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

Profile and Settings

• Name (Japanese)

  Wataru

• Name (Kana)

  Saburi

• Name

  201201038539083300

Achievement

Research Interests

• Protein engineering   Applied microbiology   Carbohydrate synthesis   Enzyme chemistry   Applied biochemistry   

Research Areas

• Life sciences / Applied biochemistry

Research Experience

• 2020/07 - Today Hokkaido University Research Faculty of Agriculture Associate Professor
• 2019/04 - 2020/06 Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
• 2010/10 - 2019/03 Hokkaido University Graduate School of Agriculture Research Faculty of Agriculture
• 2006/04 - 2010/09 日本食品化工株式会社 研究所 研究員

Education

• 2001/04 - 2006/03  Hokkaido University
• 1997/04 - 2001/03  Hokkaido University  Faculty of Agriculture  Department of Applied Bioscience

Committee Memberships

• 2021/03 - Today   Japan Society for Bioscience, Biotechnology, and Agrochemistry   Editor of Bioscience, Biotechnology, and Biochemistry

Awards

• 2020/09 日本応用糖質科学会 奨励賞
   

  受賞者: 佐分利 亘
• 2018/09 日本応用糖質科学会 ポスター賞
   

  受賞者: 佐分利 亘
• 2017/09 日本応用糖質科学会 ポスター賞
   

  受賞者: 佐分利 亘
• 2017/04 Congress on Gastrointestinal Function James Russel Award,
   

  受賞者: Saburi Wataru
• 2016/09 日本応用糖質科学会 ポスター賞
   

  受賞者: 佐分利 亘
• 2015/03 日本農芸化学会 農芸化学奨励賞
   

  受賞者: 佐分利 亘
• 2014/10 植物化学調節学会 ポスター賞
   

  受賞者: 佐分利 亘
• 2013/06 酵素応用シンポジウム 研究奨励賞
   

  受賞者: 佐分利 亘

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
• 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.
• Extraction and antioxidant capacity of mycosporine-like amino acids from red algae in Japan.

  Ryuya Yamamoto, Shigeru Toriumi, Chikara Kawagoe, Wataru Saburi, Hideki Kishimura, Yuya Kumagai

  Bioscience, biotechnology, and biochemistry 2024/04/29 [Refereed]
   

  Mycosporine-like amino acids (MAAs) are the natural UV absorbing compounds with antioxidant activity found in microalgae and macroalgae. We collected red algae Asparagopsis taxiformis, Meristotheca japonica, and Polysiphonia senticulosa from Nagasaki, where UV radiation is more intense than in Hokkaido, and investigated the effect of UV radiation on MAAs content. It was suggested that A. taxiformis and M. japonica contained shinorine and palythine, while UV absorbing compound in P. senticulosa could not be identified. The amounts of these MAAs were lower compared to those from Hokkaido. Despite an increase in UV radiation in both region from February to April, MAAs contents of red algae from Nagasaki slightly decreased, while that from Hokkaido significantly decreased. This difference was suggested the amount of inorganic nitrogen in the ocean. Antioxidant activity of MAAs increased under alkaline conditions. The extract containing MAAs from P. senticulosa showed the highest antioxidant activity among four red algae.
• 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 299 (11) 105294 - 105294 2023/09/27 [Refereed]
   

  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]
   

  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]
   

  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]
   

  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]
   

  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]
   

  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 

  β-Galactosidase (EC 3.2.1.23) hydrolyzes β-D-galactosidic linkages at the non-reducing end of substrates to produce β-D-galactose. Lacticaseibacillus casei is one of the most widely utilized probiotic species of lactobacilli. It possesses a putative β-galactosidase belonging to glycoside hydrolase family 35 (GH35). This enzyme is encoded by the gene included in the gene cluster for utilization of lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc) via the phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. casei BL23 is predicted to be 6-phospho-β-galactosidase (EC 3.2.1.85). However, its 6-phospho-β-galactosidase activity has not yet been examined, whereas its hydrolytic activity against LNB and GNB has been demonstrated. In this study, L. casei JCM1134 LBCZ_0230, homologous to GnbG, was characterized enzymatically and structurally. A recombinant LBCZ_0230, produced in Escherichia coli, exhibited high hydrolytic activity toward o-nitrophenyl β-D-galactopyranoside, p-nitrophenyl β-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophenyl 6-phospho-β-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite -1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniae β-galactosidase BgaC and not suitable for binding to 6-phospho-β-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a β-galactosidase. According to the prediction of LNB's binding mode, aromatic residues, Trp190, Trp240, Trp243, Phe244, and Tyr458, form hydrophobic interactions with N-acetyl-D-glucosamine residue of LNB at subsite +1.
• 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) 2022/12 [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.
• Functional characterization of a novel GH94 glycoside phosphorylase, 3-O-β-d-glucopyranosyl β-d-glucuronide phosphorylase, and implication of the metabolic pathway of acidic carbohydrates in Paenibacillus borealis.

