Researcher Profile and Settings

Master

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Bioscience and Chemistry

Affiliation (Master)

• Research Faculty of Agriculture　Fundamental AgriScience Research　Bioscience and Chemistry

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Profile and Settings

Degree

• Dr（Hokkaido University）

Profile and Settings

• Name (Japanese)

  Mori

• Name (Kana)

  Haruhide

• Name

  200901035995397800

Achievement

Research Interests

• 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)   

Research Areas

• Life sciences / Applied biochemistry

Research Experience

• 2013/04 - Today Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
• 2019/04 - 2021/03 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
• 2017/04 - 2019/03 Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
• 2010 - 2012 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 - 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

Education

•        - 1992  Hokkaido University
•        - 1992  Hokkaido University  Graduate School, Division of Agriculture

Awards

• 2019/09 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 Best Poster Award
  
• 2005/09 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 ベストポスター賞
  
• 2003 B.B.B.論文賞
  
• 2003 Biosci Biotechnol Biochem Paper Award(JSBBA, 2003)
  

Published Papers

• Molecular mechanism for the substrate specificity of Arthrobacter globiformis M6 α-glucosidase CmmB, belonging to glycoside hydrolase family 13 subfamily 30

  Wataru Saburi, Takayoshi Tagami, Takuya Usui, Jian Yu, Toyoyuki Ose, Min Yao, Haruhide Mori

  Food Bioscience 61 104516 - 104516 2212-4292 2024/10 [Refereed][Not invited]
  
• Comparisons of the amylolytic enzymes and malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar developed in Japan) and Kitanohoshi (currently used cultivar for beer production)

  Wataru Saburi, Haruhide Mori

  Bioscience, Biotechnology, and Biochemistry 88 (10) 1180 - 1187 2024/07/11 

  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.
• Tunable structure of chimeric isomaltomegalosaccharides with double α-(1 → 4)-glucosyl chains enhances the solubility of water-insoluble bioactive compounds

  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 0144-8617 2023/11 [Refereed][Not invited]
  
• Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-β-1,3-glucanase on β1-3/1-6-glucan.

  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 2023/09/27 [Refereed][Not invited]
   

  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.
• Hydrolysis-transglycosylation of sucrose and production of β-(2→1)-fructan by inulosucrase from Neobacillus drentensis 57N.

  Yusuke Kido, Wataru Saburi, Taizo Nagura, Haruhide Mori

  Bioscience, biotechnology, and biochemistry 87 (10) 1169 - 1182 2023/09/21 [Refereed][Not invited]
   

  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.
• Chemical synthesis of oligosaccharide derivatives with partial structure of β1-3/1-6 glucan, using monomeric units for the formation of β1-3 and β1-6 glucosidic linkages.

  Tomoya Ota, Wataru Saburi, Shiro Komba, Haruhide Mori

  Bioscience, biotechnology, and biochemistry 2023/07/05 [Refereed][Not invited]
   

  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.
• Structural insights into the substrate specificity and activity of a novel mannose 2-epimerase from Runella slithyformis.

  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 2023/07/01 [Refereed][Not invited]
   

  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.
• Identification and characterization of extracellular GH3 β-glucosidase from the pink snow mold fungus, Microdochium nivale.

  Tomoya Ota, Wataru Saburi, Linda Elizabeth Jewell, Tom Hsiang, Ryozo Imai, Haruhide Mori

  Bioscience, biotechnology, and biochemistry 87 (7) 707 - 716 2023/06/23 [Refereed][Not invited]
   

  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.
• Alteration of Substrate Specificity and Transglucosylation Activity of GH13_31 α-Glucosidase from Bacillus sp. AHU2216 through Site-Directed Mutagenesis of Asn258 on β→α Loop 5

  Waraporn Auiewiriyanukul, Wataru Saburi, Tomoya Ota, Jian Yu, Koji Kato, Min Yao, Haruhide Mori

  Molecules 28 (7) 3109 - 3109 2023/03/30 [Refereed][Invited]
   

  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.
• Function and structure of Lacticaseibacillus casei GH35 β-galactosidase LBCZ_0230 with high hydrolytic activity to lacto-N-biose I and galacto-N-biose

  Saburi Wataru, Tomoya Ota, Koji Kato, Takayoshi Tagami, Keitaro Yamashita, Min Yao, Haruhide Mori

  Journal of Applied Glycoscience 70 (2) 43 - 52 1344-7882 2023/03/11 [Refereed][Not invited]
  
• Substrate specificity of glycoside hydrolase family 1 β-glucosidase AtBGlu42 from Arabidopsis thaliana and its molecular mechanism.

  Shu Horikoshi, Wataru Saburi, Jian Yu, Hideyuki Matsuura, James R Ketudat Cairns, Min Yao, Haruhide Mori

  Bioscience, biotechnology, and biochemistry 86 (2) 231 - 245 2022/01/24 [Refereed]
   

  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.
• A practical approach to producing isomaltomegalosaccharide using dextran dextrinase from Gluconobacter oxydans ATCC 11894.

  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 2022/01 [Refereed]
   

  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%.
• Discovery of solabiose phosphorylase and its application for enzymatic synthesis of solabiose from sucrose and lactose

  Wataru Saburi, Takanori Nihira, Hiroyuki Nakai, Motomitsu Kitaoka, Haruhide Mori

  Scientific Reports 12 (1) 2022/01 [Refereed]
   

  Glycoside 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.
• A Ubiquitously Expressed UDP-Glucosyltransferase, UGT74J1, Controls Basal Salicylic Acid Levels in Rice

  Daisuke Tezuka, Hideyuki Matsuura, Wataru Saburi, Haruhide Mori, Ryozo Imai

  Plants 10 (9) 1875 - 1875 2021/09/10 [Refereed]
   

  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.
• Preliminary evaluation of colorimetric and HPLC-based methods for quantifying β-(1→4)-mannobiose in a crude material

  Kensuke Fukui, Wataru Saburi, Masahisa Ibuki, Kazunobu Tsumura, Haruhide Mori

  Food Science and Technology Research 27 (2) 249 - 257 1344-6606 2021/05/22 [Refereed][Not invited]
  
• [Review] Functions of Hydrolases, Phosphorylases, and Isomerases Acting on Carbohydrates, and their Application

  Haruhide Mori

  Bulletin of Applied Glycoscience 10 (3) 165 - 174 2185-6427 2020/08/20 [Refereed][Invited]
  
• Efficient one-pot enzymatic synthesis of trehalose 6-phosphate using GH65 α-glucoside phosphorylases.

  Yodai Taguchi, Wataru Saburi, Ryozo Imai, Haruhide Mori

  Carbohydrate research 488 107902 - 107902 2020/02 [Refereed][Not invited]
   

  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.
• Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme.

  Gao Y, Saburi W, Taguchi Y, Mori H

  Bioscience, biotechnology, and biochemistry 1 - 13 0916-8451 2019/07 [Refereed][Not invited]
  
• Enzymatic characteristics of D-mannose 2-epimerase, a new member of the acylglucosamine 2-epimerase superfamily.

  Saburi W, Sato S, Hashiguchi S, Muto H, Iizuka T, Mori H

  Applied microbiology and biotechnology 0175-7598 2019/06 [Refereed][Not invited]
  
• The rice ethylene response factor OsERF83 positively regulates disease resistance to Magnaporthe oryzae.

  Tezuka D, Kawamata A, Kato H, Saburi W, Mori H, Imai R

  Plant Physiol Biochem 135 263 - 271 0981-9428 2019/02 [Refereed][Not invited]
  
• A Transposon Mutagenesis System for Bifidobacterium longum subsp. longum Based on an IS3 Family Insertion Sequence, ISBlo11.

  Mikiyasu Sakanaka, Shingo Nakakawaji, Shin Nakajima, Satoru Fukiya, Arisa Abe, Wataru Saburi, Haruhide Mori, Atsushi Yokota

  Applied and environmental microbiology 84 (17) e00824-18  2018/09/01 [Refereed][Not invited]
   

  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.
• Function and structure of GH13_31 α-glucosidase with high α-(1→4)-glucosidic linkage specificity and transglucosylation activity.

  Auiewiriyanukul W, Saburi W, Kato K, Yao M, Mori H

  FEBS Lett 592 (13) 2268 - 2281 2018/07 [Refereed][Not invited]
  
• Biochemical and structural characterization of Marinomonas mediterranea D-mannose isomerase Marme_2490 phylogenetically distant from known enzymes

  Wataru Saburi, Nongluck Jaito, Koji Kato, Yuka Tanaka, Min Yao, Haruhide Mori

  Biochimie 144 63 - 73 6183-1638 2018/01/01 [Refereed][Not invited]
   

  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.
• Effects of mutation of Asn694 in Aspergillus niger alpha-glucosidase on hydrolysis and transglucosylation

  Min Ma, Masayuki Okuyama, Megumi Sato, Takayoshi Tagami, Patcharapa Klahan, Yuya Kumagai, Haruhide Mori, Atsuo Kimura

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 101 (16) 6399 - 6408 0175-7598 2017/08 [Refereed][Not invited]
   

  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).
• Elucidation of the biosynthetic pathway of cis-jasmone in Lasiodiplodia theobromae

  Ryo Matsui, Naruki Amano, Kosaku Takahashi, Yodai Taguchi, Wataru Saburi, Hideharu Mori, Norio Kondo, Kazuhiko Matsuda, Hideyuki Matsuura

  SCIENTIFIC REPORTS 7 (1) 6688  2045-2322 2017/07 [Refereed][Not invited]
   

  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 2017/05 [Refereed][Invited]
  
• Efficient synthesis of alpha-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining alpha-galactosidase (BtGH97b)

  Masayuki Okuyama, Kana Matsunaga, Ken-ichi Watanabe, Keitaro Yamashita, Takayoshi Tagami, Asako Kikuchi, Min Ma, Patcharapa Klahan, Haruhide Mori, Min Yao, Atsuo Kimura

  FEBS JOURNAL 284 (5) 766 - 783 1742-464X 2017/03 [Refereed][Not invited]
   

  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.
• Evaluation of acceptor selectivity of Lactococcus lactis ssp lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate

  Yodai Taguchi, Wataru Saburi, Ryozo Imai, Haruhide Mori

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 (8) 1512 - 1519 0916-8451 2017 [Refereed][Not invited]
   

  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.
• Kinetic properties and substrate inhibition of -galactosidase from Aspergillus niger

  Julan Liao, Masayuki Okuyama, Keigo Ishihara, Yoshinori Yamori, Shigeo Iki, Takayoshi Tagami, Haruhide Mori, Seiya Chiba, Atsuo Kimura

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 (9) 1747 - 1752 0916-8451 2016/09 [Refereed][Not invited]
   

  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.
• alpha-Glucosidases and alpha-1,4-glucan lyases: structures, functions, and physiological actions

  Masayuki Okuyama, Wataru Saburi, Haruhide Mori, Atsuo Kimura

  CELLULAR AND MOLECULAR LIFE SCIENCES 73 (14) 2727 - 2751 1420-682X 2016/07 [Refereed][Not invited]
   

  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.
• Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies

  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 0014-5793 2016/03 [Refereed][Not invited]
   

  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.
• Purification and characterization of a chloride ion-dependent alpha-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis)

  Yasushi Masuda, Masayuki Okuyama, Takahisa Iizuka, Hiroyuki Nakai, Wataru Saburi, Taro Fukukawa, Janjira Maneesan, Takayoshi Tagami, Tetsushi Naraoka, Haruhide Mori, Atsuo Kimura

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 (3) 479 - 485 0916-8451 2016/03 [Refereed][Not invited]
   

  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 [Refereed][Not invited]
  
• Supplemental epilactose prevents metabolic disorders through uncoupling protein-1 induction in the skeletal muscle of mice fed high-fat diets

  Yuki Murakami, Teruyo Ojima-Kato, Wataru Saburi, Haruhide Mori, Hirokazu Matsui, Soichi Tanabe, Takuya Suzuki

  BRITISH JOURNAL OF NUTRITION 114 (11) 1774 - 1783 0007-1145 2015/12 [Refereed][Not invited]
   

  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.
• Identification of rice Os4BGlu13 as a beta-glucosidase which hydrolyzes gibberellin A4 1-O-beta-D-glucosyl ester, in addition to tuberonic acid glucoside and salicylic acid derivative glucosides

  Yanling Hua, Watsamon Ekkhara, Sompong Sansenya, Chantragan Srisomsap, Sittiruk Roytrakul, Wataru Saburi, Ryosuke Takeda, Hideyuki Matsuura, Haruhide Mori, James R. Ketudat Cairns

  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 583 36 - 46 0003-9861 2015/10 [Refereed][Not invited]
   

  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.
• A Single-Nucleotide Polymorphism in an Endo-1,4-beta-Glucanase Gene Controls Seed Coat Permeability in Soybean

  Seong-Jin Jang, Masako Sato, Kei Sato, Yutaka Jitsuyama, Kaien Fujino, Haruhide Mori, Ryoji Takahashi, Eduardo R. Benitez, Baohui Liu, Tetsuya Yamada, Jun Abe

  PLOS ONE 10 (6) e0128527  1932-6203 2015/06 [Refereed][Not invited]
   

  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.
• Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-beta-d-mannosyl-d-glucose phosphorylase-like protein, UnkA

  Wataru Saburi, Yuka Tanaka, Hirohiko Muto, Sota Inoue, Rei Odaka, Mamoru Nishimoto, Motomitsu Kitaoka, Haruhide Mori

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 79 (6) 969 - 977 0916-8451 2015/06 [Refereed][Not invited]
   

  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.
• Structural analysis of the alpha-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism

  Xing Shen, Wataru Saburi, Zuoqi Gai, Koji Kato, Teruyo Ojima-Kato, Jian Yu, Keisuke Komoda, Yusuke Kido, Hirokazu Matsui, Haruhide Mori, Min Yao

  ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 71 (Pt 6) 1382 - 1391 1399-0047 2015/06 [Refereed][Not invited]
   

  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.
• Structural elements responsible for the glucosidic linkage-selectivity of a glycoside hydrolase family 13 exo-glucosidase

  Wataru Saburi, Hiroaki Rachi-Otsuka, Hironori Hondoh, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura

  FEBS LETTERS 589 (7) 865 - 869 0014-5793 2015/03 [Refereed][Not invited]
   

  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.
• Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate-binding site in GH13 dextran glucosidase

  Momoko Kobayashi, Wataru Saburi, Daichi Nakatsuka, Hironori Hondoh, Koji Kato, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Min Yao

  FEBS LETTERS 589 (4) 484 - 489 0014-5793 2015/02 [Refereed][Not invited]
   

  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.
• UDP-Glc Independent Glucosyltransferase toward Jasmonic acid Derivatives and its Biological Role.

  Takematsu Tomonori, Seto Yoshiya, Miyazawa Yoshiro, Wakuta Shinji, Ogihara Tsuyoshi, Saburi Wataru, Mori Haruhide, Takahashi Kosaku, Matsuura Hideyuki

  Symposium on the Chemistry of Natural Products, symposium papers 天然有機化合物討論会実行委員会 57 Oral14  2015 

  Plants are sessile organisms and are unable to avoid environmental stresses by changing their habitats. Therefore, plants have developed unique and sophisticated responding systems. It has been generally accepted that plant use plant hormones to give actions toward (against) environmental changes and stress. Among of the hormones, jasmonic acid(s) has pivotal roles to perform the responses. In recent years, not only the activation of JA pathway but also the deactivations of active form JA are being paid a lot of attentions such as oxidations and glucosylations.

  In previous our paper [1], we reported that Os SGT, putative salicylic acid glucosyltransferase, transferred glucosyl moiety toward 12-OHJA to afford 12-OGlcJA, and its mRNA was induced by wounding stress and JA and SA treatments. In the course of that study, we also found UDP-Glc independent glucosyl transferase activity to give preferably 12-OGlcJA in the crude extract of rice cell culture using octyl glucoside as donor molecule for supplying glucosyl moiety. There are few reports of the finding on UDP-Glc independent glucosyltransferase protein, and to our best knowledge, a report has been published by Matsuba et al. [2]. In this presentation we discuss elucidation of UDP-Glc independent glucosyl transferase toward 12-OHJA and 12-OHJA-Ile

  [1] Seto Y. et al., Phytochemistry, 70, 370-379 (2009).

  [2] Matsuba Y. et al.,Plant Cell,22, 3374-3389 (2010).

• Identity of the two dextran dextrinases produced by Gluconobacter oxydans ATCC 11894 and its localization change depending on the cell growth.

  Sadahiro J, Mori H, Saburi W, Okuyama M, Kimura A

  Biochem Biophys Res Commun 456 (1) 500 - 505 0006-291X 2015 [Refereed][Not invited]
   

  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.
• Biochemical properties and substrate recognition mechanism of GH31 alpha-glucosidase from Bacillus sp AHU 2001 with broad substrate specificity

  Wataru Saburi, Masayuki Okuyama, Yuya Kumagai, Atsuo Kimura, Haruhide Mori

  BIOCHIMIE 108 140 - 148 0300-9084 2015/01 [Refereed][Not invited]
   

  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.
• Structural Advantage of Sugar Beet alpha-Glucosidase to Stabilize the Michaelis Complex with Long-chain Substrate

  Takayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 290 (3) 1796 - 1803 0021-9258 2015/01 [Refereed][Not invited]
   

  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.
• Different molecular complexity of linear-isomaltomegalosaccharides and beta-cyclodextrin on enhancing solubility of azo dye ethyl red: Towards dye biodegradation

  Weeranuch Lang, Yuya Kumagai, Juri Sadahiro, Janjira Maneesan, Masayuki Okuyama, Haruhide Mori, Nobuo Sakairi, Atsuo Kimura

  BIORESOURCE TECHNOLOGY 169 518 - 524 0960-8524 2014/10 [Refereed][Not invited]
   

  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.
• Catalytic role of the calcium ion in GH97 inverting glycoside hydrolase

  Masayuki Okuyama, Takuya Yoshida, Hironori Hondoh, Haruhide Mori, Min Yao, Atsuo Kimura

  FEBS LETTERS 588 (17) 3213 - 3217 0014-5793 2014/08 [Refereed][Not invited]
   

  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.
• Enhancement of hydrolytic activity of thermophilic alkalophilic alpha-amylase from Bacillus sp AAH-31 through optimization of amino acid residues surrounding the substrate binding site

  Naoya Tamamura, Wataru Saburi, Atsushi Mukai, Naoki Morimoto, Toshihiko Takehana, Seiji Koike, Hirokazu Matsui, Haruhide Mori

  BIOCHEMICAL ENGINEERING JOURNAL 86 8 - 15 1369-703X 2014/05 [Refereed][Not invited]
   

  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.
• Crystallization and preliminary X-ray crystallographic analysis of α-glucosidase HaG from Halomonas sp. strain H11.

