiose, sucrose, and trehalose. YihQ exhibits the hydrolysis activity against alpha-glucosyl fluoride, and so is an alpha-glucosidase, although the natural substrates, such as alpha-glucobioses, are scarcely hydrolyzed. alpha-Glucosidase has been found for the first time in E coli. (C) 2004 Elsevier Inc. All rights reserved.
  • T Yamamoto, T Unno, Y Watanabe, M Yamamoto, M Okuyama, H Mori, S Chiba, A Kimura BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1700 (2) 189 -198 2004年08月 [査読無し][通常論文]
     
    alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 5 1,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(o)/K-m value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)- O-alpha-maltosyl-maltose were novel saccharides. (C) 2004 Elsevier B.V All rights reserved.
  • 中井博之, 谷沢茂紀, 奥山正幸, 森春英, 山本健, 佐野芳雄, 千葉誠哉, 木村淳夫 J Appl Glycosci 51 (Suppl.) 41 2004年07月20日 [査読無し][通常論文]
  • 森春英, 浜井英礼, MAR S S, 千葉誠哉, 木村淳夫 J Appl Glycosci 51 (Suppl.) 42 2004年07月20日 [査読無し][通常論文]
  • 奥山正幸, 尾瀬農之, 北村百世, 森春英, 千葉誠哉, 木村淳夫, 田中勲 J Appl Glycosci 51 (Suppl.) 40 2004年07月20日 [査読無し][通常論文]
  • 佐藤なつ子, 高橋有志, 中井博之, 光畑雅宏, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 J Appl Glycosci 51 (Suppl.) 40 2004年07月20日 [査読無し][通常論文]
  • 森春英 化学と生物 42 (3) 170-172 -172 2004年03月25日 [査読無し][通常論文]
  • 峰島希, 福田健二, 森春英, 奥山正幸, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2004 255 2004年03月05日 [査読無し][通常論文]
  • 佐分利亘, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2004 255 2004年03月05日 [査読無し][通常論文]
  • 中井博之, 奥山正幸, 森春英, 山本健, 千葉誠哉, 佐野芳雄, 木村淳夫 日本農芸化学会大会講演要旨集 2004 254 2004年03月05日 [査読無し][通常論文]
  • 浜井英礼, 森春英, SAN SAN M, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2004 108 2004年03月05日 [査読無し][通常論文]
  • 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2004 257 2004年03月05日 [査読無し][通常論文]
  • 岩井岳, 坪野真子, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2004 255 2004年03月05日 [査読無し][通常論文]
  • 八巻勉, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2004 255 2004年03月05日 [査読無し][通常論文]
  • 福原有信, 森春英, 奥山正幸, SVENSSON B, 木村淳夫 日本農芸化学会大会講演要旨集 2004 108 2004年03月05日 [査読無し][通常論文]
  • 奥山正幸, 森春英, 渡辺琴美, 木村淳夫, 千葉誠哉 日本農芸化学会誌 77 (11) 1140 -1141 2003年11月01日 [査読無し][通常論文]
  • T Naraoka, H Uchisawa, H Mori, H Matsue, S Chiba, A Kimura EUROPEAN JOURNAL OF BIOCHEMISTRY 270 (19) 4026 -4038 2003年10月 [査読無し][通常論文]
     
    Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68 993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as omicron-diphenols and to exhibit temperature-dependency of omicron-diphenolase activity like a psychrophilic enzyme.
  • SS Mar, H Mori, JH Lee, K Fukuda, W Saburi, A Fukuhara, M Okuyama, S Chiba, A Kimura BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 67 (5) 1080 -1093 2003年05月 [査読無し][通常論文]
     
    Two alpha-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a beta-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): k(cat), 71.8 and 55.5 s(-1), K-m, 0.113 and 0.097 mg /ml; for blocked 4-nitrophenyl alpha-D-maltoheptaoside: k(cat), 62.4 and 85.3 s(-1), K-m, 0.22 and 0.37 mm, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards beta-cyclodextrin Sepharose was due to some modification on /near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.
  • 福原有信, 森春英, 奥山正幸, SVENSSON B, 木村淳夫 日本農芸化学会大会講演要旨集 2003 98 2003年03月05日 [査読無し][通常論文]
  • 中井博之, 伊藤達也, 松原一樹, 奥山正幸, 森春英, 千葉誠哉, 佐野芳雄, 木村淳夫 日本農芸化学会大会講演要旨集 2003 100 2003年03月05日 [査読無し][通常論文]
  • 佐分利亘, 森春英, 奥山正幸, 木村淳夫 日本農芸化学会大会講演要旨集 2003 99 2003年03月05日 [査読無し][通常論文]
  • 森春英, SAN SAN M, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2003 98 2003年03月05日 [査読無し][通常論文]
  • 山本健, 大畑祐一郎, 海野剛裕, 小川浩一, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2003 100 2003年03月05日 [査読無し][通常論文]
  • 孫 美, 森 春英, 奥山 正幸, 木村 淳夫, 千葉 誠哉 J. Appl. Glycosci. 50 (1) 41 -44 2003年 [査読無し][通常論文]
     
    炭水化物加水分解酵素の水解反応は,その逆反応(縮合反応)が必然的に伴った反応である.従って,水解され得る基質のみが逆反応によって生成される.ミツバチα-グルコシダーゼI は,イソマルトースを全く水解できないが,他の二糖マルトース,コジビオースを水解できる.わずかではあるが,ニゲロースをも水解する.しかしながら,本酵素は高濃度のグルコースから逆反応によってマルトース,コジビオースおよびニゲロースと共にイソマルトースをも生成した.このような事実は,本酵素がイソマルトースに対して水解作用をもたないという知見と矛盾している.この特異な現象に関する矛盾の解析が試みられた.その結果,本来,グルコースから逆反応によってイソマルトースが直接生成されることはあり得ないことであるが,逆反応によって容易に生成され得るマルトースやコジビオース等の他の二糖からの分子内糖転移反応によってイソマルトースへ変換されイソマルトースが生成されると推定された.炭水化物水解酵素の反応において,通常の糖転移反応と考えられている反応には,上述のような分子内転移反応が同時に起こっていることが示唆された.
  • H Mori, KS Bak-Jensen, B Svensson EUROPEAN JOURNAL OF BIOCHEMISTRY 269 (22) 5377 -5390 2002年11月 [査読無し][通常論文]
     
    Met53 in barley alpha-amylase 1 (AMY1) is situated at the high-affinity subsite -2. While Met53 is unique to plant alpha-amy lases, the adjacent Tyr52 stacks onto substrate at subsite -1 and is essentially invariant in glycoside hydrolase family 13. These residues belong to a short sequence motif in beta-->alpha loop 2 of the catalytic (beta/alpha)(8)-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53. Compared to wild-type, Met53Glu/Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13. Towards amylose DP17 (degree of polymerization = 17) and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside the activity (k(cat)/K(m)) of all mutants was reduced to 5.5-0.01 and 1.7-0.02% of wild type, respectively. K(m) increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl alpha-D-maltohexaoside (PNPG(6)) and PNPG(5) was determined by action pattern analysis to shift to be closer to the nonreducing end. This indicated that side chain replacement at subsite -2 weakened substrate glycon moiety contacts. Thus whereas all mutants produced mainly PNPG(2) from PNPG(6) and similar amounts of PNPG(2) and PNPG(3) accounting for 85% of the products from PNPG(5), wild-type released 4-nitrophenol from PNPG(6) and PNPG and PNPG(2) in equal amounts from PNPG(5). Met53Trp affected the action pattern on PNPG(7), which was highly unusual for AMY1 subsite mutants. It was also the sole mutant to catalyze substantial transglycosylation promoted probably by slow substrate hydrolysis to produce up to maltoundecaose from PNPG(6).
  • FUKUDA K, MORI H, OKUYAMA M, KIMURA A, OZAKI H, YONEYAMA M, CHIBA S Biosci Biotechnol Biochem 66 (10) 2060 -2067 2002年10月 [査読無し][通常論文]
     
    Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of beta-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of beta-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and K. values on pH, when the substrate concentration was sufficiently lower than K-m, gave the values of 4.1 and 7.2 for the ionization constants, pK(e1) and pKe(2) of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pKe(1) and pKe(2), values shifted to higher, to + 0.60 and + 0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities A(i)'s in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive A(i) values (A(1) was 1.10kcal/mol and A(2) was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, kthe subsites 3 and 4 showed negative A(i) values (A(3) was - 0.21 kcal /mol and A(4) was - 2.8 kcal /mol).
  • M Okuyama, H Mori, K Watanabe, A Kimura, S Chiba BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 66 (4) 928 -933 2002年04月 [査読無し][通常論文]
     
    Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe alpha-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an alpha-glucosidic linkage from beta-glucosyl fluoride and 4-nitrophenyl (PNP) alpha-glucoside to produce two kinds of PNP alpha-diglucosides, alpha-isomaltoside and alpha-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29% yields of PNP alpha-isomaltoside and alpha-maltoside, respectively. PNP monoglycosides, such as alpha-xyloside, alpha-mannoside, or beta-glucoside, acted as the substrate, but PNP alpha-galactoside and maltose could not. No detectable product was observed in the combination of alpha-glucosyl fluoride and PNP alpha-glucoside. This study is the first report on an "alpha-glycosynthase"-type reaction to form an alpha-glycosidic linkage.
  • 奥山正幸, 森春英, 木村淳夫, 千葉誠哉 日本農芸化学会大会講演要旨集 2002 127 2002年03月05日 [査読無し][通常論文]
  • 佐分利亘, 奥山正幸, 森春英, 岡田厳太郎, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2002 35 2002年03月05日 [査読無し][通常論文]
  • 森春英, 佐分利亘, 尾関理香, 水上裕紀子, 木村淳夫, 千葉誠哉 日本農芸化学会大会講演要旨集 2002 126 2002年03月05日 [査読無し][通常論文]
  • 矢守美典, 石原啓吾, 奥山正幸, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2002 265 2002年03月05日 [査読無し][通常論文]
  • 中井博之, 伊藤達也, 森春英, 千葉誠哉, 木村淳夫 日本農芸化学会大会講演要旨集 2002 127 2002年03月05日 [査読無し][通常論文]
  • FUKUDA K, SHIRAKAWA K, MORI H, OKUYAMA M, KIMURA A, OZAKI H, YONEYAMA M, CHIBA S J. Appl. Glycosci. 49 (3) 265 -272 2002年 [査読無し][通常論文]
  • H Mori, K Sass Bak-Jensen, TE Gottschalk, M Saddik Motawia, Damager, I, B Lindberg Moller, B Svensson EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (24) 6545 -6558 2001年12月 [査読無し][通常論文]
     
    Enzymatic properties of barley alpha -amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites -5/-6 (Cys95 --> Ala/Thr) and +1/+2 (Met298 --> Ala/Asn/Ser) as well as in the double mutants, Cys95 --> Ala/Met298 --> Ala/Asn/Ser. Cys95 --> Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, k(cat) of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl beta -d-maltoheptaoside (Cl-PNPG(7)), respectively, but fivefold to 20-fold higher K(m). The Cys95 --> Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95 --> Ala and wild-type. Met298 --> Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas k(cat) and k(cat)/K(m) for Cl-PNPG(7) are less than or equal to 30% and less than or equal to 10% of wild-type, respectively. The activity of Cys95 --> Ala/Met298 --> Ala/Asn/Ser is 100-180% towards starch, and the k(cat)/K(m) is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG(7), respectively, emphasizing the strong impact of the Cys95 --> Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG(7) and amylose/Cl-PNPG(7) are 2.8- to 270-fold and 1.2- to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites -5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6'''-maltotriosyl-maltohexaose thus covering subsites -1 to +5, while productive binding of unbranched substrate involves subsites -3 to +3.
  • JH Lee, M Tsuji, M Nakamura, M Nishimoto, M Okuyama, H Mori, A Kimura, H Matsui, S Chiba BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 (12) 2657 -2665 2001年12月 [査読無し][通常論文]
     
    Trehalase (EC 3.2.1.28) of the bound type was purified as an electrophoretically homogeneous protein from adult honeybees by fractionation with ammonium sulfate, hydrophobic chromatography, and DEAE-Sepharose CL-6B, CM-Sepharose CL-6B, butyl-Toyopearl 650M, and p-aminophenyl beta -glucoside Sepharose 4B column chromatographies. The enzyme preparation was confirmed to be a monomeric protein containing 3.1% carbohydrate. The molecular weight was estimated to be approximately 69,000, and the optimum pH was 6.7. The Michaelis constant (K-m) was 0.66 nim, and the molecular activity (k(0)) was 86.2 s(-1). The enzyme was an "inverting" type which produced beta -glucose from alpha, alpha -trehalose. Dependence of the V and K-m values on pH gave values for the ionization constants, pKe(1) and pKe(2), of essential ionizable groups I and 2 of the free enzyme of 5.3 and 8.5, respectively. When the dielectric constant of the reaction mixture was decreased, pKe(1), and pKe(2) were shifted to higher values of + 0.2 and + 0.5 pH unit, respectively. The ionization heat (DeltaH) of ionizable group I was estimated to be + 1.8 kcal/mol, and the DeltaH value of group 2 was + 1.5 kcal/mol. These findings strongly support the notion that the essential ionizable groups of honeybee trehalase are two kinds of carboxyl groups, one being a dissociated type (-COO-, ionizable group 1) and the other a protonated type (-COOH, ionizable group 2), although the pKe(2) value is high.
  • 森 春英 化学と生物 39 (9) 588 -591 2001年09月25日
  • M Nishimoto, M Kubota, M Tsuji, H Mori, A Kimura, H Matsui, S Chiba BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 65 (7) 1610 -1616 2001年07月 [査読無し][通常論文]
     
    alpha -Glucosidase III, which was different in substrate specificity from honeybee alpha -glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of alpha -glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in alpha -glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl alpha -glucoside, maltose, and maltotriose, and by extremely high K-m for substrates, compared with those of alpha -glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k(o) values for maltose, sucrose, p-nitrophenyl alpha -glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the K-m values for these substrates, 11, 30, 13, and 10 mm, respectively. The subsite affinities (A(i)'s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the A(i) value at subsite 3 was larger than that of subsite 1.
  • M Okuyama, A Okuno, N Shimizu, H Mori, A Kimura, S Chiba EUROPEAN JOURNAL OF BIOCHEMISTRY 268 (8) 2270 -2280 2001年04月 [査読無し][通常論文]
     