  Naoto Isono, Emi Mizutani, Haruka Hayashida, Hirotaka Katsuzaki, Wataru Saburi

  Biochemical and biophysical research communications 625 60 - 65 2022/10/15 [Refereed]
   

  Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of β-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl β-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-β-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl β-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-β-d-glucopyranosyl β-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be β-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-β-d-glucopyranosyl β-d-glucuronide structure released from bacterial and plant acidic carbohydrates.
• Characterization of Antioxidant Activity of Heated Mycosporine-like Amino Acids from Red Alga Dulse Palmaria palmata in Japan.

  Yuki Nishida, Wataru Saburi, Yoshikatsu Miyabe, Hideki Kishimura, Yuya Kumagai

  Marine drugs 20 (3) 2022/03/01 [Refereed]
   

  We recently demonstrated the monthly variation and antioxidant activity of mycosporine-like amino acids (MAAs) from red alga dulse in Japan. The antioxidant activity of MAAs in acidic conditions was low compared to that in neutral and alkali conditions, but we found strong antioxidant activity from the heated crude MAA fraction in acidic conditions. In this study, we identified and characterized the key compounds involved in the antioxidant activity of this fraction. We first isolated two MAAs, palythine, and porphyra-334, from the fraction and evaluated the activities of the two MAAs when heated. MAAs possess absorption maxima at around 330 nm, while the heated MAAs lost this absorption. The heated MAAs showed a high ABTS radical scavenging activity at pH 5.8-8.0. We then determined the structure of heated palythine via ESI-MS and NMR analyses and speculated about the putative antioxidant mechanism. Finally, a suitable production condition of the heated compounds was determined at 120 °C for 30 min at pH 8.0. We revealed compounds from red algae with antioxidant activities at a wide range of pH values, and this information will be useful for the functional processing of food.
• 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%.
• A Ubiquitously Expressed UDP-Glucosyltransferase, UGT74J1, Controls Basal Salicylic Acid Levels in Rice.

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

  Plants (Basel, Switzerland) 10 (9) 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.
• β-(1→4)-Mannobiose Acts as an Immunostimulatory Molecule in Murine Dendritic Cells by Binding the TLR4/MD-2 Complex.

  Ting-Yu Cheng, Yen-Ju Lin, Wataru Saburi, Stefan Vieths, Stephan Scheurer, Stefan Schülke, Masako Toda

  Cells 10 (7) 2021/07/14 [Refereed]
   

  Some β-mannans, including those in coffee bean and soy, contain a mannose backbone with β-(1→4) bonds. Such mannooligosaccharides could have immunological functions involving direct interaction with immune cells, in addition to acting as prebiotics. This study aimed at assessing the immunological function of mannooligosaccharides with β-(1→4) bond, and elucidating their mechanism of action using bone marrow-derived murine dendritic cells (BMDCs). When BMDCs were stimulated with the mannooligosaccharides, only β-Man-(1→4)-Man significantly induced production of cytokines that included IL-6, IL-10, TNF-α, and IFN-β, and enhanced CD4+ T-cell stimulatory capacity. Use of putative receptor inhibitors revealed the binding of β-Man-(1→4)-Man to TLR4/MD2 complex and involvement with the complement C3a receptor (C3aR) for BMDC activation. Interestingly, β-Man-(1→4)-Man prolonged the production of pro-inflammatory cytokines (IL-6 and TNF-α), but not of the IL-10 anti-inflammatory cytokine during extended culture of BMDCs, associated with high glucose consumption. The results suggest that β-Man-(1→4)-Man is an immunostimulatory molecule, and that the promotion of glycolysis could be involved in the production of pro-inflammatory cytokine in β-Man-(1→4)-Man-stimulated BMDCs. This study could contribute to development of immune-boosting functional foods and a novel vaccine adjuvant.
• A model system for studying plant-microbe interactions under snow.