  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 2014/04 [Refereed][Not invited]
   

  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).
• Structural insights into the epimerization of β-1,4-linked oligosaccharides catalyzed by cellobiose 2-epimerase, the sole enzyme epimerizing non-anomeric hydroxyl groups of unmodified sugars.

  Fujiwara T, Saburi W, Matsui H, Mori H, Yao M

  The Journal of biological chemistry American Society for Biochemistry and Molecular Biology (ASBMB) 289 (6) 3405 - 3415 0021-9258 2014/02 [Refereed][Not invited]
   

  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.
• Production of 1,5-anhydro-D-fructose by an α-glucosidase belonging to glycoside hydrolase family 31.

  Maneesan J, Matsuura H, Tagami T, Mori H, Kimura A

  Biosci Biotechnol Biochem 78 (12) 2064 - 2068 2014 [Refereed][Not invited]
  
• Characterization of a thermophilic 4-O-β-D-mannosyl-D-glucose phosphorylase from Rhodothermus marinus

  Nongluck Jaito, Wataru Saburi, Rei Odaka, Yusuke Kido, Ken Hamura, Mamoru Nishimoto, Motomitsu Kitaoka, Hirokazu Matsui, Haruhide Mori

  Bioscience, Biotechnology and Biochemistry Taylor & Francis 78 (2) 263 - 270 0916-8451 2014/01/01 [Not refereed][Not invited]
   

  © 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.
• Biodecolorization of a food azo dye by the deep sea Dermacoccus abyssi MT1.1(T) strain from the Mariana Trench

  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 0301-4797 2014/01 [Refereed][Not invited]
   

  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.
• Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity

  Wataru Saburi, Momoko Kobayashi, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 288 (44) 31670 - 31677 0021-9258 2013/11 [Refereed][Not invited]
   

  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.
• Characterization of Ruminococcus albus cellodextrin phosphorylase and identification of a key phenylalanine residue for acceptor specificity and affinity to the phosphate group.

  Sawano T, Saburi W, Hamura K, Matsui H, Mori H

  The FEBS journal Wiley-Blackwell 280 (18) 4463 - 4473 1742-464X 2013/09 [Refereed][Not invited]
   

  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.
• Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis.

  Hamura K, Saburi W, Matsui H, Mori H

  Carbohydrate research Elsevier sci ltd 379 21 - 25 0008-6215 2013/09 [Refereed][Not invited]
   

  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.
• Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity

  Takatsugu Miyazaki, Megumi Ichikawa, Gaku Yokoi, Motomitsu Kitaoka, Haruhide Mori, Yoshikazu Kitano, Atsushi Nishikawa, Takashi Tonozuka

  FEBS Journal 280 4560 - 4571 1742-464X 2013/09/01 [Not refereed][Not invited]
   

  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.
• Aromatic Residue on beta ->alpha Loop 1 in the Catalytic Domain Is Important to the Transglycosylation Specificity of Glycoside Hydrolase Family 31 alpha-Glucosidase

  Kyung-Mo Song, Masayuki Okuyama, Mariko Nishimura, Takayoshi Tagami, Haruhide Mori, Atsuo Kimura

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (8) 1759 - 1765 0916-8451 2013/08 [Refereed][Not invited]
   

  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.
• Identification of rice β-glucosidase with high hydrolytic activity towards salicylic acid β-d-glucoside

  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 Japan Society for Bioscience, Biotechnology, and Agrochemistry 77 (5) 934 - 939 0916-8451 2013/06/12 [Refereed][Not invited]
   

  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.
• Molecular Basis for the Recognition of Long-chain Substrates by Plant alpha-Glucosidases

  Takayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 288 (26) 19296 - 19303 0021-9258 2013/06 [Refereed][Not invited]
   

  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.
• Crystal structure of Ruminococcus albus cellobiose 2-epimerase: structural insights into epimerization of unmodified sugar.

  Fujiwara T, Saburi W, Inoue S, Mori H, Matsui H, Tanaka I, Yao M

  FEBS letters 587 (7) 840 - 846 0014-5793 2013/04 [Refereed][Not invited]
  
• Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

  Takayoshi Tagami, Yoshiyuki Tanaka, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (2) 312 - 319 0916-8451 2013/02 [Refereed][Not invited]
   

  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.
• Identification and characterization of cellobiose 2-epimerases from various aerobes.

  Ojima T, Saburi W, Yamamoto T, Mori H, Matsui H

  Bioscience, biotechnology, and biochemistry Japan Society for Bioscience, Biotechnology, and Agrochemistry 77 (1) 189 - 193 0916-8451 2013 [Refereed][Not invited]
   

  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.
• A thermophilic alkalophilic α-amylase from Bacillus sp. AAH-31 shows a novel domain organization among glycoside hydrolase family 13 enzymes.

  Saburi W, Morimoto N, Mukai A, Kim DH, Takehana T, Koike S, Matsui H, Mori H

  Bioscience, biotechnology, and biochemistry Japan Society for Bioscience, Biotechnology, and Agrochemistry 77 (9) 1867 - 1873 0916-8451 2013 [Refereed][Not invited]
   

  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 k_cat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s⁻¹, 67.6 s⁻¹, and 5.33 s⁻¹, respectively, and the K_m 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.
• A novel mechanism for the promotion of quercetin glycoside absorption by megalo alpha-1,6-glucosaccharide in the rat small intestine

  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 0308-8146 2013/01 [Refereed][Not invited]
   

  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.
• Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 alpha-glucosidase contribute to recognition of long-chain substrates

  Takayoshi Tagami, Masayuki Okuyama, Hiroyuki Nakai, Young-Min Kim, Haruhide Mori, Kazunori Taguchi, Birte Svensson, Atsuo Kimura

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1834 (1) 329 - 335 1570-9639 2013/01 [Refereed][Not invited]
   

  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.
• Characterization of a glycoside hydrolase family 31 α-glucosidase involved in starch utilization in podospora anserina

  Kyung-Mo Song, Masayuki Okuyama, Kazuyuki Kobayashi, Haruhide Mori, Atsuo Kimura

  Bioscience, Biotechnology and Biochemistry 77 (10) 2117 - 2124 0916-8451 2013 [Refereed][Not invited]
   

  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.
• Metabolic Mechanism of Mannan in a Ruminal Bacterium, Ruminococcus albus, Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase: DISCOVERY OF A NEW CARBOHYDRATE PHOSPHORYLASE, β-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE.

  Kawahara R, Saburi W, Odaka R, Taguchi H, Ito S, Mori H, Matsui H

  The Journal of biological chemistry 50 287 (50) 42389 - 42399 0021-9258 2012/12 [Refereed][Not invited]
   

  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.
• The Delay in the Development of Experimental Colitis from Isomaltosyloligosaccharides in Rats Is Dependent on the Degree of Polymerization

  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 1932-6203 2012/11 [Refereed][Not invited]
   

  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
• Amino Acids in Conserved Region II Are Crucial to Substrate Specificity, Reaction Velocity, and Regioselectivity in the Transglucosylation of Honeybee GH-13 alpha-Glucosidases

  Lukana Ngiwsara, Gaku Iwai, Takayoshi Tagami, Natsuko Sato, Hiroyuki Nakai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 (10) 1967 - 1974 0916-8451 2012/10 [Refereed][Not invited]
   

  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.
• Bacteroides thetaiotaomicron VPI-5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions

  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 1742-464X 2012/09 [Refereed][Not invited]
   

  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.
• Purification and Characterization of a Liquefying alpha-Amylase from Alkalophilic Thermophilic Bacillus sp AAH-31

  Dae Hoon Kim, Naoki Morimoto, Wataru Saburi, Atsushi Mukai, Koji Imoto, Toshihiko Takehana, Seiji Koike, Haruhide Mori, Hirokazu Matsui

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Japan Society for Bioscience, Biotechnology, and Agrochemistry 76 (7) 1378 - 1383 0916-8451 2012/07 [Not refereed][Not invited]
   

  alpha-Amylase (EC 3.2.1.1) hydrolyzes an internal alpha-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 a-amylase with high thermostability. Extracellular alpha-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 optimum pH and temperature were 8.5 and 70 degrees C respectively. It was stable in a pH range of 6.4-10.3 and below 60 degrees C. The calcium ion did not affect its thermostability, unlike typical alpha-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 degrees C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards gamma-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.
• Structural Elucidation of Dextran Degradation Mechanism by Streptococcus mutans Dextranase Belonging to Glycoside Hydrolase Family 66

  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 0021-9258 2012/06 [Refereed][Not invited]
   

  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.
• Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

  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 0021-9258 2012/06 [Refereed][Not invited]
   

  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.
• A Novel Metabolic Pathway for Glucose Production Mediated by alpha-Glucosidase-catalyzed Conversion of 1,5-Anhydrofructose

  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 0021-9258 2012/06 [Refereed][Not invited]
   

  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.
• Enzymatic Characteristics of Cellobiose Phosphorylase from Ruminococcus albus NE1 and Kinetic Mechanism of Unusual Substrate Inhibition in Reverse Phosphorolysis

  Ken Hamura, Wataru Saburi, Shotaro Abe, Naoki Morimoto, Hidenori Taguchi, Haruhide Mori, Hirokazu Matsui

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 (4) 812 - 818 0916-8451 2012/04 [Not refereed][Not invited]
   

  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).
• Chemical constituents and free radical scavenging activity of corn pollen collected from Apis mellifera hives compared to floral corn pollen at Nan, Thailand

  Atip Chantarudee, Preecha Phuwapraisirisan, Kiyoshi Kimura, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Chanpen Chanchao

  BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 12 45  1472-6882 2012/04 [Refereed][Not invited]
   

  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.
• In vitro antiproliferative/cytotoxic activity on cancer cell lines of a cardanol and a cardol enriched from Thai Apis mellifera propolis

  Dungporn Teerasripreecha, Preecha Phuwapraisirisan, Songchan Puthong, Kiyoshi Kimura, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura, Chanpen Chanchao

  BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 12 27  1472-6882 2012/03 [Refereed][Not invited]
   

  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.
• Immobilization of a thermostable cellobiose 2-epimerase from Rhodothermus marinus JCM9785 and continuous production of epilactose.

  Sato H, Saburi W, Ojima T, Taguchi H, Mori H, Matsui H

  Bioscience, biotechnology, and biochemistry 8 76 (8) 1584 - 1587 0916-8451 2012 [Refereed][Not invited]
   

  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 [Refereed][Not invited]
  
• 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 [Refereed][Not invited]
  
• Crystallization and preliminary crystallographic analysis of dextranase from Streptococcus mutans

  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 1744-3091 2011/12 [Not refereed][Not invited]
   

  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.
• Biochemical Characterization of a Thermophilic Cellobiose 2-Epimerase from a Thermohalophilic Bacterium, Rhodothermus marinus JCM9785

  Teruyo Ojima, Wataru Saburi, Hiroki Sato, Takeshi Yamamoto, Haruhide Mori, Hirokazu Matsui

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (11) 2162 - 2168 0916-8451 2011/11 [Not refereed][Not invited]
   

  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.
• Transglycosylation by barley alpha-amylase 1

  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 1381-1177 2011/11 [Refereed][Not invited]
   

  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.
• Calcium Ion-Dependent Increase in Thermostability of Dextran Glucosidase from Streptococcus mutans

  Momoko Kobayashi, Hironori Hondoh, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 75 (8) 1557 - 1563 0916-8451 2011/08 [Not refereed][Not invited]
   

  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.
• Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity

  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 0175-7598 2011/07 [Not refereed][Not invited]
   

  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.
• Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity

  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 0175-7598 2011/07 [Refereed][Not invited]
   

  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.
• Comparison of Enzymatic Properties and Gene Expression Profiles of Two Tuberonic Acid Glucoside β-Glucosidases from Oryza sativa L.

  WAKUTA Shinji, HAMADA Shigeki, ITO Hiroyuki, IMAI Ryozo, MORI Haruhide, MATSUURA Hideyuki, NABETA Kensuke, MATSUI Hirokazu

  Journal of applied glycoscience Japanese Society of Applied Glycoscience 58 (2) 67 - 70 1344-7882 2011/04/20 [Not refereed][Not invited]
  
• Suicide Substrate-based Inactivation of Endodextranase by ω-Epoxyalkyl α-D-Glucopyranosides

  KANG Hee-Kwon, KIM Young-Min, NAKAI Hiroyuki, KANG Min-Sun, HAKAMADA Wataru, OKUYAMA Masayuki, MORI Haruhide, NISHIO Toshiyuki, KIMURA Atsuo

  Journal of Applied Glycoscience 日本応用糖質科学会 57 (4) 269 - 272 1344-7882 2010/10/20 [Not refereed][Not invited]
   

  Three kinds of ω-epoxyalkyl α-glucopyranosides (3′,4′-epoxybutyl α-D-glucopyranoside (E4G), 4′,5′-epoxypentyl α-D-glucopyranoside (E5G) and 5′,6′-epoxyhexyl α-D-glucopyranoside (E6G)), having alkyl chains of different lengths at their aglycone moieties, inactivated the endodextranase from Streptococcus mutans ATCC 25175 (SmDex) irreversibly with the pseudo-first order kinetics. Alkyl chain length-dependent inactivation was observed and the degree of activity loss was E5G, E6G and E4G, in that order, implying that the distance between epoxide group and glucosyl residue of ω-epoxyalkyl α-glucopyranoside was important in the modification of endodextranase. Inactivation by E5G followed the model of reversible intermediate-complex formation mechanism (suicide inhibitor-based mechanism). The rate constant of irreversible inactivation (k) and the dissociation constant of intermediate-complex (KR) of SmDex and E5G were 0.44 min^-1 and 1.45 mM, respectively. Hydrolytic reaction product (isomaltose) protected SmDex from E5G-inactivation, suggesting that E5G bound to the catalytic site of SmDex. This is the first report that ω-epoxyalkyl α-glucopyranoside becomes a suicide substrate for endodextranase.
• Suicide Substrate-based Inactivation of Endodextranase by .OMEGA.-Epoxyalkyl .ALPHA.-D-Glucopyranosides

  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 日本応用糖質科学会 57 (4) 269-272 (J-STAGE) - 272 1344-7882 2010 [Not refereed][Not invited]
   

  Three kinds of ω-epoxyalkyl α-glucopyranosides (3′,4′-epoxybutyl α-D-glucopyranoside (E4G), 4′,5′-epoxypentyl α-D-glucopyranoside (E5G) and 5′,6′-epoxyhexyl α-D-glucopyranoside (E6G)), having alkyl chains of different lengths at their aglycone moieties, inactivated the endodextranase from Streptococcus mutans ATCC 25175 (SmDex) irreversibly with the pseudo-first order kinetics. Alkyl chain length-dependent inactivation was observed and the degree of activity loss was E5G, E6G and E4G, in that order, implying that the distance between epoxide group and glucosyl residue of ω-epoxyalkyl α-glucopyranoside was important in the modification of endodextranase. Inactivation by E5G followed the model of reversible intermediate-complex formation mechanism (suicide inhibitor-based mechanism). The rate constant of irreversible inactivation (k) and the dissociation constant of intermediate-complex (KR) of SmDex and E5G were 0.44 min^-1 and 1.45 mM, respectively. Hydrolytic reaction product (isomaltose) protected SmDex from E5G-inactivation, suggesting that E5G bound to the catalytic site of SmDex. This is the first report that ω-epoxyalkyl α-glucopyranoside becomes a suicide substrate for endodextranase.
• Function analysis of subsite+1 mutants of isomaltooligosaccharide 6-α-glucosyltransferase

  nishimura takashi, kanegae michiyo, hondoh hironori, okuyama masayuki, mori haruhide, kimura atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2010 71 - 71 2010
• Modification of subsite +1 in α-glucosidase derived from Aspergillus niger

  Tagami Takayoshi, Okuyama Masayuki, Mori Haruhide, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2010 73 - 73 2010
• Catalytic Reaction Mechanism Based on alpha-Secondary Deuterium Isotope Effects in Hydrolysis of Trehalose by European Honeybee Trehalase

  Haruhide Mori, Jin-Ha Lee, Masayuki Okuyama, Mamoru Nishimoto, Masao Ohguchi, Doman Kim, Atsuo Kimura, Seiya Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Japan Society for Bioscience, Biotechnology, and Agrochemistry 73 (11) 2466 - 2473 0916-8451 2009/11 [Not refereed][Not invited]
   

  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.
• The first alpha-1,3-glucosidase from bacterial origin belonging to glycoside hydrolase family 31

  Min-Sun Kang, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura

  BIOCHIMIE 91 (11-12) 1434 - 1442 0300-9084 2009/11 [Refereed][Not invited]
   

  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.
• Catalytic Mechanism of Retaining alpha-Galactosidase Belonging to Glycoside Hydrolase Family 97

  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 0022-2836 2009/10 [Refereed][Not invited]
   

  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.
• Role of Ca2+ in catalysis of SusB derived from Bacteroides thetaiotaomicron

  Yoshida Takuya, Okuyama Masayuki, Hondoh Hironori, Yao Min, Mori Haruhide, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2009 38 - 38 2009
• Identification of a key residue for forming of subsites +2 and +3 in sugar beet alpha;-glucosidase

  Tagami Takayoshi, Okuyama Masayuki, Mori Haruhide, Taguchi Kazunori, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2009 37 - 37 2009
• Alteration of transglucosylation/hydrolysis ratio of Streptococcus mutans dextran glucosidase (2)

  Nakatsuka Daichi, Hondoh Hironori, Otsuka Hiroaki, Saburi Wataru, Mori Haruhide, Okuyama Masayuki, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2009 34 - 34 2009
• Improving Activity of Honeybee α-Glucosidase III by Substitution of Q349 and L350 Position.