    cDNA encoding Schizosaccharomyces pombe alpha -glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M-r 106 138. The deduced amino-acid sequence showed a high homology to those of alpha -glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the alpha -glucosidase family II. By site-directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the alpha -glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO-) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.
  • 十川詩帆, 西本完, 福士幸治, 森春英, 木村淳夫, 千葉誠哉 日本農芸化学会誌 75 65 2001年03月05日 [査読無し][通常論文]
  • 中井博之, 林正敏, 森春英, 木村淳夫, 千葉誠哉 日本農芸化学会誌 75 65 2001年03月05日 [査読無し][通常論文]
  • 水野 隆文, 森 春英, 西本 完, 伊藤 浩之, 松井 博和, 木村 淳夫, 本間 守, 千葉 誠哉 J Appl Glycosci 48 (3) 287 -291 2001年 [査読無し][通常論文]
  • H Mori, KS Bak-Jensen, B Svensson ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 220 U117 -U117 2000年08月 [査読無し][通常論文]
  • T Mizuno, H Mori, H Ito, H Matsui, A Kimura, S Chiba BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 63 (9) 1582 -1588 1999年09月 [査読無し][通常論文]
     
    The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (M-r, 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.
  • B Svensson, KS Bak-Jensen, H Mori, J Sauer, MT Jensen, B Kramhoft, TE Gottschalk, T Christensen, BW Sigurskjold, N Aghajari, R Haser, N Payre, S Cottaz, H Driguez RECENT ADVANCES IN CARBOHYDRATE BIOENGINEERING (246) 272 -281 1999年 [査読無し][通常論文]
  • 中島款冬, 大崎満, 信濃卓郎, 森春英, 但野利秋 日本土壌肥料学会講演要旨集 44 256 -256 1998年03月 [査読無し][通常論文]
  • 奥山正幸, 森春英, 木村淳夫, 千葉誠哉 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 1998 15 1998年 [査読無し][通常論文]
  • Localization of α-Glucosidase in Yeast Cells
    Oyo Toshitsu Kagaku (J. Appl. Glycosci.) 45 (3) 281 -283 1998年 [査読無し][通常論文]
  • 森 春英, 小林 哲也, 殿川 隆志, 立松 あゆみ, 松井 博和, 木村 淳夫, 千葉 誠哉 Oyo Toshitsu Kagaku (J. Appl.Glycosci) 45 (3) 261 -267 1998年 [査読無し][通常論文]
  • A Kimura, M Takata, Y Fukushi, H Mori, H Matsui, S Chiba Biosci. Biotechnol. Biochem. 61 (7) 1091 -1098 1997年07月 [査読無し][通常論文]
     
    The catalytic amino acid residue of Aspergillus niger alpha-glucosidase (ANGase) was identified by modification with conduritol B epoxide (CBE), a mechanism-based irreversible inactivator, The inactivation by CBE followed pseudo-first order kinetics, The interaction of CBE and ANGase conformed to a model with a reversible enzyme-inhibitor complex formed before covalent inactivation, A competitive inhibitor, Tris, decreased the inactivation rate, The incorporation of one mole of CBE per mole of ANGase was completely abolished the enzyme activity, A dissociated carboxyl group (-COO-) in the active site was suggested to attack the C-1 of CBE, ANGase was composed of two subunits (P1 and P2), of which P2 was modified by CBE. The labelled residue was included in a peptide (LY3) that was obtained from Lys-C protease digestion of CBE-bound P2. The sequence analysis of CBE-labelled LY3 showed that an Asp was the modified residue, that is, one of the catalytic amino acid residues of ANGase, The primary structure of LY3 was determined by analyzing the sequence of peptide fragments prepared by several proteases.
  • A Kimura, A Somoto, H Mori, O Sakai, H Matsui, S Chiba Biosci. Biotechnol. Biochem. 61 (3) 475 -479 1997年03月 [査読無し][通常論文]
     
    A kinetic study was done to identify the ionizable groups in the active site of Aspergillus niger alpha-glucosidase (ANGase). From dependence of V and K-m values on pH, we obtained the ionization constants of essential ionizable groups 1 and 2 of free enzyme; pKe(1) = 3.2 and pKe(2) = 6.4. When the dielectric constant of the reaction mixture was decreased, the pKe(1) and pKe(2) were shifted to higher values, The ionization heats (Delta H's) of ionizable groups 1 and 2 were measured to be - 0.4 kcal/mol and 0 kcal/mol, respectively, The water-soluble carbodiimide (WSC), a specific reagent for carboxyl groups, inactivated the enzyme activity completely, and maltose as substrate decreased the inactivation, The WSC did not modify the free Cys, These findings suggest that the essential ionizable groups of ANGase are two kinds of carboxyl groups: one is a charged type (-COO-, ionizable group 1), and the other is a protonated type (-COOH, ionizable group 2).
  • 水野隆文, 松井博和, 森春英, 伊藤浩之, 木村淳夫, 千葉誠哉 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨 1997 6 1997年 [査読無し][通常論文]
  • S Onodera, T Murakami, H Ito, H Mori, H Matsui, M Honma, S Chiba, N Shiomi BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 60 (11) 1780 -1785 1996年11月 [査読無し][通常論文]
     
    A cDNA and a gene encoding endo-inulinase from Penicillium purpurogenum were isolated, and were cloned for the first time. Two oligonucleotide probes, which were synthesized based on the partial amino acid sequences of the purified endo-inulinase, were used to screen a cDNA library. A 1.7-kb DNA fragment encoding endo-inulinase was isolated and analyzed. A single open reading frame, consisting of 1548-bp, was found to encode a polypeptide that comprised a 25-amino acid signal peptide and 490-amino acid mature protein. All the partial amino acid sequences of the purified enzyme were discovered in the deduced ones. The deduced amino acid sequences of endo-inulinase had similar sequences to those of fructan hydrolases. A 3.5-kb chromosomal DNA fragment encoding endo-inulinase was also isolated and analyzed. The same ORF with the cDNA clone was identified. There were no introns in the endo-inulinase gene.
  • 森一生, 岩波俊介, 森春英, 木村淳夫, 松井博和, 千葉誠哉 日本農芸化学会東北支部講演要旨 1996 (Godo Gakujutsu Koenkai) 18 1996年09月 [査読無し][通常論文]
  • 奥山正幸, 森春英, 木村淳夫, 千葉誠哉 日本農芸化学会東北支部講演要旨 1996 (Godo Gakujutsu Koenkai) 17 1996年09月 [査読無し][通常論文]
  • 岩波 俊介, 松井 博和, 伊藤 浩之, 木村 淳夫, 森 春英, 本間 守, 千葉 誠哉 日本農藝化學會誌 70 138 -138 1996年03月05日
  • 吉崎 成洋, 森 春英, 伊藤 浩之, 松井 博和, 千葉 誠哉 日本農藝化學會誌 70 137 -137 1996年03月05日
  • 奥山正幸, 森春英, 木村淳夫, 千葉誠哉 日本農芸化学会北海道支部・北海道農芸化学協会シンポジウム及び合同学術講演会講演要旨 1996 17 1996年 [査読無し][通常論文]
  • 森一生, 岩波俊介, 森春英, 木村淳夫, 松井博和, 千葉誠哉 日本農芸化学会北海道支部・北海道農芸化学協会シンポジウム及び合同学術講演会講演要旨 1996 18 1996年 [査読無し][通常論文]
  • 水野 隆文, 松井 博和, 伊藤 浩之, 森 春英, 木村 淳夫, 本間 守, 千葉 誠哉 Oyo Toshitsu Kagaku (J. Appl. Glycosci.) 43 (3) 347 -353 1996年 [査読無し][通常論文]
  • 岩波 俊介, 伊藤 浩之, 木村 淳夫, 森 春英, 松井 博和, 本間 守, 千葉 誠哉 日本農藝化學會誌 69 200 -200 1995年07月05日
  • 伊木 繁雄, 谷本 佳博, 森 春英, 木村 淳夫, 松井 博和, 千葉 誠哉 日本農藝化學會誌 69 198 -198 1995年07月05日
  • 白川 康, 森 春英, 木村 淳夫, 松井 博和, 本間 守, 千葉 誠哉, 米山 道男, 尾崎 八郎 日本農藝化學會誌 69 200 -200 1995年07月05日
  • 吉崎 成洋, 森 春英, 松井 博和, 千葉 誠哉 日本農藝化學會誌 69 12 -12 1995年07月05日
  • S IWANAMI, H MATSUI, A KIMURA, H ITO, H MORI, M HONMA, S CHIBA Biosci. Biotechnol. Biochem. 59 (3) 459 -463 1995年03月 [査読無し][通常論文]
     