  Ngiwsara Lukana, Mori Haruhide, Okuyama Masayuki, Chiba Seiya, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2009 36 - 36 2009
• Structural element for transglucosylation reaction of isomaltooligosaccharide 6-α-glucosyltransferase

  Nishimura Takashi, Kanegae Michiyo, KIM Young-Min, Hondoh Hironori, Okuyama Masayuki, Mori Haruhide, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2009 35 - 35 2009
• Structure-function relationship of substrate length specificity of dextran glucosidase from Streptococcus mutans

  Wataru Saburi, Hironori Hondoh, Young-Min Kim, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura

  BIOLOGIA 63 (6) 1000 - 1005 0006-3088 2008/12 [Not refereed][Not invited]
   

  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.
• Structural and Functional Analysis of a Glycoside Hydrolase Family 97 Enzyme from Bacteroides thetaiotaomicron

  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 0021-9258 2008/12 [Refereed][Not invited]
   

  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.
• Molecular Mechanism of α-glucosidase

  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 2008/09 [Refereed][Not invited]
   

  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.
• Rice α-glucosidase isozymes and isoforms showing different starch granules-binding and -degrading ability

  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 2008/07 [Refereed][Not invited]
  
• Substrate Recognition of Escherichia coli YicI (.ALPHA.-Xylosidase)

  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 日本応用糖質科学会 55 (2) 111-118 (J-STAGE) - 118 1344-7882 2008 [Not refereed][Not invited]
   

  Glycoside hydrolase family 31 (GH 31) is one of the most intriguing glycoside hydrolase families. This family contains α-glucosidase, α-xylosidase, α-glucan lyase and isomaltosyltransferase. Escherichia coli YicI (α-xylosidase) is a representative enzyme of GH 31 because its biochemical and structural studies have been thoroughly carried out. YicI is a strict α-xylosidase, which rigidly recognizes α-xyloside at the non-reducing terminal end, even though its amino acid sequence apparently displays similarity with α-glucosidases. Phe277, Cys307, Trp345 and Lys414 at the subsite-1 are important for α-xylosidase activity. The mutant YicI enzymes, which possesses Ile307/Asp308 instead of Cys307/Phe308 and which has a shorter β→α loop 1 of (β/α)8 barrel in place of the original longer loop, respectively, possess α-glucosidase activity. In the transxylosylation of YicI, glucose, mannose and allose are able to act as acceptors, but galactose, talose and gulose never do, implying that equatorial OH-4 of the aldopyranose is crucial for acting as an acceptor. YicI transfers α-xylosyl moieties to a specific hydroxy group in the acceptor sugar (except fructopyranose) showing 1,6 regioselectivity, which is in agreement with the structural feature of the aglycone-biding site. Among the transxylosylation products of YicI, α-D-xylopyranosyl-(1→6)-D-mannopyranose, α-D-xylopyranosyl-(1→6)-D-fructofuranose, and α-D-xylopyranosyl-(1→3)-D-fructopyranose are novel sugars. α-D-Xylopyranosyl-(1→6)-D-mannopyranose and α-D-xylopyranosyl-(1→6)-D-fructofuranose have the ability to inhibit rat intestinal α-glucosidases.
• The structural factor for transglucosylation of isomaltooligosaccharide 6-α-glucosyltrasferase(I6GT) from Bacillus sp.

  Kanegae Michiyo, KIM Young-Min, Hondoh Hironori, Okuyama Masayuki, Mori Haruhide, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2008 104 - 104 2008
• Catalytic mechanism of retaining glycosidase in glycoside hydrolase family 97

  OKUYAMA MASAYUKI, YAO MIN, Hondoh Hironori, KITAMURA MOMOYO, MORI HARUHIDE, TANAKA ISAO, KIMURA ATSUO

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2008 139 - 139 2008
• Cloning and Expression of α-glucosidase derived from ripening seed of Sugar Beet

  Tagami Takayoshi, Okuyama Masayuki, Mori Haruhide, Taguchi Kazunori, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2008 94 - 94 2008
• Coversion of Lactobacillus johnsonii novel α-glucosidase into glycosynthase

  Kang Min-Sun, Okuyama Masayuki, Mori Haruhide, Kimura Atsuo

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2008 96 - 96 2008
• Glycoside hydrolase family 31 Escherichia coli alpha-xylosidase

  M-S. Kang, M. Okuyama, K. Yaoi, Y. Mitsuishi, Y-M. Kim, H. Mori, A. Kimura

  BIOCATALYSIS AND BIOTRANSFORMATION 26 (1-2) 96 - 103 1024-2422 2008 [Not refereed][Not invited]
   

  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.
• Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans.

  Hondoh H, Saburi W, Mori H, Okuyama M, Nakada T, Matsuura Y, Kimura A

  J Mol Biol 378 (4) 913 - 922 2008 [Refereed][Not invited]
  
• Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are alpha-glucosidase and alpha-xylosidase

  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 Japanese Biochemical Society 142 (4) 491 - 500 0021-924X 2007/10 [Not refereed][Not invited]
   

  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.
• Crystallization and preliminary X-ray analysis of Streptococcus mutans dextran glucosidase

  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 1744-3091 2007/09 [Not refereed][Not invited]
   

  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.
• Molecular cloning of cDNA for trehalase from the European honeybee, Apis mellifera L., and its heterologous expression in Pichia pastoris

  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 0916-8451 2007/09 [Not refereed][Not invited]
   

  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.
• Molecular cloning of cDNAs and genes for three alpha-glucosidases from European honeybees, Apis mellifera L., and heterologous production of recombinant enzymes in Pichia pastoris

  Mamoru Nishimoto, Haruhide Mori, Tsuneharu Moteki, Yukiko Takamura, Gaku Iwai, Yu Miyaguchi, Masayuki Okuyama, Jintanart Wongchawalit, Rudee Surarit, Jisnuson Svasti, Atsuo Kimura, Seiya Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Japan Society for Bioscience, Biotechnology, and Agrochemistry 71 (7) 1703 - 1716 0916-8451 2007/07 [Not refereed][Not invited]
   

  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.
• Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare)

  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 Elsevier Masson SAS 89 (1) 49 - 62 0300-9084 2007/01 [Not refereed][Not invited]
   

  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.
• Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA

  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 Japan Society for Bioscience, Biotechnology, and Agrochemistry 70 (12) 2889 - 2898 0916-8451 2006/12 [Not refereed][Not invited]
   

  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.
• Interactions between Barley α-Amylases, Substrates, Inhibitors and Regulatory Proteins

  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 日本応用糖質科学会 53 (2) 163 - 169 1344-7882 2006/04/20 [Not refereed][Not invited]
   

  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 K_d 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.
• Plant α-Glucosidase : Molecular Analysis of Rice α-Glucosidase and Degradation Mechanism of Starch Granules in Germination Stage

  NAKAI Hiroyuki, ITO Tatsuya, TANIZAWA Shigeki, MATSUBARA Kazuki, YAMAMOTO Takeshi, OKUYAMA Masayuki, MORI Haruhide, CHIBA Seiya, SANO Yoshio, KIMURA Atsuo

  Journal of applied glycoscience 日本応用糖質科学会 53 (2) 137 - 142 1344-7882 2006/04/20 [Not refereed][Not invited]
   

  In germination of plant seeds, storage starch is principally degraded by the combination of amylolytic enzymes. As starch is an insoluble granule, a conventional view of the degradation pathway is that the initial attack is performed by α-amylase having the starch granule-binding ability. Plant α-glucosidase was also capable of adsorbing and hydrolyzing starch granules directly, indicating a possible second pathway: the direct liberation of glucose from starch granules by plant α-glucosidase rather than the α-amylase-mediated system. We found that the starch-binding site of plant α-glucosidase was situated in its C-terminal region, of which function was independent of the catalytic domain. Site-directed mutagenesis analysis on the aromatic amino acid residues conserved in this region revealed that Trp803 and Phe895 of rice α-glucosidase were responsible for binding to starch granules. Mold α-glucosidases were devoid of the ability to attack starch granules. In plant seeds, multiple α-glucosidases have been observed. Two types of α-glucosidases, insoluble and soluble enzymes, were found in the germinating stage of rice. Expression patterns of their activities classified 14 rice varieties into two groups (Groups 1 and 2). In Group 1 varieties, insoluble enzyme decreased immediately after germination. The soluble enzyme increased by de novo synthesis. Group 2 maintained a constant activity level of insoluble and soluble α-glucosidases in germination. From Groups 1 and 2, we selected varieties of Akamai and Nipponbare, respectively, of which analysis elucidated interesting molecular mechanisms of insoluble and soluble enzymes: i) isoform and isozyme formations by post-translational proteolysis as well as by chromosomal gene expression; ii) characterization of purified enzymes exhibiting different activities to starch granules.
• Identification and Manipulation of Subsite Structure and Starch Granule Binding Site in Plant .ALPHA.-Amylase

  MORI HARUHIDE

  J Appl Glycosci 53 (1) 51 - 56 1344-7882 2006/01/20 [Not refereed][Not invited]
  
• Developmental regulation of photosynthate distribution in leaves of rice

  T Shinano, K Nakajima, J Wasaki, H Mori, T Zheng, M Osaki

  PHOTOSYNTHETICA 44 (1) 1 - 10 0300-3604 2006 [Not refereed][Not invited]
   

  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.
• Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase

  F Sato, M Okuyama, H Nakai, H Mori, A Kimura, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Japan Society for Bioscience, Biotechnology, and Agrochemistry 69 (10) 1905 - 1913 0916-8451 2005/10 [Not refereed][Not invited]
   

  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.
• Oligosaccharide binding to barley alpha-amylase 1

  Robert, X, R Haser, H Mori, B Svensson, N Aghajari

  JOURNAL OF BIOLOGICAL CHEMISTRY 280 (38) 32968 - 32978 0021-9258 2005/09 [Refereed][Not invited]
   

  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."
• Binding of carbohydrates and protein inhibitors to the surface of α-amylases

  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 1335-6399 2005 [Refereed][Not invited]
   

  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.
• Molecular analysis of α-glucosidase belonging to GH-family 31

  Nakai H, Okuyama M, Kim YM, Saburi W, Wongchawalit J, Mori H, Chiba S, Kimura A

  Biologia, Bratislava 60 131 - 135 2005 [Refereed][Not invited]
  
• Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey

  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 Japan Society for Bioscience, Biotechnology, and Agrochemistry 68 (11) 2346 - 2352 0916-8451 2004/11 [Not refereed][Not invited]
   

  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.
• An improved method for deleting large regions of Escherichia coli K-12 chromosome using a combination of Cre/loxP and lambda Red.

  Fukiya S, Mizoguchi H, Mori H

  FEMS microbiology letters 2 234 (2) 325 - 331 0378-1097 2004/05 [Refereed][Not invited]
  
• Barley Proteome Analysis, Starch Degrading Eznymes and Proteinaceous Inhibitors

  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 日本応用糖質科学会 50 (2) 277 - 282 1344-7882 2003/07/14 [Not refereed][Not invited]
   

  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...
• Impact on Substrate Specificity of Mutational Subsite Isozyme Mimicry in Barley α-Amylase

  SVENSSON Birte, MORI Haruhide, BAK-JENSEN Kristian Sass, JENSEN Morten Tovborg

  Journal of Applied Glycoscience 日本応用糖質科学会 50 (2) 143 - 145 1344-7882 2003/07/14 [Not refereed][Not invited]
   

  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) ...
• Purification, characterization, and sequence analysis of two alpha-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards beta-cyclodextrin sepharose

  SS Mar, H Mori, JH Lee, K Fukuda, W Saburi, A Fukuhara, M Okuyama, S Chiba, A Kimura

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 67 (5) 1080 - 1093 0916-8451 2003/05 [Not refereed][Not invited]
   

  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.
• Evidence of Intramolecular Transglucosylation Cathlyzed by an α-Glucosidase

  SON Mee, MORI Haruhide, OKUYAMA Masayuki, KIMURA Atsuo, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 50 (1) 41 - 44 1344-7882 2003/01/20 [Not refereed][Not invited]
   

  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 toward isomaltose. However, the conflict for the peculiar phenomenon on the reaction was rationally explained by the evidence that isomaltose might be formed by the intramolecular transglucosylation via other α-glucobioses that are easily produced from glucose by the condensation reaction. It is suggested that the usual transglycosylation of carbohydrate-hydrolase may be accompanied by an intramolecular transfer reaction.
• Evidence of Intramolecular Transglucosylation Catalyzed by an .ALPHA.-Glucosidase.

  Son Mee, Mori Haruhide, Okuyama Masayuki, Kimura Atsuo, Chiba Seiya

  Journal of Applied Glycoscience 日本応用糖質科学会 50 (1) 41 - 44 1344-7882 2003 [Not refereed][Not invited]
   

  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 ...
• Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F-1 from a Streptomyces sp.

  K Fukuda, H Mori, M Okuyama, A Kimura, H Ozaki, M Yoneyama, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 66 (10) 2060 - 2067 0916-8451 2002/10 [Not refereed][Not invited]
   

  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).
• Kinetic Studies on Substrate Specificity and Active Site of β-_D-Glucosidase F_1 from Streptomyces sp.

  FUKUDA Kenji, SHIRAKAWA Kou, MORI Haruhide, OKUYAMA Masayuki, KIMURA Atsuo, OZAKI Hachiro, YONEYAMA Michio, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 49 (3) 265 - 272 1340-3494 2002/07/18 [Not refereed][Not invited]
   

  The substrate specificity and the active site of β-D-glucosidase F^-1 (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_m, 0.72 mM; k₀, 63 s^-1) but also ρ-nitrophenyl β-fucoside (K_m, 0.19 mM; k₀, 44 s^-1) and laminaribiose (K_m, 1.6 mM; k₀, 70 s^-1). The kinetic study was made as to whether the hydrolyses of these synthetic glycosides were catalyzed at a single active site or at dual active sites in the β-D-glucosidase. In the experiments with the mixed substrates of p-nitrophenyl β-glucoside (PNPG) and ρ-nitrophenyl β-fucoside (PNPF) or ρ-nitrophenyl β-galactoside (PNPGal), the kinetic features, the linearity of Lineweaver-Burk plots and the dependence of the apparent maximal veloities and K_m value on the mole fraction (f) of PNPG in the mixed substrate, f = [PNPG/([PNPG] + [PNPF or PNPGal]) agreed very closely with those theoretically predicted for a single catalytic site mechanism. The findings strongly support the notion that the β-D-glucosidase attacks the synthetic glycosides at a common active site.
• Study on Three .ALPHA.-Glucosidase Isozymes from Honeybee, Apis mellifera L.

  NISHIMOTO MAMORU, MORI HARUHIDE, KIMURA ATSUO, CHIBA SEIYA

  J Appl Glycosci The Japanese Society of Applied Glycoscience 49 (2) 191 - 197 1344-7882 2002/04/01 [Not refereed][Not invited]
   

  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.
• Catalytic Amino Acid Residue Providing Proton Donor in .ALPHA.-Glucosidase Family II.

  OKUYAMA MASAYUKI, MORI HARUHIDE, KIMURA ATSUO, CHIBA SEIYA

  J Appl Glycosci The Japanese Society of Applied Glycoscience 49 (2) 211 - 219 1344-7882 2002/04/01 [Not refereed][Not invited]
   

  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.
• Catalytic Amino Acid Residue Providing Proton Donor in α-Glucosidase Family II

  OKUYAMA Masayuki, MORI Haruhide, KIMURA Atsuo, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 49 (2) 211 - 219 1344-7882 2002/04/01 [Not refereed][Not invited]
   

  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.
• Study on Three α-Glucosidase Isozymes from Honeybee, Apis mellifera L.

  NISHIMOTO Mamoru, MORI Haruhide, KIMURA Atsuo, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 49 (2) 191 - 197 1344-7882 2002/04/01 [Not refereed][Not invited]
   

  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.
• alpha-glucosidase mutant catalyzes "alpha-glycosynthase"-type reaction

  M Okuyama, H Mori, K Watanabe, A Kimura, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 66 (4) 928 - 933 0916-8451 2002/04 [Not refereed][Not invited]
   

  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.
• 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 [Not refereed][Not invited]
  
• 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 [Not refereed][Not invited]
  
• 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 [Not refereed][Not invited]
  
• Kinetic Studies on Substrate Specificity and Active Site of .BETA.-D-Glucosidase F1 from Streptomyces sp.

  Fukuda Kenji, Shirakawa Kou, Mori Haruhide, Okuyama Masayuki, Kimura Atsuo, Ozaki Hachiro, Yoneyama Michio, Chiba Seiya

  Journal of Applied Glycoscience 日本応用糖質科学会 49 (3) 265 - 272 1344-7882 2002 [Not refereed][Not invited]
   

  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>,...
• (Gluco)amylases, what have we learned so far?

  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 0260-6291 2002 [Refereed][Not invited]
  
• Purification and identification of the essential ionizable groups of honeybee, Apis mellifera L., trehalase

  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 0916-8451 2001/12 [Not refereed][Not invited]
   

  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.
• Isolation and Sequence of a Putative α-Glucosidase Gene from Brevibacterium fuscum var. dextranlyticum Strain 0407

  MIZUNO Takafumi, MORI Haruhide, NISHIMOTO Mamoru, ITO Hiroyuki, MATSUI Hirokazu, KIMURA Atsuo, HONMA Mamoru, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 48 (3) 287 - 291 1340-3494 2001/07/01 [Not refereed][Not invited]
   

  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 were two other open reading frames, of which the deduced amino acid sequences showed a similarity with those of oligosaccharides membrane transporter proteins. The gene cluster consisting of the membrane transporter protein genes, dexG, and dexT, seems to participate in the degradation and utilization of dextran in this bacterium.
• Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III

  M Nishimoto, M Kubota, M Tsuji, H Mori, A Kimura, H Matsui, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 65 (7) 1610 - 1616 0916-8451 2001/07 [Not refereed][Not invited]
   

  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.
• Isolation and Sequence of a Putative .ALPHA.-Glucosidase Gene from Brevibacterium fuscum var. dextranlyticum Strain 0407.

  Mizuno Takafumi, Mori Haruhide, Nishimoto Mamoru, Ito Hiroyuki, Matsui Hirokazu, Kimura Atsuo, Honma Mamoru, Chiba Seiya

  Journal of Applied Glycoscience 日本応用糖質科学会 48 (3) 287 - 291 1344-7882 2001 [Not refereed][Not invited]
   

  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...
• Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli

  T Mizuno, H Mori, H Ito, H Matsui, A Kimura, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 63 (9) 1582 - 1588 0916-8451 1999/09 [Not refereed][Not invited]
   

  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.
• Localization of α-Glucosidase in Yeast Cells

  SAEKI Takeshi, OKUYAMA Masayuki, MORI Haruhide, KIMURA Atsuo, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 45 (3) 281 - 283 1340-3494 1998/08/31 [Not refereed][Not invited]
  
• Molecular Cloning of an α-Amylase cDNA from Germinating Cotyledons of Kidney Bean (Phaseolus vulgaris L. cv. Toramame)

  MORI Haruhide, KOBAYASHI Tetsuya, TONOKAWA Takashi, TATEMATSU Ayumi, MATSUI Hirokazu, KIMURA Atsuo, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 45 (3) 261 - 267 1340-3494 1998/08/31 [Not refereed][Not invited]
  
• Amylases and Branching Enzyme of Developing Kidney Bean Seeds on Native Electrophoretic Gel

  NOZAKI Kouichi, MATSUI Hirokazu, TONOKAWA Takashi, MORI Haruhide, ITO Hiroyuki, HONMA Mamoru, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 45 (2) 117 - 122 1340-3494 1998/06/30 [Not refereed][Not invited]
  
• A catalytic amino acid and primary structure of active site in Aspergillus niger alpha-glucosidase

  A Kimura, M Takata, Y Fukushi, H Mori, H Matsui, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 61 (7) 1091 - 1098 0916-8451 1997/07 [Not refereed][Not invited]
   

  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.
• Substrate Specificity and Primary Structure of Sugar Beet α-Glucosidase

  MATSUI Hirokazu, IWANAMI Shunsuke, ITO Hiroyuki, KIMURA Atsuo, MORI Haruhide, HONMA Mamoru, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 44 (2) 245 - 252 1340-3494 1997/06/30 [Not refereed][Not invited]
  
• Primary Structure and Sugar Chains of Crystalline α-Glucosidase from Aspergillus niger

  KIMURA Atsuo, TAKAYANAGI Tsutomu, MORI Haruhide, MATSUI Hirokazu, UOZUMI Takeshi, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 44 (2) 233 - 243 1340-3494 1997/06/30 [Not refereed][Not invited]
  
• Substrate Specificity and Primary Structure of Sugar Beet .ALPHA.-Glucosidase.

  MATSUI HIROKAZU, IWANAMI SHUNSUKE, ITO HIROYUKI, KIMURA ATSUO, MORI HARUHIDE, HONMA MAMORU, CHIBA SEIYA

  応用糖質科学 44 (2) 245-252  1340-3494 1997/06 [Not refereed][Not invited]
  
• Primary Structure and Sugar Chains of Crystalline .ALPHA.-Glucosidase from Aspergillus niger.

  KIMURA ATSUO, TAKAYANAGI TSUTOMU, MORI HARUHIDE, UOZUMI TAKESHI, CHIBA SEIYA, MATSUI HIROKAZU

  応用糖質科学 44 (2) 233-243  1340-3494 1997/06 [Not refereed][Not invited]
  
• Cloning and sequencing of a cDNA encoding alpha-glucosidase from sugar beet

  H Matsui, S Iwanami, H Ito, H Mori, M Honma, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 61 (5) 875 - 880 0916-8451 1997/05 [Not refereed][Not invited]
   

  A cDNA encoding sugar beet alpha-glucosidase was cloned from a library constructed from mRNA of suspension-cultured cells, The cDNA, 3056 bp 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 alpha-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 alpha-glucosidase, The primary structure indicated relatively high homology in the range of 28.2 to 54.3% to those for other alpha-glucosidases, The highest homology was found in barley alpha-glucosidase.
• Identification of essential ionizable groups in active site of Aspergillus niger alpha-glucosidase

  A Kimura, A Somoto, H Mori, O Sakai, H Matsui, S Chiba

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 61 (3) 475 - 479 0916-8451 1997/03 [Not refereed][Not invited]
   

  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).
• Molecular cloning and nucleotide sequences of cDNA and gene encoding endo-inulinase from Penicillium purpurogenum

  S Onodera, T Murakami, H Ito, H Mori, H Matsui, M Honma, S Chiba, N Shiomi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 60 (11) 1780 - 1785 0916-8451 1996/11 [Not refereed][Not invited]
   

  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.
• Reinvestigation of Isomaltotrio-Dextranase from Brevibacterium fuscum var. dextranlyticum

  MIZUNO Takafumi, MATSUI Hirokazu, ITO Hiroyuki, MORI Haruhide, KIMURA Atsuo, HONMA Mamoru, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 43 (3) 347 - 353 1340-3494 1996/08/31 [Not refereed][Not invited]
  
• Substrate Specificity and Subsite Affinities of α-Amylase from Germinating Cotyledons of Phaseolus vulgaris L. cv Toramame

  MORI Haruhide, TATEMATSU Ayumi, SAITO Akiko, MATSUI Hirokazu, KIMURA Atsuo, CHIBA Seiya

  Journal of applied glycoscience 日本応用糖質科学会 42 (4) 387 - 394 1340-3494 1995/12/01 [Not refereed][Not invited]
  
• CHEMICAL MODIFICATION AND AMINO-ACID-SEQUENCE OF ACTIVE-SITE IN SUGAR-BEET ALPHA-GLUCOSIDASE

  S IWANAMI, H MATSUI, A KIMURA, H ITO, H MORI, M HONMA, S CHIBA

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 社団法人日本農芸化学会 59 (3) 459 - 463 0916-8451 1995/03 [Not refereed][Not invited]
   

  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.
• A sialic acid-binding lectin from the mashroom Hericium erinaceum.

  Kawagishi H, Mori H, Uno A, Kimura A, Chiba S

  FEBS Lett 340 (1) 56 - 58 1994 [Refereed][Not invited]
  
• STARCH-HYDROLYZING ENZYMES IN GERMINATING KIDNEY BEAN

  H MORI, A TATEMATSU, H MATSUI, T TAKAYANAGI, M HONMA, S CHIBA

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 56 (9) 1499 - 1500 0916-8451 1992/09 [Not refereed][Not invited]
  
• NUCLEOTIDE AND DERIVED AMINO-ACID-SEQUENCE OF A CATALASE CDNA ISOLATED FROM RICE IMMATURE SEEDS

  H MORI, K HIGO, H HIGO, Y MINOBE, H MATSUI, S CHIBA

  PLANT MOLECULAR BIOLOGY 18 (5) 973 - 976 0167-4412 1992/03 [Not refereed][Not invited]
  

MISC

• β1-3グルカン分解酵素群の多様な切断様式とその分子基盤 ラミナリンを端から切る酵素と内から切る酵素の共通点と相違点

  太田 智也, 佐分利 亘, 森 春英  化学と生物  62-  (8)  362  -364  2024/08  [Not refereed][Not invited]
  
• 新規酵素ガラクトース2-エピメラーゼの諸性質ならびにX線結晶構造の解析

  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
• GH55β-1,3-グルカナーゼのエンド様式の作用と構造

  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  [Not refereed][Invited]
  
• Chemical synthesis of oligosaccharide with partial structure of β1-3/1-6 glucan.

  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
• GH3β-グルコシダーゼMnBG3Aの反応速度と基質阻害に対する単糖の影響

  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
• 雪腐病菌由来GH3β-グルコシダーゼMnBG3AのpNP-Glc加水分解速度への各種配糖体および単糖の影響

  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
• Laminarin degradation mechanism of GH55 laminarinase from Microdochium nivale

  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
• GH55β-1,3-グルカナーゼのエンド様式の作用と構造

  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
• 紅色雪腐病菌Microdochium nivale由来GH3β-グルコシダーゼの酵素化学的諸性質とオリゴ糖の合成

  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
• Functional analysis of the extracellular β-glucosidase from phytopathogenic fungus Microdochium nivale

  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
• 植物病原性真菌Microdochium nivale由来菌体外ラミナリナーゼの機能解析

  太田智也, 佐分利亘, 今井亮三, 森春英  応用糖質科学  9-  (3)  2019
• ビフィズス菌のトランスポゾン変異株を用いたα-グルコシダーゼ遺伝子の機能解明

  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
• セロビオース2‐エピメラーゼによるマンノビオースのエピメリ化機構

  佐分利亘, 加藤公児, 于健, 姚閔, 森春英  応用糖質科学  8-  (3)  (40)  2018/08/20  [Not refereed][Not invited]
  
• ゲノム編集を用いたサリチル酸高蓄積イネ変異体の作出

  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
• 転写因子に着目したトレハロース誘導抵抗性のシグナル伝達機構の解析

  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
• Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes

  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  2018/01  

  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.
• アルドース‐ケトース間の異性化反応を利用したセロビオース2‐エピメラーゼの反応機構の解析

  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)  2017/03/05  [Not refereed][Not invited]
  
• イネのトレハロース誘導抵抗性に関与する新たなERF転写因子の機能解析

  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  2016/10/07  [Not refereed][Not invited]
  
• トレハロース6‐リン酸ホスホリラーゼを用いたトレハロース6‐リン酸および新規糖リン酸の合成

  田口陽大, 佐分利亘, 今井亮三, 森春英  応用糖質科学  6-  (3)  46  2016/08/20  [Not refereed][Not invited]
  
• 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  6-  (3)  65  -65  2016/08/20  [Not refereed][Not invited]
  
• マルトースを出発物質とするトレハロース6‐リン酸の酵素合成法の確立

  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)  2016/03/05  [Not refereed][Not invited]
  
• 乳中ラクトースのエピラクトースへの直接変換を指向した各種セロビオース2‐エピメラーゼの機能評価

  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)  2016/03/05  [Not refereed][Not invited]
  
• Gluconobacter oxydans由来の菌体外酵素dextran dextrinaseは自らの生成物デキストランによって細胞表層から遊離する.

  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)  2016/03/05  [Not refereed][Not invited]
  
• 嫌気性細菌によるエピラクトース代謝の多様性

  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)  2016/03/05  [Not refereed][Not invited]
  
• Ruminococcus albus由来4‐O‐β‐D‐マンノシル‐D‐グルコースホスホリラーゼのサブサイト+1に保存されるArg92の基質結合への関与

  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)  2016/03/05  [Not refereed][Not invited]
  
• P055 Molecular basis of Trehalose-induced Systemic Resistance

  Tezuka Daisuke, Wakuta Shinji, Kato Hideki, Matsuura Hideyuki, Saburi Wataru, Mori Haruhide, Matsui Hirokazu, Imai Ryozo  植物化学調節学会研究発表記録集  50-  (0)  73  -73  2015/10/01  [Not refereed][Not invited]
  
• Trehalose‐induced Systemic Resistance(TSR)の分子基盤

  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  2015/10/01  [Not refereed][Not invited]
  
• S3-2 長鎖阻害剤の利用による植物α-グルコシダーゼの機能構造相関の解明(応用糖質科学シンポジウム,一般社団法人日本応用糖質科学会平成27年度大会(第64回))

  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  2015/08/20
• Ba-3 Isomaltooligosaccharide 6-α-glucosyltransferaseの構造と触媒部位の機能性アミノ酸残基の同定(澱粉関連酵素,一般講演,一般社団法人日本応用糖質科学会平成27年度大会(第64回))

  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  2015/08/20
• Ba-9 Ruminococcus albus由来4-O-β-D-mannosyl-D-glucose phosphorylase RaMP1の無機リン酸に対するホモトロピック相互作用(ホスホリラーゼ関連,一般講演,一般社団法人日本応用糖質科学会平成27年度大会(第64回))

  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  2015/08/20  [Not refereed][Not invited]
  
• Bacillus sp.AHU2001由来GH31α‐グルコシダーゼの生化学的諸性質と基質認識に重要な構造因子の解析

  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)  2015/03/05  [Not refereed][Not invited]
  
• Rhodothermus marinus JCM9785由来セロビオース2‐エピメラーゼの基質結合部位周辺アミノ酸残基の機能解析

  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)  2015/03/05  [Not refereed][Not invited]
  
• グライコシンターゼ反応を利用したシロイヌナズナ由来β‐グルコシダーゼBglu15のアグリコン特異性の解析

  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)  2015/03/05  [Not refereed][Not invited]
  
• テンサイα-グルコシダーゼの高重合度基質特異性はらせん構造の長鎖基質に適したサブサイト構造に起因する

  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
• 19. Reduction of GA levels in response to chilling enhances chilling tolerance of rice

  Takahashi Naoki, Kawamata Aya, Tezuka Daisuke, Saburi Wataru, Matsuura Hideyuki, Mori Haruhide, Imai Ryozo  植物化学調節学会研究発表記録集  49-  (0)  37  -37  2014/10/01  [Not refereed][Not invited]
  
• 43. Glucosylation of tuberonic acid using salicylic acid glucoside in wound rice

  Takematsu Tomonori, Seto Yoshiya, Miyazawa Yoshiroh, Wakuta Shinji, Saburi Wataru, Mori Haruhide, Takahashi Kosaku, Matsuura Hideyuki  植物化学調節学会研究発表記録集  49-  (0)  61  -61  2014/10/01  [Not refereed][Not invited]
  
• 52. Jasmonic acid is involved in the trehalose-induced systemic disease resistance in rice

  Tezuka Daisuke, Sakai Shiho, Wakuta Shinji, Kato Hideki, Matsuura Hideyuki, Saburi Wataru, Mori Haruhide, Matsui Hirokazu, Imai Ryozo  植物化学調節学会研究発表記録集  49-  (0)  70  -70  2014/10/01  [Not refereed][Not invited]
  
• 62. A possible function of a salicylic acid β-glucosidase in rice wound response

  Takeda Ryosuke, Saburi Wataru, Himeno Nami, Wakuta Shinji, Matsuura Hideyuki, Imai Ryozo, Matsui Hirokazu, Mori Haruhide  植物化学調節学会研究発表記録集  49-  (0)  80  -80  2014/10/01  [Not refereed][Not invited]
  
• イネの傷害応答におけるサリチル酸グルコシド加水分解酵素の機能

  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  2014/10/01  [Not refereed][Not invited]
  
• イネのトレハロースにより誘導されるシステミックな病害抵抗性にはジャスモン酸が関与する

  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  2014/10/01  [Not refereed][Not invited]
  
• 傷害時のイネにおけるサリチル酸グリコシドを糖ドナーとするツベロン酸への糖転移反応

  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  2014/10/01  [Not refereed][Not invited]
  
• 低温に応答したGA量の抑制によりイネの耐性を高めることができる

  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  2014/10/01  [Not refereed][Not invited]
  
• Halomonas sp. H11株由来α‐グルコシダーゼの糖転移機構の解析

  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  2014/09/22  [Not refereed][Not invited]
  
• Bp-6 Amphiphilic function of linear-isomaltomegalosaccharides (L-IMS) on ethyl red (ER) solubility

  Lang Weeranuch, Kumagai Yuya, Sadahiro Juri, Okuyama Masayuki, Mori Haruhide, Sakairi Nobuo, Kimura Atsuo  Bulletin of applied glycoscience  4-  (3)  B42  2014/08/20
• イネ由来スクロースシンターゼ3のヌクレオチド二リン酸特異性に関わるアミノ酸残基の決定

  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)  2014/08/20  [Not refereed][Not invited]
  
• 難消化性二糖エピラクトースによる抗メタボリックシンドローム作用の解析

  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  2014/04/30  [Not refereed][Not invited]
  
• Halomonas sp.H11株由来α‐グルコシダーゼD274G変異酵素の一価カチオンによる活性化機構の解析

  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)  2014/03/05  [Not refereed][Not invited]
  
• アクセプタ結合部位を改変したRuminococcus albus由来マンノシドホスホリラーゼの基質特異性と反応特性の解析

  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)  2014/03/05  [Not refereed][Not invited]
  
• Ba-3 Dextran dextrinaseのPhe602の生成物特異性に関する影響(その他の糖質関連酵素,一般講演,日本応用糖質科学会平成26年度大会(第63回))

  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  [Not refereed][Not invited]
  
• Acidophilic β-Galactosidase from Aspergillus niger AHU7120 with Lactose Hydrolytic Activity Under Simulated Gastric Conditions