    The modification of amino acid residues in sugar beet alpha-glucosidase with conduritol B epoxide (CBE), an affinity labeling reagent, inactivated the enzyme. The inactivation followed pseudo-first-order kinetics. The enzyme was protected from inactivation by a competitive inhibitor, Tris, and the partially inactivated enzymes showed only the decrease of V values and no change in K-m value. An H-3-CBE labeled peptide isolated from the digest of the inactivated enzyme with Lys-C protease was sequenced. The -COO- group of Asp was found to be specifically labeled, implicating that it is a catalytic group of the enzyme. The sequence around the essential Asp was determined to be-DGIWIDMNE-, which showed a high homology with those of other alpha-glucosidases.
  • Oyo Toshitsu Kagaku (J. Appl. Glycosci.) 42 (4) 387 -394 1995年 [査読無し][通常論文]
  • A IWAI, H ITO, T MIZUNO, H MORI, H MATSUI, M HONMA, G OKADA, S CHIBA JOURNAL OF BACTERIOLOGY 176 (24) 7730 -7734 1994年12月 [査読無し][通常論文]
     
    The gene encoding an extracellular isomalto-dextranase, designated imd, was isolated from the chromosomal DNA of Arthrobacter globiformis T6 and cloned and expressed in Escherichia coli. A single open reading frame consisting of 1,926 base pairs that encoded a polypeptide composed of a signal peptide of 39 amino acids and a mature protein of 602 amino acids (M(r), 65,900) was found. The primary structure had no significant homology with the structures of any other reported carbohydrases, including two other dextranases. Transformed E. cell cells carrying the 2.3-kb fragment overproduced isomalto dextranase into the periplasmic space under control of the promoter of the imd gene itself.
  • 岩波俊介, 伊藤浩之, 木村淳夫, 松井博和, 森春英, 本間守, 千葉誠哉 日本農芸化学会誌 68 (3) 430 1994年03月 [査読無し][通常論文]
  • 谷本佳博, 木村淳夫, 森春英, 松井博和, 千葉誠哉 日本農芸化学会誌 68 (3) 430 1994年03月 [査読無し][通常論文]
  • 水野隆文, 岩井淳, 伊藤浩之, 森春英, 松井博和, 本間守, 千葉誠哉, 岡田厳太郎 日本農芸化学会誌 68 (3) 427 1994年03月 [査読無し][通常論文]
  • 森春英, 小林哲也, 伊藤浩之, 松井博和, 本間守, 千葉誠哉 日本農芸化学会誌 68 (3) 428 1994年03月 [査読無し][通常論文]
  • 小林哲也, 森春英, 伊藤浩之, 松井博和, 本間守, 千葉誠哉 日本農芸化学会誌 68 (3) 428 1994年03月 [査読無し][通常論文]
  • 邑上豊隆, 小野寺秀一, 塩見徳夫, 伊藤浩之, 森春英, 松井博和, 本間守, 千葉誠哉 日本農芸化学会誌 68 (3) 436 1994年03月 [査読無し][通常論文]
  • 伊木繁雄, 谷本佳博, 森春英, 松井博和, 千葉誠哉 日本農芸化学会北海道支部・北海道農芸化学協会シンポジウム及び合同学術講演会講演要旨 1994 34 1994年 [査読無し][通常論文]
  • H MORI, A TATEMATSU, H MATSUI, T TAKAYANAGI, M HONMA, S CHIBA BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 56 (9) 1499 -1500 1992年09月 [査読無し][通常論文]

所属学協会

  • 日本農芸化学会   日本応用糖質科学会   

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2019年06月 -2022年03月 
    代表者 : 森 春英
     
    加リン酸分解酵素に関し，新規活性の探索，機能構造相関解析，および糖質合成を実施した．新規酵素としてGlcβ1-3Gal特異的な酵素を見出し，新たな代謝経路の可能性と実用的な同二糖合成法を示した．澱粉加水分解酵素群のマルトシドホスホリラーゼでは，基質特異性などの解析に加え，加リン酸分解と糖転移の反応機構に基づく評価，加水分解を加えた3活性の寄与残基の特定を実施した．改変により糖転移や加水分解に偏る残基の特定など，関連加水分解酵素等への加リン酸分解活性付与に向けた機能構造相関の基盤的知見が得られた．また本酵素の利用による分岐鎖の伸長した不溶性のα-グルカン多糖が合成された．
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 森 春英
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2009年 -2012年 
    代表者 : 木村 淳夫, 森 春英
     
    植物が光合成で作る同化澱粉は、ショ糖に転換される。植物はショ糖を色々な組織から分泌し、微生物や昆虫など(環境生物)とクロストークするが、最近、その量が膨大であることが分った(この系を「スクロース・ワールド」と仮称)。本研究の目的は、花や根を対象にスクロース・ワールドのメンバー(環境生物と酵素)を調査することであり、特に糖質に着眼した研究を光量の大きなタイで行った。その結果、根から放出されたショ糖に関し「ショ糖→多糖→二次・三次転換体」に関わる菌株や酵素を明らかにできた。花バチやミツアリについては「ショ糖→蜜・オリゴ糖」の生成酵素を調べた。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2008年 -2009年 
    代表者 : 木村 淳夫, 森 春英, 奥山 正幸
     