  Saburi Wataru, M. Ueno Hiroshi, Matsui Hirokazu, Mori Haruhide  Journal of Applied Glycoscience  61-  (2)  53  -57  2014  [Not refereed][Not invited]
   

  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.
• Colorimetric Quantification of β-(1→4)-Mannobiose and 4-O-β-D-Mannosyl-D-glucose

  Jaito Nongluck, Saburi Wataru, Muto Hirohiko, Matsui Hirokazu, Mori Haruhide  Journal of Applied Glycoscience  61-  (4)  117  -119  2014  [Not refereed][Not invited]
   

  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.
• シロイヌナズナβ‐グルコシダーゼ様タンパク質の生化学的機能解析

  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  2013/11/27  [Not refereed][Not invited]
  
• Gluconobacter oxydansが産生する2つのデキストラン合成酵素の同一性に関する解析

  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  2013/11/27  [Not refereed][Not invited]
  
• イネ胚乳ADP‐グルコースピロホスホリラーゼの酵素化学的諸性質

  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  2013/11/27  [Not refereed][Not invited]
  
• シロイヌナズナβ‐グルコシダーゼ様タンパク質の生化学的機能解析

  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  2013/11/27  [Not refereed][Not invited]
  
• Cellvibrio vulgaris NCIMB8633のマンナン代謝オペロンの機能解析

  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  2013/11/27  [Not refereed][Not invited]
  
• Ca-2 Schizosaccharomyces pombe由来グルコシダーゼIIα-サブユニットの大腸菌生産および機能解析(α-グルコシダーゼ・その他の糖質関連酵素1,一般講演,日本応用糖質科学会平成25年度大会(第62回))

  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  2013/08/20
• Cp-15 糖鎖の長さを制御できる新規両親媒性プライマーの調製(多糖の構造・物性・合成,一般講演,日本応用糖質科学会平成25年度大会(第62回))

  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  2013/08/20
• Ca-3 Streptococcus mutans由来dextran glucosidaseのβ→α loop 6への変異導入による鎖長特異性の改変(α-グルコシダーゼ・その他の糖質関連酵素1,一般講演,日本応用糖質科学会平成25年度大会(第62回))

  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  2013/08/20
• Halomonas sp.H11株由来α‐グルコシダーゼの一価陽イオンによる活性化機構の解析

  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  2013/08/20  [Not refereed][Not invited]
  
• Bacillus sp.AAH‐31株由来α‐アミラーゼの一次構造の解析とN末端ドメインの機能解析

  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  2013/08/20  [Not refereed][Not invited]
  
• セロビオース2‐エピメラーゼの異性化反応機構の解明

  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  2013/08/20  [Not refereed][Not invited]
  
• セロビオース2‐エピメラーゼを用いたエピラクトースの実用的酵素合成法の開発

  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  2013/05/20  [Not refereed][Not invited]
   

  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。
• Megalo types of alpha-1,6-glucosaccharide enhance absorption of quercetin glycosides in rats

  Aki Shinoki, Weeranuch Lang, Haruhide Mori, Atsuo Kimura, Satoshi Ishizuka, Hiroshi Hara  FASEB JOURNAL  27-  2013/04  [Not refereed][Not invited]
  
• イネ由来ツベロン酸β‐グルコシドグルコシダーゼのアグリコン認識メカニズムの解析

  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)  2013/03/05  [Not refereed][Not invited]
  
• デキストラン合成酵素dextran dextrinaseの触媒残基およびその機能の決定

  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)  2013/03/05  [Not refereed][Not invited]
  
• イネ由来ツベロン酸β‐グルコシドグルコシダーゼアイソザイム1の機能解析

  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)  2013/03/05  [Not refereed][Not invited]
  
• Halomonas sp.H11株由来α‐グルコシダーゼ組換え酵素の大腸菌による生産と諸性質

  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)  2013/03/05  [Not refereed][Not invited]
  
• Da-10 Gluconobacter oxydans由来dextran dextrinaseによる直鎖イソマルトメガロ糖の酵素合成(糖質の合成・生産,一般講演,日本応用糖質科学会平成25年度大会(第62回))

  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  [Not refereed][Not invited]
  
• Bacillus sp.AAH‐31株由来耐熱性アルカリα‐アミラーゼの高機能化に関する研究

  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  2012/11/01  [Not refereed][Not invited]
  
• ツベロン酸グルコシド加水分解酵素の基質認識機構の解明

  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  2012/11/01  [Not refereed][Not invited]
  
• 組換えツベロン酸グルコシドグルコシダーゼの酵素化学的諸性質の解析

  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  2012/11/01  [Not refereed][Not invited]
  
• Bp1-10 cellobiose phosphorylaseのepilactose phosphorylaseへの改変(ホスホリラーゼおよび関連酵素,一般講演,日本応用糖質科学会平成24年度大会(第61回))

  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  2012/08/20  [Not refereed][Not invited]
  
• S3-4 セロビオース2-エピメラーゼを用いたエピラクトースの実用的酵素合成法の開発(応用糖質科学シンポジウム(旧糖質関連酵素化学シンポジウム),日本応用糖質科学会平成24年度大会(第61回))

  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  2012/08/20  [Not refereed][Not invited]
  
• Ruminococcus albus由来セロビオース 2‐エピメラーゼ(RaCE)のX線結晶構造解析

  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)  2012/08/20  [Not refereed][Not invited]
  
• Acarviosyl‐maltooligsaccharideの酵素合成とテンサイα‐glucosidaseに対する基質アナログとしての利用

  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)  2012/08/20  [Not refereed][Not invited]
  
• cellobiose phosphorylaseのepilactose phosphorylaseへの改変

  羽村健, 佐分利亘, 森春英, 松井博和  応用糖質科学  2-  (3)  (37)  2012/08/20  [Not refereed][Not invited]
  
• セロビオース2‐エピメラーゼを用いたエピラクトースの実用的酵素合成法の開発

  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)  2012/08/20  [Not refereed][Not invited]
  
• Ruminococcus albus由来セロデキストリンホスホリラーゼの諸性質とリン酸結合部位の解析

  澤野達也, 佐分利亘, 森春英, 松井博和  応用糖質科学  2-  (3)  (38)  2012/08/20  [Not refereed][Not invited]
  
• Ruminococcus albus NE1株由来マンノシルグルコースホスホリラーゼ(RaMP1)の一般酸触媒残基の解析

  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)  2012/08/20  [Not refereed][Not invited]
  
• 固定化セロビオース2‐エピメラーゼによるエピラクトースの合成

  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)  2012/03/05  [Not refereed][Not invited]
  
• Ruminococcus albus NE1株由来cellobiose phosphorylaseのアクセプター特異性の改変

  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)  2012/03/05  [Not refereed][Not invited]
  
• Streptococcus mutans由来デキストラナーゼの結晶構造に基づく触媒機構の解明

  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  [Not refereed][Not invited]
  
• Biochemical Characterization of a Thermophilic Cellobiose 2-Epimerase from a Thermohalophilic Bacterium, Rhodothermus marinus JCM9785

  Teruyo Ojima, Wataru Saburi, Hiroki Sato, Takeshi Yamamoto, Haruhide Mori, Hirokazu Matsui  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  75-  (11)  2162  -2168  2011/11  [Not refereed][Not invited]
   

  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.
• デキストラングルコシダーゼとイソマルトオリゴ糖6‐α‐グルコシル転移酵素:基質認識と糖転移反応のメカニズム

  森春英  飯島記念食品科学振興財団年報  2009-  348  2011/08  [Not refereed][Not invited]
  
• Calcium Ion-Dependent Increase in Thermostability of Dextran Glucosidase from Streptococcus mutans

  Momoko Kobayashi, Hironori Hondoh, Haruhide Mori, Wataru Saburi, Masayuki Okuyama, Atsuo Kimura  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  75-  (8)  1557  -1563  2011/08  [Not refereed][Not invited]
   

  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.
• Ruminococcus albus NE1におけるマンナン資化性の解析とβ‐1,4‐マンナナーゼの同定

  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)  2011/07/20  [Not refereed][Not invited]
  
• Ruminococcus albus NE1株由来cellobiose phosphorylaseの諸性質の解析

  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)  2011/07/20  [Not refereed][Not invited]
  
• Isomaltooligosaccharide 6‐α‐glucosyltransferaseサブサイト+1変異酵素の機能解析

  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)  2011/07/20  [Not refereed][Not invited]
  
• Pichia pastorisによる組換えツベロン酸グルコシドβ‐glucosidaseの諸性質の解明

  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)  2011/07/20  [Not refereed][Not invited]
  
• 好熱性好気性細菌Rhodothermus marinus JCM 9785株より単離したcellobiose 2‐epimeraseの諸性質

  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)  2011/07/20  [Not refereed][Not invited]
  
• Dextran glucosidase(DexB)ウォーターパス改変体の機能と構造

  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)  2011/07/20  [Not refereed][Not invited]
  
• GH family 31に属するAspergillus niger由来α‐glucosidaseの鎖長特異性の改変

  田上貴祥, 奥山正幸, 森春英, 木村淳夫  応用糖質科学  1-  (3)  (36)  2011/07/20  [Not refereed][Not invited]
  
• Ruminococcus albus NE1株由来マンノシルグルコース加リン酸分解酵素の諸性質と一次構造の解析

  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)  2011/07/20  [Not refereed][Not invited]
  
• イネ培養細胞を用いたスクロース誘導性タンパク質の網羅的解析

  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)  2011/07/20  [Not refereed][Not invited]
  
• アノマー反転型トレハラーゼ変異体により合成された新規非還元二糖の構造

  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)  2011/07/20  [Not refereed][Not invited]
  
• Glycoside Hydrolase Family 63に属する酵素YgjKのグライコシンターゼ化

  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)  2011/07/20  [Not refereed][Not invited]
  
• 環状イソマルトオリゴ糖グルカノトランスフェラーゼのβ→αループ2はCI‐8の優先的生成に寄与する

  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)  2011/07/20  [Not refereed][Not invited]
  
• Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity

  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  2011/07  [Not refereed][Not invited]
   

  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.
• Ruminococcus albus NE1のマンナン代謝の解析

  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  2011/03/05  [Not refereed][Not invited]
  
• Ruminococcus albus NE1由来cellobiose phosphorylaseの一次構造の解析と大腸菌による組換え酵素の諸性質

  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  2011/03/05  [Not refereed][Not invited]
  
• ホタテガイ(Pastinopecton yessoensis)中腸腺由来α‐glucosidase—Cl⁻による反応速度の上昇と基質特異性の変化—

  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  2011/03/05  [Not refereed][Not invited]
  
• Dextran glucosidase(DexB)のウォーターパス改変酵素の機能解析

  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  2011/03/05  [Not refereed][Not invited]
  
• アノマー反転型トレハラーゼ変異体によるオリゴ糖合成

  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  2011/03/05  [Not refereed][Not invited]
  
• 環状イソマルトオリゴ糖グルカノトランスフェラーゼが示すCI‐8の優先的生成に寄与する構造因子

  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  2011/03/05  [Not refereed][Not invited]
  
• Ruminococcus albus NE1株由来cellodextrin phosphorylaseの酵素化学的諸性質

  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  [Not refereed][Not invited]
  
• 中等度好熱性アルカリ細菌Bacillus sp.AAH‐31株由来アミラーゼ遺伝子のクローニングおよび大腸菌組換え酵素の諸性質

  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  [Not refereed][Not invited]
  
• Rhodothermus marinus JCM9785由来耐熱性セロビオース2‐エピメラーゼのエピラクトースの効率的合成への応用

  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  [Not refereed][Not invited]
  
• Comparison of Enzymatic Properties and Gene Expression Profiles of Two Tuberonic Acid Glucoside β-Glucosidases from Oryza sativa L.

  WAKUTA Shinji, HAMADA Shigeki, ITO Hiroyuki, IMAI Ryozo, MORI Haruhide, MATSUURA Hideyuki, NABETA Kensuke, MATSUI Hirokazu  Journal of applied glycoscience  58-  (2)  67  -70  2011  [Not refereed][Not invited]
  
• Oligo‐1,6‐glucosidaseがCl⁻依存性を示すために必要なアミノ酸残基の決定

  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  2010/09/27  [Not refereed][Not invited]
  
• 加水分解反応の抑制により縮合反応の生成物を蓄積させるグルコアミラーゼ変異体

  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  2010/09/27  [Not refereed][Not invited]
  
• isomaltooligosaccharide6‐α‐glucosyltransferaseのサブサイト+1変異酵素の機能解析

  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  2010/07/20  [Not refereed][Not invited]
  
• Aspergillus niger α‐glucosidaseのサブサイト+1の改変

  田上貴祥, 奥山正幸, 森春英, 木村淳夫  J Appl Glycosci  57-  (Suppl.)  44  2010/07/20  [Not refereed][Not invited]
  
• Oligo‐1,6‐glucosidaseの変異導入によるCl⁻イオン依存型への改変

  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  2010/03/05  [Not refereed][Not invited]
  
• 大腸菌組換えデキストリンデキストラナーゼのオリゴ糖に対する作用と触媒アミノ酸残基の解析

  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  2010/03/05  [Not refereed][Not invited]
  
• GH family 31 α‐グルコシダーゼにおける基質の鎖長認識機構の解明

  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  2010/03/05  [Not refereed][Not invited]
  
• Suicide Substrate-based Inactivation of Endodextranase by ω-Epoxyalkyl α-D-Glucopyranosides

  KANG Hee-Kwon, KIM Young-Min, NAKAI Hiroyuki, KANG Min-Sun, HAKAMADA Wataru, OKUYAMA Masayuki, MORI Haruhide, NISHIO Toshiyuki, KIMURA Atsuo  Journal of Applied Glycoscience  57-  (4)  269  -277  2010  [Not refereed][Not invited]
   

  Three kinds of ω-epoxyalkyl α-glucopyranosides (3′,4′-epoxybutyl α-D-glucopyranoside (E4G), 4′,5′-epoxypentyl α-D-glucopyranoside (E5G) and 5′,6′-epoxyhexyl α-D-glucopyranoside (E6G)), having alkyl chains of different lengths at their aglycone moieties, inactivated the endodextranase from Streptococcus mutans ATCC 25175 (SmDex) irreversibly with the pseudo-first order kinetics. Alkyl chain length-dependent inactivation was observed and the degree of activity loss was E5G, E6G and E4G, in that order, implying that the distance between epoxide group and glucosyl residue of ω-epoxyalkyl α-glucopyranoside was important in the modification of endodextranase. Inactivation by E5G followed the model of reversible intermediate-complex formation mechanism (suicide inhibitor-based mechanism). The rate constant of irreversible inactivation (k) and the dissociation constant of intermediate-complex (KR) of SmDex and E5G were 0.44 min^-1 and 1.45 mM, respectively. Hydrolytic reaction product (isomaltose) protected SmDex from E5G-inactivation, suggesting that E5G bound to the catalytic site of SmDex. This is the first report that ω-epoxyalkyl α-glucopyranoside becomes a suicide substrate for endodextranase.
• The first alpha-1,3-glucosidase from bacterial origin belonging to glycoside hydrolase family 31

  Min-Sun Kang, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura  BIOCHIMIE  91-  (11-12)  1434  -1442  2009/11  [Not refereed][Not invited]
   

  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.
• Catalytic Reaction Mechanism Based on alpha-Secondary Deuterium Isotope Effects in Hydrolysis of Trehalose by European Honeybee Trehalase

  Haruhide Mori, Jin-Ha Lee, Masayuki Okuyama, Mamoru Nishimoto, Masao Ohguchi, Doman Kim, Atsuo Kimura, Seiya Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  73-  (11)  2466  -2473  2009/11  [Not refereed][Not invited]
   

  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.
• Streptococcus mutans由来dextran glucosidaseの部位特異的変異導入による糖転移活性の改変(2)

  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  2009/07/20  [Not refereed][Not invited]
  
• isomaltooligosaccharide6‐α‐glucosyltransferaseの糖転移反応に関わる構造因子

  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  2009/07/20  [Not refereed][Not invited]
  
• テンサイα‐glucosidaseのサブサイト+2および+3の形成に重要なアミノ酸残基の決定

  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  2009/07/20  [Not refereed][Not invited]
  
• Bacteroides thetaiotaomicron由来SusBの触媒反応におけるCa²⁺の役割

  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  2009/07/20  [Not refereed][Not invited]
  
• Streptococcus mutans由来dextran glucosidaseの部位特異的変異導入による糖転移活性の改変(1)

  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  2009/07/20  [Not refereed][Not invited]
  
• Structural Comparison of Streptococcus mutans Dextran Glucosidase with Glucoside Hydrolases in GH13

  HONDOH Hironori, OTSUKA RACHI Hiroaki, SABURI Wataru, MORI Haruhide, OKUYAMA Masayuki, KIMURA Atsuo  Journal of applied glycoscience  56-  (2)  111  -117  2009/04/20  [Not refereed][Not invited]
   

  In glycoside hydrorase family (GH) 13, α-glucosidase, oligo-1,6-glucosidase and dextran glucosidase, which hydrolyze the non-reducing end glucosidic linkages of maltooligo- and/or isomaltoolligosaccharides, are categorized as α-glucoside hydrolase. Despite a high similarity in the sequence and overall structure of those family enzymes, GH 13 α-glucoside hydrolases show a wide range of substrate specificity. Until now, three crystal structures of α-glucoside hydrolase, dextran glucosidase from Streptococcus mutans (DGase), oligo-1,6-glucosidase from Bacillus cereus (O16G), and α-glucosidase from Geobacillus sp. HTA-462 (GSJ) have been determined. In this study, we have performed the structural comparison of these α-glucoside hydrolases. Their overall structures are generally similar, and consist of three major domains A, B and C as found in many α-amylase family enzymes. The significant structural differences in these enzymes are mainly found in loop regions. GSJ has a shorter β→α loop 6 in a different orientation in addition to the disordered regions, whereas DGase and O16G show high similarity in their tertiary structures. Though these enzymes have the different substrate preference, they all possess the completely conserved configuration at subsite -1. Therefore, the substrate preference will be originated from the structure at subsite for the reducing end side of substrate. The substrate binding modes of these glucoside hydrolases were predicted by superimposing the substrate molecule of substrate-complex structures of DGase and α-amylase. DGase and O16G are thought to have a similar manner in substrate binding with conserved amino acid residues. The substrate recognition of GSJ at subsite -1 and +1 would be similar to that of α-amylases since the key residues in substrate binding are conserved in both primary and tertiary structures.
• Isomal tooligosaccharide 6‐α‐glucosyltransferase(I6GT)のイソマルトオリゴ糖への作用と転移に関する構造因子

  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  2009/03/05  [Not refereed][Not invited]
  
• アズキα‐アミラーゼ(VAA)のMet398は過酸化水素により特異的に酸化される

  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  2009/03/05  [Not refereed][Not invited]
  
• 加水分解反応の抑制によりトレハロースを縮合蓄積させるトレハラーゼ変異酵素

  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  2009/03/05  [Not refereed][Not invited]
  
• Preliminary Structure Determination of Polysaccharide Extracted from Rhizobium sp.

  Wongchawalit Jintanart, Hashidoko Yasuyuki, Okuyama Masayuki, Mori Haruhide, Chiba Seiya, Kimura Atsuo  Journal of Applied Glycoscience Supplement  2009-  (0)  39  -39  2009  [Not refereed][Not invited]
  
• Structural Comparison of Streptococcus mutans Dextran Glucosidase with Glucoside Hydrolases in GH13

  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  [Not refereed][Not invited]
   

  In glycoside hydrorase family (GH) 13, α-glucosidase, oligo-1,6-glucosidase and dextran glucosidase, which hydrolyze the non-reducing end glucosidic linkages of maltooligo- and/or isomaltoolligosaccharides, are categorized as α-glucoside hydrolase. Despite a high similarity in the sequence and overall structure of those family enzymes, GH 13 α-glucoside hydrolases show a wide range of substrate specificity. Until now, three crystal structures of α-glucoside hydrolase, dextran glucosidase from Streptococcus mutans (DGase), oligo-1,6-glucosidase from Bacillus cereus (O16G), and α-glucosidase from Geobacillus sp. HTA-462 (GSJ) have been determined. In this study, we have performed the structural comparison of these α-glucoside hydrolases. Their overall structures are generally similar, and consist of three major domains A, B and C as found in many α-amylase family enzymes. The significant structural differences in these enzymes are mainly found in loop regions. GSJ has a shorter β→α loop 6 in a different orientation in addition to the disordered regions, whereas DGase and O16G show high similarity in their tertiary structures. Though these enzymes have the different substrate preference, they all possess the completely conserved configuration at subsite -1. Therefore, the substrate preference will be originated from the structure at subsite for the reducing end side of substrate. The substrate binding modes of these glucoside hydrolases were predicted by superimposing the substrate molecule of substrate-complex structures of DGase and α-amylase. DGase and O16G are thought to have a similar manner in substrate binding with conserved amino acid residues. The substrate recognition of GSJ at subsite -1 and +1 would be similar to that of α-amylases since the key residues in substrate binding are conserved in both primary and tertiary structures.
• Structural and Functional Analysis of a Glycoside Hydrolase Family 97 Enzyme from Bacteroides thetaiotaomicron

  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  2008/12  [Not refereed][Not invited]
   

  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.
• Dextran glucosidaseの加水分解および糖転移反応における基質特異性の変換

  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  2008/07/20  [Not refereed][Not invited]
  
• 大腸菌trehalaseの触媒アミノ酸残基の決定とglycosynthase反応

  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  2008/07/20  [Not refereed][Not invited]
  
• α‐1,4グルコシド結合特異的なdextran glucosidase変異体の基質認識機構

  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  2008/07/20  [Not refereed][Not invited]
  
• Glycoside hydrolase family97アノマー保持型酵素の触媒機構

  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  2008/07/20  [Not refereed][Not invited]
  
• デキストリンデキストラナーゼ遺伝子の異宿主発現および組換え酵素の解析

  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  2008/07/20  [Not refereed][Not invited]
  
• Bacillus sp.由来isomaltooligosaccharide6‐α‐glucosyltrasferase(I6GT)の転移反応に関与する構造因子

  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  2008/07/20  [Not refereed][Not invited]
  
• テンサイα‐glucosidaseのcDNAクローニングとpichia pastorisによる組換え酵素の生産

  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  2008/07/20  [Not refereed][Not invited]
  
• Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans

  Hironori Hondoh, Wataru Saburi, Haruhide Mori, Masayuki Okuyama, Toshitaka Nakada, Yoshiki Matsuura, Atsuo Kimura  JOURNAL OF MOLECULAR BIOLOGY  378-  (4)  913  -922  2008/05  [Not refereed][Not invited]
   

  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.
• Substrate Recognition of Escherichia coli YicI (α-Xylosidase)

  OKUYAMA Masayuki, KANG Min-Sun, YAOI Katsuro, MITSUISHI Yasushi, MORI Haruhide, KIMURA Atsuo  Journal of applied glycoscience  55-  (2)  111  -118  2008/04/20  [Not refereed][Not invited]
   

  Glycoside hydrolase family 31 (GH 31) is one of the most intriguing glycoside hydrolase families. This family contains α-glucosidase, α-xylosidase, α-glucan lyase and isomaltosyltransferase. Escherichia coli YicI (α-xylosidase) is a representative enzyme of GH 31 because its biochemical and structural studies have been thoroughly carried out. YicI is a strict α-xylosidase, which rigidly recognizes α-xyloside at the non-reducing terminal end, even though its amino acid sequence apparently displays similarity with α-glucosidases. Phe277, Cys307, Trp345 and Lys414 at the subsite-1 are important for α-xylosidase activity. The mutant YicI enzymes, which possesses Ile307/Asp308 instead of Cys307/Phe308 and which has a shorter β→α loop 1 of (β/α)8 barrel in place of the original longer loop, respectively, possess α-glucosidase activity. In the transxylosylation of YicI, glucose, mannose and allose are able to act as acceptors, but galactose, talose and gulose never do, implying that equatorial OH-4 of the aldopyranose is crucial for acting as an acceptor. YicI transfers α-xylosyl moieties to a specific hydroxy group in the acceptor sugar (except fructopyranose) showing 1,6 regioselectivity, which is in agreement with the structural feature of the aglycone-biding site. Among the transxylosylation products of YicI, α-D-xylopyranosyl-(1→6)-D-mannopyranose, α-D-xylopyranosyl-(1→6)-D-fructofuranose, and α-D-xylopyranosyl-(1→3)-D-fructopyranose are novel sugars. α-D-Xylopyranosyl-(1→6)-D-mannopyranose and α-D-xylopyranosyl-(1→6)-D-fructofuranose have the ability to inhibit rat intestinal α-glucosidases.
• 新しく見出されたイソマルトオリゴ糖不均化酵素の遺伝子クローニングと異種宿主発現

  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  2008/03/05  [Not refereed][Not invited]
  
• アクセプタ結合部位への変異導入によるdextran glucosidaseの糖転移生成物の制御

  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  2008/03/05  [Not refereed][Not invited]
  
• Schwanniomyces occidentalis由来GH31 α‐glucosidaseへの変異導入によるグルコシド結合選択性の変換

  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  2008/03/05  [Not refereed][Not invited]
  
• デキストリンデキストラナーゼの一次構造決定および組換え酵素の解析

  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  [Not refereed][Not invited]
  
• 新規酵素isomal tool igosaccharide6‐α‐glucosyl transferase(I6GT)の機能解析

  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  [Not refereed][Not invited]
  
• Aglycone specificity of Escherichia coli alpha-xylosidase investigated by transxylosylation

  Min-Sun Kang, Masayuki Okuyama, Katsuro Yaoi, Yasushi Mitsuishi, Young-Min Kim, Haruhide Mori, Doman Kim, Atsuo Kimura  FEBS JOURNAL  274-  (23)  6074  -6084  2007/12  [Not refereed][Not invited]
   

  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.
• Aglycone specificity of Escherichia coli alpha-xylosidase investigated by transxylosylation

  Min-Sun Kang, Masayuki Okuyama, Katsuro Yaoi, Yasushi Mitsuishi, Young-Min Kim, Haruhide Mori, Doman Kim, Atsuo Kimura  FEBS JOURNAL  274-  (23)  6074  -6084  2007/12  [Not refereed][Not invited]
   

  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.
• Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are alpha-glucosidase and alpha-xylosidase

  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  2007/10  [Not refereed][Not invited]
   

  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.
• Molecular cloning of cDNA for trehalase from the European honeybee, Apis mellifera L., and its heterologous expression in Pichia pastoris

  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  2007/09  [Not refereed][Not invited]
   

  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.
• Crystallization and preliminary X-ray analysis of Streptococcus mutans dextran glucosidase

  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  2007/09  [Not refereed][Not invited]
   

  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.
• 新規なイソマルトオリゴ糖生成酵素の単離と性質

  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  2007/07/20  [Not refereed][Not invited]
  
• Streptococcus mutans由来dextran glucosidaseの保存領域IVに存在するAsnの機能解析

  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  2007/07/20  [Not refereed][Not invited]
  
• 大腸菌Yicl(α‐xylosidase)の基質認識の解析

  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  2007/07/20  [Not refereed][Not invited]
  
• ホタテガイ由来塩素イオン依存性α‐amylaseの構造と機能

  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  2007/07/20  [Not refereed][Not invited]
  
• Molecular cloning of cDNAs and genes for three alpha-glucosidases from European honeybees, Apis mellifera L., and heterologous production of recombinant enzymes in Pichia pastoris

  Mamoru Nishimoto, Haruhide Mori, Tsuneharu Moteki, Yukiko Takamura, Gaku Iwai, Yu Miyaguchi, Masayuki Okuyama, Jintanart Wongchawalit, Rudee Surarit, Jisnuson Svasti, Atsuo Kimura, Seiya Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  71-  (7)  1703  -1716  2007/07  [Not refereed][Not invited]
   

  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.
• Streptococcus mutans由来dextran glucosidase(DGase)の基質特異性の変換:α‐1,4結合加水分解酵素への改変

  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  2007/03/05  [Not refereed][Not invited]
  
• デキストラン関連酵素が有するデキストラン結合ドメインの機能解析

  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  2007/03/05  [Not refereed][Not invited]
  
• B.thetaiotaomicron BT1871の求核触媒残基変異酵素の性質

  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  2007/03/05  [Not refereed][Not invited]
  
• ホタテガイ閉殻筋由来α‐glucosidaseのマルチプルフォームの構造解析

  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  2007/03/05  [Not refereed][Not invited]
  
• Streptococcus mutans由来Dextran Glucosidase M198Wの結晶構造解析

  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  [Not refereed][Not invited]
  
• クロマルハナバチ(Bombus terrestris)α‐グルコシダーゼ:アロステリック酵素の性質解明と遺伝子発現

  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  [Not refereed][Not invited]
  
• Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare)

  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  2007/01  [Not refereed][Not invited]
   

  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.
• Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA

  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  2006/12  [Not refereed][Not invited]
   

  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.
• ホタテガイ閉殻筋に存在するイオン要求性α‐glucosidaseの精製とその諸性質

  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  2006/08/30  [Not refereed][Not invited]
  
• Bacillus subtills 168S由来α‐glucosidase(YugT)の基質特異性に関わるアミノ酸残基の推定

  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  2006/08/30  [Not refereed][Not invited]
  
• Bacteroides thetaiotaomicron由来SusBの触媒残基解析

  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  2006/08/30  [Not refereed][Not invited]
  
• Dextran glucosidaseのα‐1,6グルコシド結合認識機構

  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  2006/08/30  [Not refereed][Not invited]
  
• クロマルハナバチ,Bombus ignitus,α‐GlucosidaseアイソザイムIの長鎖基質認識に関わるアミノ酸残基の決定

  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  2006/08/30  [Not refereed][Not invited]
  
• GST融合タンパク質を用いたイネα‐glucosidaseの澱粉粒吸着および分解機構の解析

  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  2006/08/30  [Not refereed][Not invited]
  
• Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase

  M Okuyama, A Kaneko, H Mori, S Chiba, A Kimura  FEBS LETTERS  580-  (11)  2707  -2711  2006/05  [Not refereed][Not invited]
   

  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.
• Structural elements in dextran glucosidase responsible for high specificity to long chain substrate

  Wataru Saburi, Haruhide Mori, Saori Saito, Masayuki Okuyama, Atsuo Kimura  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS  1764-  (4)  688  -698  2006/04  [Not refereed][Not invited]
   

  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.
• Glycoside hydrolase family 97酵素BT1871の反応機構

  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  2006/03/05  [Not refereed][Not invited]
  
• Bacillus subtilis168S由来α‐glucosidase YugTの機能解析

  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  2006/03/05  [Not refereed][Not invited]
  
• セイヨウミツバチα‐glucosidaseアイソザイムIIのβ→αループ8変異酵素の機能解析

  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  2006/03/05  [Not refereed][Not invited]
  
• アズキα‐amylaseのデンプン粒吸着能はMet398改変により変化する

  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  2006/03/05  [Not refereed][Not invited]
  
• Bacillus subtilis 168S由来β‐N‐acetylglucosaminidase(YbbD)の求核触媒残基を酸素酸化型システインに置換した変異酵素の性質

  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  2006/03/05  [Not refereed][Not invited]
  
• Identification and Manipulation of Subsite Structure and Starch Granule Binding Site in Plant α-Amylase

  MORI Haruhide  Journal of applied glycoscience  53-  (1)  51  -56  2006/01/20  [Not refereed][Not invited]
   

  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" Tyr380_AMY1 was proven to be involved in starch granule binding in adzuki bean α-amylase.
• Purification and characterization of the hyper-glycosylated extracellular alpha-glucosidase from Schizosaccharomyces pombe

  M Okuyama, Y Tanimoto, T Ito, A Anzai, H Mori, A Kimura, H Matsui, S Chiba  ENZYME AND MICROBIAL TECHNOLOGY  37-  (5)  472  -480  2005/10  [Not refereed][Not invited]
   

  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.
• Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase

  F Sato, M Okuyama, H Nakai, H Mori, A Kimura, S Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  69-  (10)  1905  -1913  2005/10  [Not refereed][Not invited]
   

  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.
• Oligosaccharide binding to barley alpha-amylase 1

  Robert, X, R Haser, H Mori, B Svensson, N Aghajari  JOURNAL OF BIOLOGICAL CHEMISTRY  280-  (38)  32968  -32978  2005/09  [Not refereed][Not invited]
   

  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."
• 植物α‐アミラーゼのデンプン結合部位の同定と改変に関する研究

  森春英  飯島記念食品科学振興財団年報  2003-  90-95  2005/08  [Not refereed][Not invited]
  
• セイヨウミツバチα‐Glucosidase IIのV167N変異酵素は高いK_mおよびk₀を与え,マルハナバチα‐Glucosidase IIの性質を示した

  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  2005/07/20  [Not refereed][Not invited]
  
• Acetobacter capsulatum由来dextrin dextranaseの一次構造の解析

  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  2005/07/20  [Not refereed][Not invited]
  
• —ホタテガイ中腸腺由来イオン依存性α‐glucosidase—

  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  2005/07/20  [Not refereed][Not invited]
  
• 植物α‐GlucosidaseのC末端領域は単独で澱粉粒に吸着する‐GST融合タンパク質による解析‐

  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  2005/07/20  [Not refereed][Not invited]
  
• 植物α‐Glucosidase,特にイネ酵素の分子解析と発芽時の澱粉粒分解に関する研究

  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  2005/07/20  [Not refereed][Not invited]
  
• Bacteroides thetaiotaomicron SusBの機能解析

  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  2005/07/20  [Not refereed][Not invited]
  
• Molecular analysis of alpha-glucosidase isozymes from Japanese honeybees (Apis cerana)

  J Wongchawalit, T Yamamoto, M Okuyama, H Mori, R Surarit, J Svasti, S Chiba, AK Kimura  FEBS JOURNAL  272-  101  -101  2005/07  [Not refereed][Not invited]
  
• Bacteroides thetaiomicron VPI‐5482由来Glycoside hydrolase family 66様タンパク質の発現と精製および機能解析

  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  2005/03/05  [Not refereed][Not invited]
  
• Pichia pastorisを用いたアズキα‐Amylase組換酵素の高生産条件の検討

  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  2005/03/05  [Not refereed][Not invited]
  
• Glycoside hydrolase family 31酵素の構造と機能

  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  2005/03/05  [Not refereed][Not invited]
  
• イネGlycoside Hydrolase Family 31様遺伝子の発現解析

  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  2005/03/05  [Not refereed][Not invited]
  
• クロマルハナバチ,Bombus ignitus,α‐Glucosidaseアイソザイムの精製・諸性質および一次構造の解析

  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  2005/03/05  [Not refereed][Not invited]
  
• イネGlycoside hydrolase family 31様タンパク質の機能解析

  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  2005/03/05  [Not refereed][Not invited]
  
• ミツバチ由来結合型トレハラーゼのcDNAクローニングと組換え酵素の生産

  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  2005/03/05  [Not refereed][Not invited]
  
• Bacillus subtilis 168S由来YbbDはβ‐N‐acetylglucosaminidase活性を示す。

  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  2005/03/05  [Not refereed][Not invited]
  
• Streptococus mutans由来dextran glucosidaseの基質特異性の改変

  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  2005/03/05  [Not refereed][Not invited]
  
• ミツバチα‐グルコシダーゼIIIの蜂蜜生産への適応:Tyr210の高K_nへの寄与

  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  2005/03/05  [Not refereed][Not invited]
  
• Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase

  B Kramhoft, KS Bak-Jensen, H Mori, N Juge, J Nohr, B Svensson  BIOCHEMISTRY  44-  (6)  1824  -1832  2005/02  [Not refereed][Not invited]
   

  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.
• Crystallization and preliminary X-ray analysis of alpha-xylosidase from Escherichia coli

  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  2005/02  [Not refereed][Not invited]
  
• 大腸菌Trehalase(TreA)の触媒残基の部位特異的変異導入による解析

  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  [Not refereed][Not invited]
  
• Streptococcus mutans由来dextran glucosidaseのMet198変異酵素の機能

  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  [Not refereed][Not invited]
  
• Glycoside hydrolase family31酵素の活性中心に保存されているArg残基の役割

  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  [Not refereed][Not invited]
  
• Enzymatic synthesis of alkyl alpha-2-deoxyglucosides by alkyl alcohol resistant alpha-glucosidase from Aspergillus niger

  YM Kim, M Okuyama, H Mori, H Nakai, W Saburi, S Chiba, A Kimura  TETRAHEDRON-ASYMMETRY  16-  (2)  403  -409  2005/01  [Not refereed][Not invited]
   

  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.
• Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey

  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  2004/11  [Not refereed][Not invited]
   

  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.
• Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli

  M Okuyama, H Mori, S Chiba, A Kimura  PROTEIN EXPRESSION AND PURIFICATION  37-  (1)  170  -179  2004/09  [Not refereed][Not invited]
   

  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.
• Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage

  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  2004/08  [Not refereed][Not invited]
   

  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.
• 植物α‐Glucosidaseの生澱粉吸着に関与するアミノ酸残基の解析

  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  2004/07/20  [Not refereed][Not invited]
  
• アズキα‐アミラーゼの澱粉粒結合部位に関する研究

  森春英, 浜井英礼, MAR S S, 千葉誠哉, 木村淳夫  J Appl Glycosci  51-  (Suppl.)  42  2004/07/20  [Not refereed][Not invited]
  
• 大腸菌yicl産物の機能およびX線構造解析

  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  2004/07/20  [Not refereed][Not invited]
  
• クロマルハナバチ,Bombus ignitus,α‐Glucosidase Iの精製と諸性質

  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  2004/07/20  [Not refereed][Not invited]
  
• Search for a mechanism by which barley .ALPHA.-amylase recongnizes the substrate.