    1残基のアミノ酸置換で「多糖の加水分解酵素(デキストラナーゼ)を合成酵素に転換できる現象」を見出した。この反応機構を分子解析することが、本申請の目的である。このような合成反応は例がなく、世界で初めての現象である。また、産業利用への発展にも期待したい。この残基は触媒アミノ酸と考えられる。本現象は試験管内の観察であるが、このような点突然変異した酵素が実際に生物で機能している可能性を得た。進化の過程においてアミノ酸置換は容易に生じ、1つのアミノ酸を変異させることで酵素分子を「加水分解→合成」にする戦略は、進化的に効率が良い。この戦略の検証も本申請の目的である。本年度は次の結果を得た。多糖合成の分子解析(デキストラナーゼ):1)酵素の結晶化と立体構造解析:昨年度に大量精製した親酵素とGly置換体を用いて結晶化条件を検討した。良好な結晶化条件を確定でき、X線構造解析を進行中である。2)他のアミノ酸による変異酵素:Asp→Gly置換体が合成反応を示したが、より効率の良いアミノ酸置換も想定されたため、他の残基への点変異を試みた。その結果、Gly置換体が最も高い反応効率を与えた。Glyは最もサイズの小さな残基であり、Asp→Gly置換で生じた大きな空間が重要と考えられた。すなわち、このサイズの大きい空間に陰イオンが侵入し合成反応が進行したと考えられた。3)陰イオンの解析:アザイドイオンが最も反応効率の良い陰イオンであった。従って本イオンのサイズ・強度が合成反応に最適であることが分かった。反応の至適pHを確定でき、pK_a値に大きな変化がないと予想できた。4)生成物の構造解析:生成多糖はデキストラン様の構造であった。触媒残基の変異酵素の解析(ウニ酵素):5)遺伝子の発現:酵素遺伝子の異種宿主発現を行った。酵素蛋白質は封入体を形成せず発現しているが、塩存在下であっても酵素活性が極めて低かった。
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2007年 -2008年 
    代表者 : 森 春英
     
    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.
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2007年 
    代表者 : 木村 淳夫, 森 春英, 奥山 正幸
     
    近年の糖質酵素の分類は、構造の類似性からなされる。研究対象は、α-グルコシダーゼと相同する酵素グループであり、産業界で重要な酵素が属する。すなわちα-グルコシダーゼ、グルカンリアーゼ、環状4糖合成酵素とα-キシロシダーゼであり、4つの反応(加水分解・糖転移・環状化・脱離反応)を触媒する。 本研究の目的は、我々が解析した本グループの立体構造に基づき、多様な反応の分子機構を明らかにし、得られた知見を応用研究に結びつけることにある。具体的には、X線解析法で得られた酵素-基質の分子認識を検討し、基質とアミノ酸の相互作用を解明;触媒アミノ酸の置換酵素の反応を解析し、触媒残基の機能を究明;4酵素は構造が類似するが全く異なる作用を示すので、個々の反応を発揮させる構造因子を解明;得られた知見から産業に重要な酵素を作製、である。 本研究で得られた成果を述べる。(1)酵素-基質の立体構造から、α-キシロシダーゼの反応に重要なアミノ酸残基を予想した。その予想に基づいたアミノ酸置換をα-キシロシダーゼに行い、α-キシロシダーゼ活性を消失させ、α-グルコシダーゼ活性を発現させることに成功した。(2)解離型とプロトン化型の触媒基を決定し、機能を究明した(中間体安定とグルコシド結合切断)。(3)転移環状化と脱離反応の2つの作用は、反応中間体に水分子が攻撃(加水分解)しないことで生じると仮定し、加水分解型の酵素であるα-グルコシダーゼの触媒水(水解反応の水分子)を除去するようなアミノ酸変異を導入した。変異酵素は加水分解活性を失い転移作用のみを示すことが認められ、転移環状化と脱離の反応機構に関し初発ステップを実証できた。残り半分[分子内転移(環状化)と基質C2水素の引抜(脱離)]の解析が期待される。(4)α-グルコシダーゼのサブサイト+1にあるアミノ酸に変異導入すると、糖転移の結合生成能が変わり、新規な転移能力を示す酵素が得られた。有用なオリゴ糖の調製に成功した。
  • 文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2005年 -2006年 
    代表者 : 森 春英
     
    本申諸研究は,糖質加水分解酵素α-グルコシグーゼとその関速酵素を利用して,α型オリゴ糖の合成に適した新たなグリコシレーションテクノロジーの開発を目的とした.開発技術は具体的に次の2点,1)酵素の糖転移酵素化,2)特異性の改変,である.第1点により効率的なオリゴ糖合成マシーナリーを獲得し,第2点により合成オリゴ糖を多様化できる.得られた研究成果は以下の通りである.1.転移酵素化:オオムギa-アミラーゼ,Streptococcus mutansデキストラングルコシダーゼ,枯草菌由来オリゴ-1,6-グルコシグーゼ,などの酵素の触媒に必煩であり高度に保存されたアミノ酵残基の改変酵素を作製し,合成反応に使用した.反応条件の最適化により,一例として,有機合成したα型フッ化グルコースとマルトースから,三糖パノースを合成するシステムを開発した.野生型酵素で収率16%に対して,本システムでは71%と高い収率を示した.本システムに使用する変異酵素は加水分解活性を示さないので,安定的に高収率を得られる点に特徴がある.2.特異性の改変:基質特異性の改変酵素を作出した.デキストラングルコシグーゼでは,得られた立体構造のデータも併せて,基質の鎖長特異性,結合特異性(α1,6結合からα1,4結合へ)の変換に成功した.大腸菌YicIでは,キシロシドからグルコシドヘ特異性を改変した.3.その他,多様な酵素を得...
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2004年 -2005年 
    代表者 : 木村 淳夫, 森 春英, 奥山 正幸
     
    糖質の加水分解酵素は、2つの酸性アミノ酸(AspやGlu)を触媒残基とし、それぞれが-COO^-と-COOHの荷電状態を形成し、協奏的に加水分解を触媒する。応用性の高い糖転移作用も示すが、分解と転移は2つの酸性アミノ酸でなされ分割できない。最近、α-グルコシダーゼにある-COO^-型の触媒基であるAspをCysに置換した。本酵素(Asp→Cys)には活性はないが、温和な酸化で活性を発揮した。Cysの-SHが酸化され-SOOHとなり、活性中心内で-SOO^-に解離し-COO^-の代わりを行うと考えている。この酵素は分解能を失い、糖転移能が上昇し95%の収率を与えた。本研究の目的は、-SOO^-酵素に見出された「非分解・高転移」の現象を解析することである。具体的には、1)酸化したCys残基の構造決定、2)糖転移反応の解析、3)他の酵素を合成酵素にする先駆けとして、触媒基を-SOO^-にした糖質酵素の構築と機帯解析、である。計画は順調に進行し、1)と2)が完了した。本年度は、この現象の応用を図るために3)の課題を中心に研究を進行させた。レバン合成酵素とキチン分解酵素を取り上げ、触媒残基をCysに置換し、酸化処理を行った。両酵素のCys変異体には活性がなかったが、穏やかな酸化により活性が回復した。導入したSH基が-SOOHに変化したことを確認した。Cys酸化酵素は、親酵素と異なる性質を示した(レバン合成酵素:至適pHや転移作用の変化、キチン分解酵素:至適pHや協同性の変化)。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2004年 -2005年 
    代表者 : 木村 淳夫, 森 春英, 奥山 正幸
     
    我々は、西洋ミツバチを対象に蜂蜜生成のキー酵素であるショ糖分解酵素(α-グルコシダーゼ)を研究してきた。その理由は「蜂蜜の生成が極度に高い基質濃度・生成物濃度(85%)の条件」で行われるからである。その結果、異常な反応条件に適応した3つの酵素を見出した。研究の最終の到達地点は、1)ミツバチ属における蜂蜜生成の分子機構を究明、2)活性化を招くアロステリック構造を決定、である。しかし、蛋白構造の比較対象がないため作業は難航しており、アジア原産ミツバチの酵素データに期待している。本申請の目的は、タイ・韓国の4種ミツバチについて、A)基質・生成物-活性化型のアロステリック酵素が存在すること;B)各ミツバチ種が行う蜂蜜の生成機構の特徴を比較;C)アイソザイム発現に時空間制御があることの確認、を調査することである。 アジア原産ミツバチは、西洋ミツバチと同様に3酵素あるは2酵素が存在することを調査できた。特に、東洋ミツバチについては3つの酵素をクロマト操作により分離に成功し、アロステリック酵素の照合(基質に対する活性化から判定)を行った。現在は活性化の分子解析を計画している。これらの3アイソザイムの発現に関し、時空間制御を示唆する結果を得た。小ミツバチにも基質活性化型酵素とさらにもう2種の酵素を確認できた。両ミツバチの蜂蜜にも活性があり、東洋ミツバチでは成虫酵素の1つと一致するデータを得た。クロ小ミツバチと大ミツバチの蜂蜜中にも活性があり、成虫酵素との照合を行った。これらには2種の酵素が存在する可能性が高い。本調査の解析から3アイソザイム系と2アイソザイム系の2タイプに分類できる結果を得た。本研究とは別個に行ったマル花バチの実験では2アイソザイム系を支持し、さらなる解析を行うことは花バチのショ糖分解システムの究明に重要である。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2002年 -2004年 
    代表者 : 木村 淳夫, 森 春英, 奥山 正幸, 玖村 朗人
     
    グリコシラーゼは糖質のグリコシド結合を加水分解する酵素の総称である。これらの酵素は加水分解の他に、基質のグリコシド残基をアクセプターに転移する反応(糖転移反応)を示す。転移反応は応用性が高く、i)清涼飲料水などの食品に含まれる機能性オリゴ糖の生産やii)多彩な生理活性をもつ複合糖鎖の合成に利用されている。しかし、加水分解と糖転移反応は切り離すことはできず、同時に進行する。従って、転移糖の生成条件であっても基質の加水分解が行われ、かつ、転移反応の生成物も分解されてしまう。我々は、グリコシラーゼの構造と機能を解析してきた。この研究過程で糖転移反応のみを抽出できる現象を見出した。本課題の目的は、本現象の機構を分子解析し応用に結びつけることであり、成果を以下に示す。1)対象としたグリコシラーゼについて自殺基質の手法で求核残基を推定後、本残基の点置換酵素(合成酵素)を発現・精製した。フルオライド基質について合成法を確立させ、大量調製を行った。得られた合成酵素は、いずれも分解活性を示さず、フルオライド基質とアクセプター基質を合成させる反応のみを行った。合成の至適pHはアルカリ側へシフトし、本現象は一般的に観察された。アクセプター特異性を調べたところ、アリールなどの疎水基を有する合成配糖体が最も高い収率を与えた。これは、サブサイト+2による疎水グループへの認識能が高いことを意味している。2)「歪み発生のアミノ酸変異酵素」を作製・精製・性質解析を行い、オリゴ糖合成試験を行った。収率が低いことが判明したので、反応条件を検討し、基質濃度の変化に効果を認めた。また、有機溶媒の添加も有効であったが、高濃度では活性低下が生じた。有機溶媒に耐性な糖質分解酵素が得られているので、これを歪み発生型酵素にするような発展研究を行いたい。
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2000年 -2002年 
    代表者 : 千葉 誠哉, 森 春英, 森 春英, 木村 淳夫, 福士 幸治
     
    本研究は、α-グルコシダーゼが一次構造・基質認識を異にする2つのファミリー(I型とII型)からなること(収斂進化)、さらに各々の相同酵素が存在すること(発散進化)に着目した。この事実は、両ファミリーの構造・機能に関する知見が、他のグリコシダーゼに適用できることを暗示している。本課題の目的は、ファミリー間の基質認識・機作の相違をアミノ酸レベルから分子解剖し、得た知見を相同酵素に発展させることである。1)I型酵素に自殺酵素法と点突然変異法を適用させ、解離型の触媒基を含む3つの触媒アミノ酸の推定に成功した。II型酵素においても同手法で解離型触媒基を決定し、さらにプロトン供与型の触媒残基を初めて明らかにした。II型酵素の相同酵素であるα-キシラナーゼの遺伝子発現に成功した。前述の触媒基が作動していると判断された。2)II型酵素において、基質のグリコンやアグリコンの認識を行う数個のアミノ酸を解明した。グリコンの水酸基への認識を解析するため、各々の水酸基のデオキシ基質を合成した。II型酵素のみが2-デオキシ基質に作用できた。グルカール水和反応の相違(II型のみが水和)からも2位水酸基の認識が両酵素群で異なることが推定できた。I型酵素と相同のα-アミラーゼにおいて基質結合に重要なMet残基を決定した。3)立体構造が未知のII型酵素の結晶を得た。4)C-1水素を重水素にした基質を合成し、同位...
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2001年 -2001年 
    代表者 : 木村 淳夫, 森 春英
     
    α-グルコシダーゼは、α-グルコシド結合をもつ基質に作用し、グルコースを遊離させる酵素である。転移反応では、α-グルコシル基を移し、有用な二糖類であるイソマルトースやニゲロースが工業的に生産されている。本酵素は、一次構造や基質認識が異なる2つのグループ(ファミリーIとII)に分類できる。触媒反応は、2つのカルボキシル基(_-COO^-と_-COOH)でなされ、この点では両ファミリーともに共通である。我々は、ファミリーI・II酵素の触媒残基(酸性アミノ酸)を自殺基質法や点突然変異法で決定した。II型酵素の_-COO^-である触媒基AspをAsn・Ala・Glyに置換した変異酵素は、加水分解反応を触媒できないが、活性中心の構造に大きな変化はない。最近、我々は触媒基AspのGly組換え酵素に糖転移活性が存在することを見い出した。本変異酵素は糖転移のみを一方的に行い、転移生成物を分解しない。本研究の目的は、新しく見い出されたこの現象を解析することであり、次に示す研究成果が得られた。(1)Asp→Ala酵素では本現象が認められず、Asp→Gly酵素のみが本反応を触媒した。Alaより小さなアミノ酸残基への置換が有効であった。(2)β-グルコシルフルオリドが第一基質となったが、α-グルコシルフルオリドでは反応が生じなかった。野生型酵素はα-型基質に作用するので、変異酵素の基質認識は逆転していた。従って、反応機構は縮合であると考えられた。(3)第二基質には、p-ニトロフェニル(PNPと略)α-グルコシド、α-キシロシド、α-マンノシドおよびβ-グルコシドが利用された。マルトースやPNPα-ガラクトシドには作用しなかった。PNPα-グルコシドの場合、約70%の高収率でPNPα-マルトシドとα-イソマルトシドが得られた。(4)現在、I型α-グルコシダーゼ、β-グルコシダーゼやα-ガラクトシダーゼについても本現象の解析を行っている。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1998年 -2000年 
    代表者 : 木村 淳夫, 森 春英, 千葉 誠哉
     