  MORI HARUHIDE  化学と生物  42-  (3)  170-172  -172  2004/03/25  [Not refereed][Not invited]
  
• Streptomyces sp. No.35由来β‐glucosidase F₃の活性化現象

  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  2004/03/05  [Not refereed][Not invited]
  
• 触媒残基をcysteineに置換したdextran glucosidaseは酸化処理により活性が回復する

  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  2004/03/05  [Not refereed][Not invited]
  
• 植物α‐Glucosidaseに見出された新規な生澱粉結合部位の解析

  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  2004/03/05  [Not refereed][Not invited]
  
• アズキα‐Amylaseのデンプン粒に対する吸着能は翻訳後修飾によって低下する

  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  2004/03/05  [Not refereed][Not invited]
  
• Glycosidase様タンパク質のglycosyl fluorideに対する反応の解析

  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  2004/03/05  [Not refereed][Not invited]
  
• ミツバチα‐glucosidase III触媒残基変異酵素D206Gはアノマー反転型反応を行う。

  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  2004/03/05  [Not refereed][Not invited]
  
• Bacillus subtilis 168S^r由来α‐glucosidase Phe144の機能解析

  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  2004/03/05  [Not refereed][Not invited]
  
• オオムギα‐アミラーゼ1 Asn209変異酵素の機能解析

  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  2004/03/05  [Not refereed][Not invited]
  
• α‐Glucosidase変異酵素がα‐Glycosynthase反応を触媒する

  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  2003/11/01  [Not refereed][Not invited]
  
• Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus

  T Naraoka, H Uchisawa, H Mori, H Matsue, S Chiba, A Kimura  EUROPEAN JOURNAL OF BIOCHEMISTRY  270-  (19)  4026  -4038  2003/10  [Not refereed][Not invited]
   

  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.
• Purification, characterization, and sequence analysis of two alpha-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards beta-cyclodextrin sepharose

  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  2003/05  [Not refereed][Not invited]
   

  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.
• オオムギα‐アミラーゼ1(AMY1)の部位特異的変異による基質特異性の改変

  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  2003/03/05  [Not refereed][Not invited]
  
• 赤米および日本晴で発芽時に作用するα‐Glucosidaseの一次構造の解析

  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  2003/03/05  [Not refereed][Not invited]
  
• Streptococcus mutans由来Dextran glucosidase変異酵素の長鎖基質に対する作用

  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  2003/03/05  [Not refereed][Not invited]
  
• β‐Cyclodextrinに異なる親和力を示すアズキα‐amylaseアイソフォーム

  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  2003/03/05  [Not refereed][Not invited]
  
• 糸状菌Acremonium implicatum IF030538由来α‐Glucosidase遺伝子のクローニング

  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  2003/03/05  [Not refereed][Not invited]
  
• Evidence of Intramolecular Transglucosylation Catalyzed by an .ALPHA.-Glucosidase.

  Son Mee, Mori Haruhide, Okuyama Masayuki, Kimura Atsuo, Chiba Seiya  Journal of Applied Glycoscience  50-  (1)  41  -44  2003  [Not refereed][Not invited]
   

  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 toward isomaltose. However, the conflict for the peculiar phenomenon on the reaction was rationally explained by the evidence that isomaltose might be formed by the intramolecular transglucosylation via other α-glucobioses that are easily produced from glucose by the condensation reaction. It is suggested that the usual transglycosylation of carbohydrate-hydrolase may be accompanied by an intramolecular transfer reaction.
• Barley alpha-amylase Met53 situated at the high-affinity subsite-2 belongs to a substrate binding motif in the beta ->alpha loop 2 of the catalytic (beta/alpha)(8)-barrel and is critical for activity and substrate specificity

  H Mori, KS Bak-Jensen, B Svensson  EUROPEAN JOURNAL OF BIOCHEMISTRY  269-  (22)  5377  -5390  2002/11  [Not refereed][Not invited]
   

  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).
• Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F-1 from a Streptomyces sp.

  K Fukuda, H Mori, M Okuyama, A Kimura, H Ozaki, M Yoneyama, S Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  66-  (10)  2060  -2067  2002/10  [Not refereed][Not invited]
   

  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).
• alpha-glucosidase mutant catalyzes "alpha-glycosynthase"-type reaction

  M Okuyama, H Mori, K Watanabe, A Kimura, S Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  66-  (4)  928  -933  2002/04  [Not refereed][Not invited]
   

  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.
• α‐Glucosidase変異酵素はGlycosynthase反応を触媒する

  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  2002/03/05  [Not refereed][Not invited]
  
• Acetobacter capsulatum由来Dextrin Dextranaseの初期反応の解析とその測定法の確立

  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  2002/03/05  [Not refereed][Not invited]
  
• Bacillus subtilis 168S^r α‐glucosidase組換え体の大腸菌での生産

  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  2002/03/05  [Not refereed][Not invited]
  
• Aspergillus niger α‐Galactosidase遺伝子のPichia pastorisでの発現

  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  2002/03/05  [Not refereed][Not invited]
  
• 赤米α‐Glucosidaseの限定分解とそれによる諸性質への影響

  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  2002/03/05  [Not refereed][Not invited]
  
• Kinetic Studies on Substrate Specificity and Active Site of beta-D-Glucosidase F1 from Streptomyces sp.

  FUKUDA K, SHIRAKAWA K, MORI H, OKUYAMA M, KIMURA A, OZAKI H, YONEYAMA M, CHIBA S  J. Appl. Glycosci.  49-  (3)  265  -272  2002  [Not refereed][Not invited]
  
• Modulation of activity and substrate binding modes by mutation of single and double subsites+1/+2 and-5/-6 of barley alpha-amylase 1

  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  2001/12  [Not refereed][Not invited]
   

  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.
• Purification and identification of the essential ionizable groups of honeybee, Apis mellifera L., trehalase

  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  2001/12  [Not refereed][Not invited]
   

  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.
• ビールの国でのオオムギα-アミラーゼ研究 デンマーク・カールスバーグ研究所に滞在して

  森 春英  化学と生物  39-  (9)  588  -591  2001/09/25
• Purification and substrate specificity of honeybee, Apis mellifera L., alpha-glucosidase III

  M Nishimoto, M Kubota, M Tsuji, H Mori, A Kimura, H Matsui, S Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  65-  (7)  1610  -1616  2001/07  [Not refereed][Not invited]
   

  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.
• Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe

  M Okuyama, A Okuno, N Shimizu, H Mori, A Kimura, S Chiba  EUROPEAN JOURNAL OF BIOCHEMISTRY  268-  (8)  2270  -2280  2001/04  [Not refereed][Not invited]
   

  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.
• アイソトープ効果に基づいたアミラーゼの反応機構

  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  2001/03/05  [Not refereed][Not invited]
  
• 赤米α‐Glucosidaseの精製と諸性質

  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  2001/03/05  [Not refereed][Not invited]
  
• Isolation and Sequence of Putative alpha-Glucosidase Gene from Brevibacterium fuscum var. dextranlyticum Strain 0407

  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  [Not refereed][Not invited]
  
• Protein engineering of substrate binding subsites in amylolytic enzymes.

  H Mori, KS Bak-Jensen, B Svensson  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY  220-  U117  -U117  2000/08  [Not refereed][Not invited]
  
• Molecular cloning of isomaltotrio-dextranase gene from Brevibacterium fuscum var. dextranlyticum strain 0407 and its expression in Escherichia coli

  T Mizuno, H Mori, H Ito, H Matsui, A Kimura, S Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  63-  (9)  1582  -1588  1999/09  [Not refereed][Not invited]
   

  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.
• The engineering of specificity and stability in selected starch degrading enzymes

  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  [Not refereed][Not invited]
  
• Expression characteristic of an enzyme related to carbon and nitrogen balance in the leaves of rice plants.

  NAKAJIMA MASAKAZU, OSAKI MITSURU, SHINANO TAKURO, MORI HARUHIDE, TANNO TOSHIAKI  日本土壌肥料学会講演要旨集  44-  256  -256  1998/03  [Not refereed][Not invited]
  
• Analysis of amino acid residue concerning catalytic activity of .ALPHA.-glucosidase in Schizosaccharomyces pombe.

  OKUYAMA MASAYUKI, MORI HARUHIDE, KIMURA ATSUO, CHIBA SEIYA  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  [Not refereed][Not invited]
  
• Localization of α-Glucosidase in Yeast Cells

  Oyo Toshitsu Kagaku (J. Appl. Glycosci.)  45-  (3)  281  -283  1998  [Not refereed][Not invited]
  
• Molecular Cloning of an α-Amylase cDNA from Germinating Cotyledons of Kidney Bean (Phaseolus vulgaris L. cv. Toramame)

  MORI Haruhide, KOBAYASHI Tetsuya, TONOKAWA Takashi, TATEMATSU Ayumi, MATSUI Hirokazu, KIMURA Atsuo, CHIBA Seiya  Journal of applied glycoscience  45-  (3)  261  -267  1998  [Not refereed][Not invited]
  
• A catalytic amino acid and primary structure of active site in Aspergillus niger alpha-glucosidase

  A Kimura, M Takata, Y Fukushi, H Mori, H Matsui, S Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  61-  (7)  1091  -1098  1997/07  [Not refereed][Not invited]
   

  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.
• Identification of essential ionizable groups in active site of Aspergillus niger alpha-glucosidase

  A Kimura, A Somoto, H Mori, O Sakai, H Matsui, S Chiba  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  61-  (3)  475  -479  1997/03  [Not refereed][Not invited]
   

  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).
• Brevibacterium fuscum. Cloning and analysis of primary structure.of var. dextranlyticum derived isomaltotrio-dextranase gene

  MIZUNO TAKAFUMI, MATSUI HIROKAZU, MORI HARUHIDE, ITO HIROYUKI, KIMURA ATSUO, CHIBA SEIYA  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  [Not refereed][Not invited]
  
• Molecular cloning and nucleotide sequences of cDNA and gene encoding endo-inulinase from Penicillium purpurogenum

  S Onodera, T Murakami, H Ito, H Mori, H Matsui, M Honma, S Chiba, N Shiomi  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  60-  (11)  1780  -1785  1996/11  [Not refereed][Not invited]
   

  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.
• Amino acid sequence analysis of the active center vicinity of buckwheat seed .ALPHA.-glucosidase.

  MORI ISSEI, IWANAMI SHUNSUKE, MORI HARUHIDE, KIMURA ATSUO, MATSUI HIROKAZU, CHIBA MASAYA  日本農芸化学会東北支部講演要旨  1996-  (Godo Gakujutsu Koenkai)  18  1996/09  [Not refereed][Not invited]
  
• Schizosaccharomyces pombe. Inference of the primary structure and active free radical of .ALPHA.-glucosidase.

  OKUYAMA MASAYUKI, MORI HARUHIDE, KIMURA ATSUO, CHIBA MASAYA  日本農芸化学会東北支部講演要旨  1996-  (Godo Gakujutsu Koenkai)  17  1996/09  [Not refereed][Not invited]
  
• テンサイα-Glucosidaseの一次構造について : 酵素

  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  1996/03/05
• トラマメ(Phaseolus vulgaris L.)の発芽子葉及び緑葉で発現するα-amylaseについて : 酵素

  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  1996/03/05
• Schizosaccharomyces pombe. Estimation of primary structure and active dissociated group of .ALPHA.-glucosidase.

  OKUYAMA MASAYUKI, MORI HARUHIDE, KIMURA ATSUO, CHIBA SEIYA  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  [Not refereed][Not invited]
  
• Amino acid sequence analysis near active center of buckwheat seed .ALPHA.-glucosidase.

  MORI KAZUO, IWANAMI SHUNSUKE, MORI HARUHIDE, KIMURA ATSUO, MATSUI HIROKAZU, CHIBA SEIYA  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  [Not refereed][Not invited]
  
• Reinvestigation of Isomaltotrio-Dextranase from Brevibacterium fuscum var. dextranlyticum

  MIZUNO Takafumi, MATSUI Hirokazu, ITO Hiroyuki, MORI Haruhide, KIMURA Atsuo, HONMA Mamoru, CHIBA Seiya  Journal of applied glycoscience  43-  (3)  347  -353  1996  [Not refereed][Not invited]
  
• 植物α-Glucosidaseの活性中心 : 特にテンサイについて : 酵素

  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  1995/07/05
• Aspergillus nigerの生産するα-galactosidaseに関する研究 : 酵素

  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  1995/07/05
• Streptomyces sp. No.35菌体内β-glucosidaseの基質特異性 : 酵素

  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  1995/07/05
• トラマメ(Phaseolus vulgaris L.)緑葉α-amylaseの精製と諸性質 : 酵素

  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  1995/07/05
• CHEMICAL MODIFICATION AND AMINO-ACID-SEQUENCE OF ACTIVE-SITE IN SUGAR-BEET ALPHA-GLUCOSIDASE

  S IWANAMI, H MATSUI, A KIMURA, H ITO, H MORI, M HONMA, S CHIBA  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  59-  (3)  459  -463  1995/03  [Not refereed][Not invited]
   

  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.
• Substrate Specificity and Subsite Affinities of α-Amylase from Germinating Cotyledons of Phaseolus vulgaris L. cv Toramame.

  Oyo Toshitsu Kagaku (J. Appl. Glycosci.)  42-  (4)  387  -394  1995  [Not refereed][Not invited]
  
• MOLECULAR-CLONING AND EXPRESSION OF AN ISOMALTO-DEXTRANASE GENE FROM ARTHROBACTER-GLOBIFORMIS T6

  A IWAI, H ITO, T MIZUNO, H MORI, H MATSUI, M HONMA, G OKADA, S CHIBA  JOURNAL OF BACTERIOLOGY  176-  (24)  7730  -7734  1994/12  [Not refereed][Not invited]
   

  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.
• テンサイα‐Glucosidaseの活性中心のアミノ酸配列

  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  1994/03  [Not refereed][Not invited]
  
• Schizosaccharomyces pombe菌体外分泌α‐Glucosidaseの精製と諸性質

  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  1994/03  [Not refereed][Not invited]
  
• Arthrobacter globiformis T6のIsomalto‐dextranase: 大腸菌による発現酵素の性質

  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  1994/03  [Not refereed][Not invited]
  
• トラマメ(Phaseolus vulgaris L)緑葉に発現するα‐amylase

  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  1994/03  [Not refereed][Not invited]
  
• トラマメ(Phaseolus vulgaris L.)α‐amylase遺伝子の発現について

  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  1994/03  [Not refereed][Not invited]
  
• Penicillium purpurogenum由来endo‐inulinaseの遺伝子構造

  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  1994/03  [Not refereed][Not invited]
  
• Aspergillus nigerの生産するα‐galactosidaseに関する研究

  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  [Not refereed][Not invited]
  
• STARCH-HYDROLYZING ENZYMES IN GERMINATING KIDNEY BEAN

  H MORI, A TATEMATSU, H MATSUI, T TAKAYANAGI, M HONMA, S CHIBA  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  56-  (9)  1499  -1500  1992/09  [Not refereed][Not invited]
  

Association Memberships

• Japan Society of Bioscience, Biotechnology, and Agrochemistry   Japanese Society of Applied Glycoscience   

Research Projects

• Diversification of carbohydrate phosphorylases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/06 -2022/03 

  Author : MORI Haruhide

   

  To develop the enzymatic production of carbohydrates, phosphorylases were investigated in this study. A new activity, solabiose phosphorylase, was found. A possible metabolism involving the enzyme and a practical enzymatic synthesis of solabiose using this enzyme were shown. Maltoside phosphorylase, as a member of starch-hydrolyzing enzyme family, was analysed intensively. Kinetic analysis revealed that the reaction followed the two-steps reaction, and phosphorolysis, transglycosylation, and hydrolysis occurred through competitive binding of the second substrates on the glycosyl enzyme intermediate. Significant change of the proportion of the three activities caused by mutations in possible binding residues suggested appropriate residues for the activities. Using the enzyme activity, insoluble α-glucans were produced though extension of their branched chains.
• 糖質の多様化高機能化に向けた酵素法アプローチの新展開

  文部科学省：科学研究費補助金(基盤研究(B))

  Date (from‐to) : 2018/04 -2021/03 

  Author : 森 春英
• Survey on members of sucrose-world in Asian tropical reef, namely in Thailand

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2009 -2012 

  Author : KIMURA Atsuo, MORI Haruhide

   

  Plants synthesize the starch in the leaves by photosynthesis. Leaf-starch is converted to sucrose, and transported to storage organ (e.g., tubers and fruit-bodies), followed by re-formation of starch (storage-starch). From many sites of bodies, plants secret sucrose, by which plants perform the cross-talk with environmental organisms (e.g., microorganisms and insects). Recently, it was found that huge amount of sucrose was secreted (this system is call as sucrose-world). The purpose of this research is survey of sucrose-world-members (environmental organisms and their enzymes) in Thailand, where high sunshine is available. As a result, the system of "sucrose -> polysaccharide -> secondary or ternary sugar" was investigated at sucrose secreted from roots. About flower bee, the enzyme converting sucrose to oligosaccharide was also investigated.
• 1残基のアミノ酸置換で「糖質分解酵素を多糖合成酵素に変換」する現象の分子機構

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2008 -2009 

  Author : 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。
• Promotion of reverse reaction of carbohydrolase and its applicationfor oligosaccharide synthesis

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2007 -2008 

  Author : Aruhide MORI

   

  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.
• Molecular Analysis of Carbohydrases Showing Different Reaction by Novel Structure and Its Application

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2005 -2007 

  Author : KIMURA Atsuo, MORI Haruhide, OKUYAMA Masayuki

   

  This project is about the enzymes having the structure similar to α-glucosidase; i.e. α-xylosidase, glucan lyase, cyclic-tetrasaccharide-forming enzyme, and α-glucosidase, each of which catalyzes the different reaction. Three-dimensional structure available recently allows us to analyze the molecular mechanism of reactions exhibited by four enzymes. The purposes of research are 1) to elucidate the relationship between substrate and amino acid residue(s) in the catalytic site; 2) to analyze the function of catalytic residues; 3) to elucidate the structural element(s) to display the above-described different reactions; 4) to synthesize the useful enzyme. Results are as follows. (1) We analyzed the amino acid residues in the catalytic site of α-xylosidase to recognize α-xyloside-structure, and succeeded in conversion of α-xylosidase into α-glucosidase by the mutagenesis of its structural elements. (2) The catalytic residues were identified and their functions were investigated. (3) Amino acid replacement of α-glucosidase (a hydrolyzing enzyme) lost its hydrolytic activity and enhanced the transglucosidation ability, meaning the conversion of hydrolyzing enzyme into transferring enzyme. (4) We have succeed in change of transferring products.
• Synthesis of a-linked oligosaccharides by engineered enzymes

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(C))

  Date (from‐to) : 2005 -2006 

  Author : Haruhide MORI

   

  The aim of this project was to establish new methods to synthesize alpha-linked oligosaccharides using engineered carbohydrate-hydrolysing enzymes. I focused the following two points : 1. establishment the new reaction suitable for oligosaccharide synthesis using catalytic residue-mutated "inactive" hydrolases. 2. changing specificities of the enzyme and screening enzymes showing novel specificities. In the point-1, a new basic method effective for oligosaccharide synthesis was developed, and through the point-2, wide varieties of oligosaccharide would be prepared.Point-1, catalytic residue...
• 触媒アミノ酸を置換した酵素が、発揮する新たな機能とその応用

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2004 -2005 

  Author : 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)。
• Investigation on Molecular Mechanism of Sucrose-degrading Enzyme in Asian Honeybee.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2004 -2005 

  Author : KIMURA Atsuo, MORI Haruhide, OKUYAMA Masayuki

   

  We have analyzed three sucrose-degrading isozymes (α-glucosidase I,II, and III) in European honeybees, since these enzymes exhibited the interesting activity to sucrose of high concentration. α-Glucosidase I was a unique allosteric enzyme to display negative cooperativity in extremely high sucrose concentration. α-Glucosidase II also exhibited the allosteric property of positive cooperativity. α-Glucosidase III was a Michaelis-Menten-type enzyme to be secreted to nectar (bees gathered from flower) and to contribute to the formation of honey. Purpose of this project is i)existence of allosteric α-glucosidase in Asian honeybees, ii)investigation of honey-forming mechanism, and iii)regulation in expression of three α-glucosidase isozymes. Four kinds of honeybee species were investigated in this research. From typical Asian honeybees (living in Thailand, Korea and Japan), three isozymes were isolated by chromatographic method. One of them was an allosteric enzyme having similar properties to European honeybee α-glucosidase I, allowing to molecular analysis of negative cooperativity. The second enzyme was α-glucosidase II-type isozyme. Enzyme in honey was also investigated, and showed similar to one of enzymes (α-glucosidase III-type isozyme). cDNAs of these three enzyme were cloned. It was found that the expression of three isozymes was regulated. In small honeybees living in Thailand, three α-glucosidases were also found, in which two α-glucosidases were isolated. Their properties investigated were almost identical to those of α-glucosidases I and II. Enzyme in honey was compared with adult bee enzymes. All honeybee species studied in this project contained allosteric enzyme. Asian honeybees were divided into two types : i)having three α-glucosidase isozymes and ii)having two α-glucosidase isozymes. Recently, we found that bumblebees also had two α-glucosidase isozymes. Further research will elucidate the interesting molecular mechanism of sucrose-degrading enzymes in flower bees.
• Research on Glycosylases which Obtained New Functions by Mutation on Catalytic Residue.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2002 -2004 

  Author : KIMURA Atsuo, MORI Haruhide, OKUYAMA Masayuki

   

  Glycosylases are enzymes that hydrolyze the glycosidic linkage. These enzymes also catalyze the transglycosidation, in which the glycosyl residue is transferred to the acceptor substrate. The transglycosidation is an important reaction i)to produce oligosaccharides valuable for foods and ii)to synthesize bio-active sugar-chains. Transglycosidation and hydrolysis proceed in the same time, meaning that the substrate for transglycosidation as well as its product(s) is cleaved by hydrolysis even under conditions of transglycosidation. We have studied the reactions of glycosylase, and have found the phenomena that catalyzed the transglycosidation only. In this study, we analyze the mechanism of valuable phenomena and perform their application. The results are summarized as follows. 1)We have determined the catalytic residue of negatively charged by the method using suicide substrate. Mutant enzyme (synthase), of which catalytic residue was replaced, was produce and purified. The enzyme showed no hydrolytic reaction, only catalyzed the synthesis of oligosaccharide(s) from fluoride-substrate and acceptor. Acceptor of aryl glycoside is a good substrate, meaning that the hydrophobic interaction between aryl-group and subsite +2 is important. 2)Mutant enzyme, which recognized the plane-shaped substrate, a mimic compound of reaction intermediate, was constructed, and its ability of oligosaccharide-synthesis was studied. The low production was observed. We changed the substrate concentration, and succeeded in the improvement of yield. Addition of alcohol to reaction mixture was also effective, but the high concentration of alcohol decreased the production of oligosaccharide. We have found a glycosidase resistant for alcohol. Currently, the conversion of alcohol-stable enzyme to mutant enzyme of same type is trying.
• Molecular Mechanism and Molecular Evolution of Glycosidases

  Ministry of Education, Culture, Sports, Science and Technology：Grants-in-Aid for Scientific Research(基盤研究(B))

  Date (from‐to) : 2000 -2002 

  Author : 千葉 誠哉, Haruhide MORI, 森 春英, 木村 淳夫, 福士 幸治

   

  α-Glucosidase (EC 3.2.1.20), an exo-type glycosidase releasing α-glucose from substrate, is divided into two types of groups (family I and family II) that have different structures and substrate recognition. There are also the glycosidases, of which structures are homologous to each of family enzyme, such as α-amylase for family I and α-xylosidase for family II. The purpose of the project is to investigate the relationship of structure and function of two α-glucosidase families, and to understand the molecular mechanism of glycosidases. 1)The catalytic amino acid residues of two family enzy...
• 活性中心の改変による新しいグリコシラーゼの作製

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2001 -2001 

  Author : 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。
• Application Studies on Product-Immobilization Phenomenon Newly Discovered

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -2000 

  Author : KIMURA Atsuo, MORI Haruhide, CHIBA Seiya

   

  Recently we have found the "immobilization of product" in the enzyme-digestion of starch granule, in which the product precipitates with granule. The phenomenon is of importance in i) the easy recovery of product under the less energy and ii) the inclusion of effective compounds into the polysaccharide. In this project, the fundamental study was done to learn the phenomenon, and the application of unutilized or low-utilized polysaccharides to the food industry was tried to be established. The results obtained were as follows. (1) Cellulose. i) The product-immobilization was also found in the degradation of cellulose by cellulase. ii) Products in the cellulose of precipitate were water-soluble oligosaccharides of short-chain. iii) The inside product increased by addition of ethanol to reaction mixture. Saccharides in the cellulose was easily recovered under the low concentration of ethanol. iv) Production of oligosaccharides was done using ethanol. After enzyme reaction the supernatant was discarded, and then water was added to the precipitate, releasing oligosaccharides of different size. Isolation was done by gel-filtration and HPLC.(2) Chitin and xylan. i) In the enzymatic degradation, the product-immobilization was also found. However, the included amount was small as compared with starch granule and cellulose. ii) The addition of ethanol increased products in the two polysaccharides. (3) Porous starch granule as inclusion material. i) Porous starch granules of various plant origins were prepared by enzyme treatment. There were two types of starches which formed pores or no pore on the surface of granule. ii) The pore size could be controlled by amount and treatment time of enzyme. iii) The smallporous granule was able to incorporate the short-chain oligosaccharides, and the long-chain one entered the starch of large pore. iv) Ascorbic acid was also incorporated into the porous starch granule. After the granule including ascorbic acid was transferred into water, the compound gradually released into water.
• Molecular Mechanism of Novel N-Linked Sugar Chain Having α-Galactofuranosyl Structure

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -1999 

  Author : KIMURA Atsuo, MORI Haruhide, CHIBA Seiya

   

  In microorganisms there are the N- and O-linked sugar chains which are not found in animals and plants. Their biological function and synthesis have not been elucidated. In this project the structural determination of novel O-linked sugar chains was done, and the enzymes concerned with the formation of α-galactofuranosyl structure (Galf) in N-linked oligosaccharide, UDP-Galf synthetase and Galf transferase, were analyzed. (1) Five kinds of O-linked sugar chains, which were separated chemically from Aspergillus niger α-glu-cosidase, were purified, and the following structures were determined by monosaccharide analysis, exo-glycosidase treatment, and MS, 1D- and 2D-NMR: I) mannose, ii) mannobiose having α-1,2-linkage, iii) glucosylmannobiose of branched type, iv) two mannotrioses of branched (iv-a) and linear (iv-b) structures. Sugar chains of iii and iv-b were novel ones. (2) The substrate and product for Galf transferase were prepared from A .niger α-gluco-sidase. Since the activities of UDP-Galf synthetase and Galf transferase in cell extract of A .niger were low, the cultivation conditions were examined. The addition of maltose or starch to culture broth increased the both enzyme activities with induction of amylases (secretory proteins). When disruption of cells, the loss of activities was observed. It was found that the detergent stabilized the Galf transferase. The transferase preparation of high purity, which did not give a single band in electophoretic analysis, was obtained after several chromatographies. The purification is now doing to figure out the amino acid sequence and to separate the enzyme gene.
• Joint Studies on Dextran-Producing and -Degrading Enzymes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -1999 

  Author : CHIBA Seiya, MORI Haruhide, KIMURA Atsuo

   

  Dextransucrase, one of glucansucrases, catalyzes the formation of dextran from sucrose. Recently Dr. Kim has established a novel mutation technique, Vacuum UV radiation. The mutant strains obtained hyper-produced dextransucrase constitutively, giving a possibility to produce the large amount of dextran. Dextran and its oligosaccharides have been found to have several useful functions to human. In the international project we tried to elucidate the relationship between structures and functions of dextran-producing and -degrading enzymes, and prepared the dextran and isomaltooligosaccharides by both enzymes. (1) We succeeded in isolation of a hyper-produced dextransucrase gene and its expression in Escherichia coli. The mutated position was found in the promoter region. (2) A gene of isomaltotrio-dextranase was cloned and expressed in E. coli. The α-glucosidase gene was found in the upstream region of this gene. Both genes made a cluster structure, which was controlled by one promoter. (3) The large amount of dextran was prepared by hyper-produced dextransucrase. We examined the effective production method of pure isomaltotriose from dextran using isomaltotrio-dextranase. (4) An enzyme giving tetra- and penta-saccharides from dextran was purified and the properties were investigated. (5) Mechanism-based inactivator for dextranase was designed. Kinetic studies on inactivation indicated that compounds synthesized were found to be novel suicide substrate for dextranase.
• Structures and Substrate Recognitions of Animal, Botanical and Microbial α-Glucosidases and Their Molecular Evolution

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1997 -1999 

  Author : CHIBA Seiya, MORI Haruhide, KIMURA Atsuo

   

  α-Glucosidase (EC 3.2.1.20) is a typical exo-type glycosidase that releases the α-glucose from non-reducing side of substrate. We are interested in the relationship between catalytic action and the structure, since the substrate specificity differs greatly with the source of enzyme. There are at least two types of α-glucosidases which show different substrate recognitions, suggesting that enzymes can be classified into two groups (family I and family II). However, the structural information is not enough to learn that structures of enzymes belonging to each group are homologous or not. In this project, we analyzed the primary structures of α-glucosidases from animal, botanical and microbial origins, and found that the enzymes of family I and II had different primary amino acid sequences. α-Glucosidases from insect and bacteria belonged to family I, of which molecular weight was about 70 kDa. Four catalytic regions found were similar to those of α-amylase. The activity toward heteroside (sucrose or p-nitrophenyl α-glucoside) was higher than holoside (maltooligosaccharides). Members of family II were from animal, botanical and mold origins, and showed the opposite substrate specificity, high activity to holoside and low to heteroside. The molecular sizes were about 100 kDa. We analyzed the primary structures of six kinds of α-glucosidases belonging to this group. The sequences obtained were homologous each other, but no similarity was observed with family I enzymes. The findings suggest that the α-glucosidase was evolved from two different ancestral proteins.
• 双子葉植物α-amylaseの細胞内局在性とそれに関与する因子の同定

  文部科学省：科学研究費補助金(奨励研究(A))

  Date (from‐to) : 1997 -1998 

  Author : 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...
• 植物の栄養ストレス下における糖代謝およびストレス関連酵素

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1995 -1995 

  Author : 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。
• New Technique for High Yield Production of Glucose by Using of Mutarotase

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1993 -1995 

  Author : CHIBA Seiya, MORI Haruhide, ITO Hiroyuki, KIMURA Atsuo

   

  (1) We kinetically analyzed the glucoamylase-catalyzed condensation of beta-glucose (two substrates reaction), and obtained the rate parameters and reaction rate equation which makes an accurate estimate of condensation product formation in any beta-glucose concentration. Addition of alpha-glucose to beta-glucose reaction system activated the rate of condensation. We analyzed this activation, and found that the subsite 1 of glucoamylase did not bind to alpha-glucose, meaning no inhibition to condensation, and that alpha-glucose had higher affinity to the subsite 2 than beta-glucose. We changed the composition of alpha-and beta-glucose with keeping the total glucose concentration constant, and measured the reaction rate. The velocity was reduced with decreasig in the molar fraction of beta-glucose, suggesting that the decrease of substrate (beta-glucose) is more effective than the activation by alpha-glucose and that the mutarotase suppresses the formation of condensation products. (2) It was found that the mutarotase reduced the amount of disaccharides in 30% which was the products from 30% beta-glucose by glucoamylase. We investigated the effect of mutarotase on glucose production by two methods. The first was the butch-typed method which is presently used in the industrial glucose production system. The second was the immobilized enzyme technique where glucoamylase was linked to matrix, and then reaction was done in the column by running of maltodextrin with mutarotase. In both tests mutarotase gave the effective results, the increase of glucose production and the decrease of condensation products. (3) We analyzed the amino acid sequence of porcine kidney mutarotase for developing the extensive project in the cloning of its gene and over-production of enzyme. The N-terminus of mutarotase was found to be blocked. We purified the N-terminal-blocked peptide fragment which was prepared by protease digestion, and determined its amino acid sequence by tandem mass spectrometry, elucidating that the acetyl group blocked the N-terminus of mutarotase.
• 植物の栄養ストレス下における糖代謝酵素の生化学・分子生物学的解析

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1994 -1994 

  Author : 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。

Industrial Property Rights

• 特開2019-033702:エピメリ化活性を有するタンパク質  2019/03/07

  佐分利亘, 森春英, 飯塚貴久, 藤本佳則, 高木宏基
• 特許6417061:α－１，６－グルコシル転移活性を有する酵素    2018/10/31

  森春英, 佐分利亘, 金井研太, 相沢健太, 飯塚貴久, 竹地紀昭, 谷美生夏
• 特開2018-134058:マンノオリゴ糖合成酵素およびこれを用いたマンノオリゴ糖の製造法  2018/08/30

  森春英, 佐分利亘, 伊吹昌久, 津村和伸, 吉田靖彦