    最近、見い出された"生成物の固定化"現象(不溶性多糖類の酵素分解時に生成物が多糖と共沈)には重要な利点がある(生成物を容易かつ低エネルギー下に回収できる。優良な低分子化合物を多糖類に包接できる)。本研究は、この現象自体の基礎的知見をさらに集積し、利用方法が未開発・低利用な多糖類の食品産業への応用を意図して計画され、次に述ベる研究成果が得られた。(1)セルロースi)セルラーゼを作用させ、生成物固定化現象を確認した(反応初期に約50-70%の生成物が基質とともに沈殿)。ii)沈殿中の生成物は、可溶性の短鎖オリゴ糖であった。iii)反応液にエタノールを加えると、沈殿中の生成物量が増加した。反応後にアルコール濃度を下げることで、沈殿画分からオリゴ糖を容易に回収できた。iv)本手法を用いて、オリゴ糖の調製を試みた。エタノール存在下で酵素分解を行い、上清を除き、沈殿物に水を加えることで鎖長の異なるオリゴ糖を得た。分離をゲル濾過法とHPLC法で行った。(2)キチンとキシランi)生成物固定化現象が認められたが、セルロースや澱粉粒の場合に比べ、高い保持量が得られなかった。ii)エタノール存在下や低温で処理すると、固定化量が向上した。(3)澱粉多孔体(包接体)i)分解酵素を各種植物起源の澱粉粒に作用させ、多孔体の調製を行った。起源により多孔体になるもの(コムギなど)とならないもの(馬鈴薯など)が存在した。ii)酵素量や処理時間で孔径の大きさを調節できた。iii)孔径の異なる多孔体を用いて、短鎖や長鎖のオリゴ糖に対する包接能を比較した。小孔径の多孔体は短鎖オリゴ糖を包接でき、長鎖のものは大孔径の多孔体に包接された。iv)アスコルビン酸は粒内部に移行・包接され、酸化から保護された。包接後、水溶液中に放置すると徐々に遊離し、大孔径の多孔体は保持率が低かった。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1998年 -1999年 
    代表者 : 木村 淳夫, 森 春英, 千葉 誠哉
     
    微生物には、哺乳動物や植物に見られないN-あるいはO-型糖鎖が存在する。が、その生物機能や生合成機構については解明されていない。本研究は、その新規なO-型糖鎖ならびに5員環ガラクトース(以下、Galfと略す)がα型で結合する新規なN-型糖鎖の生合成に関与する酵素(UDP-Galf合成酵素とGalf転移酵素)の解析を目的として行われ、以下に示す成果が得られた。1)Aspergillus nigerのα-グルコシダーゼに存在するO-型糖鎖の精製・構造解析を行った。本酵素を還元条件下でβ-エリミネーション反応によりO-型糖鎖を化学的に切り出し、ゲル濾過法と逆相HPLCで単離した。中性糖からなる5種類の糖鎖(単糖類と二糖類が1種ずつ、三糖類が3種)が精製された。ミリマス解析・単糖分析・エキソグリコジダーゼ処理・1Dや2D-NMRにより、単糖としてマンノース、2残基のマンノースがα-1,2-結合した二糖類、マンノ二糖類の構造にグルコースが分岐結合する三糖類(新規糖鎖)、分岐型と直鎖型のマンノトリオースであった(直鎖型は新規糖鎖)。 2)Aspergillus nigerのαグルコシダーゼから、Galf転移酵素の基質ならび生成物となる糖鎖を調製した。A.niger抽出液の UDP-Galf合成酵素とGalf転移酵素の活性は微弱であるため、菌体の培養条件を検討した。マルトースやデンプンの添加で糖化酵素(分泌蛋白)の誘導とともに両酵素活性も上昇した。菌体抽出の際に失活が大きいことが認められたが、界面活性剤の共存で特にGalf転移酵素酵素の安定化が認められた。各種クロマト操作で電気泳動的に単一ではないが、純度の高い標品を得た。アミノ酸配列解析を目指して両酵素の大量調製を行っており、得られた配列から遺伝子の単離を図りたい。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1998年 -1999年 
    代表者 : 千葉 誠哉, 森 春英, 木村 淳夫, KIM Doman, ROBYT John F
     
    200nm以下の紫外線は高度な真空が要求され(真空紫外線)、微生物変異への利用が困難であった。近年、共同研究者のKim準教授は技術的問題を克服し、デキストラン合成酵素の生産株を変異させ、酵素高発現に成功した。一方、デキストランとその短鎖オリゴ糖は多彩な生理機能を示す。本研究は、デキストラン合成・分解酵素の構造と機能の関係究明ならびに合成・分解酵素を利用したデキストランや短鎖オリゴ糖の生産研究を最終目的とした国際共同研究であり、以下の成果が得られた。(1)変異株は合成酵素生産能を上昇させ、ショ糖誘導能の欠損を示した。酵素遺伝子を単離後、塩基配列を解析し、プロモータ領域に変異を見い出した。この変異は酵素高発現あるいはショ糖誘導欠損に関与する可能性が考えられた。(2)イソマルトトリオ-デキストラナーゼ遺伝子を単離した。上領域にはα-グルコシダーゼ遺伝子が存在し、同一プロモーターに支配されるクラスター構造を形成していた。本酵素遺伝子を大腸菌で大量発現させた。(3)変異株から高発現する合成酵素を用いてデキストランを大量に調製した。本糖の分岐構造を温和な酸処理で切断した。イソマルトトリオ-デキストラナーゼを作用させ、直鎖三糖類であるイソマルトトリオースの生産条件を決定した。(4)デキストランから四糖類の生成酵素を精製した。安定性などの性質を調べ、基質特異性や生成物の解析を行った。精製酵素をデキストランに作用させると、四糖類のみならず五-八糖類も調製でき、より長鎖のオリゴ糖を獲得できることが見い出された。(5)デキストラン分解酵素に対する新規なmechanism-based inactivatorのデザイン・合成を行い、自殺基質的に酵素を失活させていることを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1997年 -1999年 
    代表者 : 千葉 誠哉, 森 春英, 木村 淳夫
     
    本研究は、糖代謝に重要な役割を担うα-グルコシダーゼの基質認識や構造を総合的に解析し、"大きく2つのファミリー(ファミリーIとII)に大別できること"の実証を意図したものである。両グループの触媒部位構造の詳細な解析も行い、反応機作の究明も目的に加えた。1.植物酵素のうち、ソバ、イネ、テンサイのものについて、遺伝子単離に成功した。アミノ酸一次配列を決定し、典型的なファミリーIIの酵素であることが認められた。基質認識もこれと符合する。単・双子葉植物起源の酵素は、いずれもII型であることが判明した。2.動物酵素は、ミツバチに関して遺伝子単離を終了させた。I型の基質認識を示すものであり、構造もこれを支持する。ブタ血液酵素に関しては活性解離基(カルボキシル基)を調べた。部分アミノ酸配列からファミリーII型酵素であると推定した。3.微生物酵素のうち、好熱性Bacillus属酵素は、基質認識とN末端アミノ酸配列からファミリーIに属すと考えられた。速度論的解析からカルボキシル基が活性解離基であることを見い出した(至適pHは中性域にあり、His残基の関与が報告されていたが、カルボキシル基であった)。分裂酵母の酵素は遺伝子の単離に成功した。推定されたアミノ酸一次配列からII型であることを確認した。構造中にI型ファミリーの活性部位配列があったので、点突然変異法で検討した。いずれの変異体も活性を有し、これらの配列は機能していない。グルコアミラーゼと報じられているSchanniomyces ocidentalis酵素はα-グルコシダーゼであった。エドマン法で解析したアミノ酸配列から、II型ファミリーに属すと考えられる。4.本計画の知見からα-グルコシダーゼは二つのファミリーに分類可能と思われる。これらの結果から、α-グルコシダーゼの収斂進化(異なる祖先蛋白から別々な進化を行い、生物機能が同一の蛋白になる)が予想された。
  • 文部科学省：科学研究費補助金(奨励研究(A))
    研究期間 : 1997年 -1998年 
    代表者 : 森 春英
     
    双子葉植物であるPhaseolus vulgarisにおいて、α-amylaseにアイソザイムは存在せず、発芽子葉と緑葉において同一の酵素が発現する。この同一のタンパク質が器官特異的な因子により異なる細胞内オルガネラに標的されている可能性を確認するために、まずPhaseolus vulgarisにおいてα-amylaseの細胞内局在性の確認と、発現制御の観点から遺伝子のクローニングを行った。(1) Phaseolus vulgarisにおける細胞内局在性の確認:Phaseolus vulgaris緑葉および発芽子葉における酵素の細胞内局在性を明らかにすることを目的に、ショ糖密度勾配法を用いた細胞内器官分画を行った。これにより、本酵素は子葉においてはプラスチド画分にあることが示唆され、一方緑葉においては少なくとも葉緑体画分に酵素は検出されなかった。さらに明確にするために、金コロイドによる免疫電子顕微鏡像を作製する。抗体を調製した。(2) Arabidopsisの形質転換(減圧浸潤法)をpBl121を用いて行った。また、pBl121上のGUSのN型糖鎖付加配列(-)の変異体GUS N356Sをレポーターに持つpBl124を創出した。これを用いて、本酵素上の器官特異的細胞内局在性因子を解析する。(3) α-Amylase遺伝子のクローニング:P.vulgans α-amylase遺...
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1995年 -1995年 
    代表者 : 松井 博和, 森 春英, 伊藤 浩之
     
    本研究では、低栄養素ストレス下でも期待する収量が得られるよう、栄養素の利用効率を高めた作物の作出が急務と考え、作物が多量に集積するデンプンに着目した。サイトウやイネを材料に、デンプン合成・分解に関わる酵素と、いわゆるストレス関連酵素であるperoxidaseについて得られた成果を概説する。 1.デンプン枝付け酵素(Branching enzyme: BEと略)の精製と諸性質……トラマメ登熟種子よりBEを精製し、一般的な諸性質を明らかにした。本酵素はZn^<2+>やHg^<2+>などの金属イオンにより阻害されるばかりでなく、Ca^<2+>によっても強い阻害を受けた。N末端配列とプロテアーゼ消化による幾つかのペプチド断片の配列を解析したところ、イネおよびトウモロコシ起源のBEに高い相同性を示した。 2.α-Amylaseの精製とcDNAの解析……トラマメ発芽期と登熟期のcDNA解析を行った。両者には3'末端側のpolyA部分のみに相違が認められた。一方、緑葉中のα-Amylaseを単一に精製し、発芽種子α-Amylaseと性質や一次配列が酷似していることを明らかにした。 3.ストレス酵素Peroxidase遺伝子……イネperoxidaseをコードするprxRPN cDNAに対応する遺伝子(poxN)を単離し構造を解析した。さらに、プロモーター領域を5段階に削り込み、その下流にβ-グルクロニダーゼ(GUS)遺伝子を導入した各プラスミドを用いてタバコを形質転換し、得られた形質転換タバコ葉に幾つかの処理を施しGUS活性を測定した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1993年 -1995年 
    代表者 : 千葉 誠哉, 森 春英, 伊藤 浩之, 木村 淳夫
     
    1.グルコアミラーゼの縮合本能速度の動的解析---本酵素の縮合反応を基質であるβ-グルコースを用い、二基質反応として取り扱い、速度パラメータを算出し、反応速度式を導いた。β-グルコースによる縮合反応系にα-グルコースを共存させれると、拮抗せず、反応促進がみられた。この現象を解析した結果、α-グルコースが本酵素の第一サブサイトに結合せず、専ら第二サブサイトに結合し、その親和力がβ-グルコースより強いことが判明した。一方、全グルコース濃度を一定にし、グルコースアノマーの様々な混合比率における縮合反応速度を測定したところ、βアノマーの比率が低いと速度が減少した。これは、基質であるβ-グルコースの濃度低下による速度減少効果の寄与がα-グルコースによる反応促進より大きいことを意味し、ムタロターゼの効果が期待された。 2.ムタロターゼによる縮合反応の抑制とグルコース製造試験---30%β-グルコースに対するグルコアミアラーゼの縮合反応はムタロターゼの存在により30%低下した。現行のバッチ方式によるグルコース製造法に対し、ムタロターゼの効果を調べた。次に、グルコアミアラーゼ固定化カラムを作製し、ムタロターゼ存在下でマルトデキストリン(液化デンプンの代用)の送液・糖化試験をカラム内で行った。両試験ともに、ムタロターゼの共存によりグルコースの収量向上と縮合反応による二糖類生成量の低下が認められ、ムタロターゼの良好な効果を確認した。 3.ブタ腎臓ムタロターゼの蛋白構造解析---本酵素の遺伝子取得と大量発現を意図し、アミノ酸配列の解析を行った。本酵素はN末端がブロックされていた。プロテアーゼ消化で得られたN末端閉塞ペプチドの配列をタンデム質量分析法で決定し、アセチル基によるブロックであることを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 1994年 -1994年 
    代表者 : 松井 博和, 森 春英, 伊藤 浩之
     
    低栄養条件下でも期待する収量が得られるよう、栄養素の利用効率を高めた作物を作出する観点から、本研究ではイネ科およびマメ科作物の栄養利用効率の差異を明らかにし、炭素および糖代謝化合物分配系、澱粉合成関連酵素ならびにストレス一般に関わる幾つかの酵素について、生化学・分子生物学的に解析することを目的とし、以下の成果を得た。 1。C-Nバランス酵素系:イネとダイズを標準培養液で水耕栽培し、種々の窒素条件下での呼吸速度を調べたところ、光呼吸および暗呼吸のいずれの速度をダイズの方が高かった。このような条件下における両作物のPEPCおよびSPS活性を測定したところ、窒素量とPEPC活性に相関が認められ、ダイズではいずれの活性もイネのそれら活性より低かったが、PEPC/SPSは高く、ダイズSPSはイネ酵素よりも窒素含有量に敏感には感応していないものと判断された。 2。初期光合成産物の同定:作物に^<14>CO_2を10分間吸収させ、その直後と30分後の葉を採取し、^<14>Cの分配を調べた。その結果、イネでは糖画分に多く分配されるのに対し、ダイズでは有機酸やアミノ酸に多く分配された。 3。澱粉合成時の酵素系:トラマメ登熟種子には少なくとも2種類のBranching Enzymeが存在することを明らかにした。DEAE-SepharoseおよびBio-Gel P-200を用いたクロマトグラフィーにより、その1つをSDS-PAGE的に単一に精製した。 4。ストレス酵素系:peroxidase isozymesの発現誘導機構を明らかにする目的で、その遺伝子断片を単離し、構造を解析した。また、5^1上流プロモーター領域の解析を、レポーターとしてβ-glucuronidase(GUS)遺伝子を用いた形質転換タバコで行った。

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