  Chikako Kuwabara, Kentaro Sasaki, Natsuki Umeki, Tamotsu Hoshino, Wataru Saburi, Hirokazu Matsui, Ryozo Imai

  Plant physiology 185 (4) 1489 - 1494 2021/02/02 [Refereed]
  
• 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 [Refereed]
  
• 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.
• Two binding proteins of the ABC transporter that confers growth of Bifidobacterium animalis subsp. lactis ATCC27673 on β-mannan possess distinct manno-oligosaccharide-binding profiles.

  M Ejby, A Guskov, M J Pichler, G C Zanten, E Schoof, W Saburi, D J Slotboom, M Abou Hachem

  Molecular microbiology 112 (1) 114 - 130 2019/07 [Refereed]
   

  Human gut bifidobacteria rely on ATP-binding cassette (ABC) transporters for oligosaccharide uptake. Multiple oligosaccharide-specific solute-binding protein (SBP) genes are occasionally associated with a single ABC transporter, but the significance of this multiplicity remains unclear. Here, we characterize BlMnBP1 and BlMnBP2, the two SBPs associated to the β-manno-oligosaccharide (MnOS) ABC transporter in Bifidobacterium animalis subsp. lactis. Despite similar overall specificity and preference to mannotriose (Kd ≈80 nM), affinity of BlMnBP1 is up to 2570-fold higher for disaccharides than BlMnBP2. Structural analysis revealed a substitution of an asparagine that recognizes the mannosyl at position 2 in BlMnBP1, by a glycine in BlMnBP2, which affects substrate affinity. Both substitution types occur in bifidobacterial SBPs, but BlMnBP1-like variants prevail in human gut isolates. B. animalis subsp. lactis ATCC27673 showed growth on gluco and galactomannans and was able to outcompete a mannan-degrading Bacteroides ovatus strain in co-cultures, attesting the efficiency of this ABC uptake system. By contrast, a strain that lacks this transporter failed to grow on mannan. This study highlights SBP diversification as a possible strategy to modulate oligosaccharide uptake preferences of bifidobacterial ABC-transporters during adaptation to specific ecological niches. Efficient metabolism of galactomannan by distinct bifidobacteria, merits evaluating this plant glycan as a potential prebiotic.
• 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 83 2097 - 2109 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]
  
• Enzymatic production of xylooligosaccharides from red alga dulse (Palmaria sp.) wasted in Japan {IF:2.616]

  Y.Yamamoto, H.Kishimura, Y.Kinoshita, W.Saburi, Y.Kumagai, H.Yasui, T.Ojima

  Process Biochemistry 2019/04 [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 physiology and biochemistry : PPB 135 263 - 271 0981-9428 2019/02 [Refereed][Not invited]
  
• Functional modulation of caecal fermentation and microbiota in rat by feeding bean husk as a dietary fibre supplement.

  Myint H, Kishi H, Iwahashi Y, Saburi W, Koike S, Kobayashi Y

  Beneficial microbes 9 (6) 1 - 12 1876-2883 2018/09 [Refereed][Not invited]
   

  A feeding study using rats was conducted to evaluate the utility of lablab bean husk and soya bean husk as sources of potential prebiotic fibre. Twenty 5-week-old Sprague Dawley rats were divided into 4 groups and fed one of the following diets for 3 weeks: purified diet (AIN93 G) containing 5% cellulose (CEL), or the same diet in which cellulose was replaced by corn starch (STA), lablab bean husk (LBH), or soya bean husk (SBH). Rats were sacrificed at 8 weeks of age and caecal digesta were collected. Feed intake, body weight, anatomical parameters, and caecal ammonia level did not differ significantly among diets. Rats on LBH and SBH showed higher concentrations of caecal short-chain fatty acid and lactate than those on CEL. Rats on CEL, SBH, and LBH exhibited lower caecal indole and skatole levels. LBH yielded increased caecal abundance of Akkermansia muciniphila and Oscillibacter relatives, as demonstrated by either qPCR, MiSeq, or clone library analysis. SBH favoured the growth of lactobacilli as assessed by both qPCR and MiSeq, and favoured the growth of bifidobacteria as assessed by MiSeq. In comparison with STA, LBH and SBH yielded lower caecal abundance of bacteria related to Dorea massiliensis, as demonstrated by qPCR, MiSeq, and clone library analysis. Both types of bean husk were found to contain oligosaccharides that might selectively stimulate the growth of beneficial bacteria. Based on these results, the two species of bean husk tested are considered potentially functional for promoting the gut health of monogastric animals.
• 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) 0099-2240 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 letters 592 (13) 2268 - 2281 0014-5793 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.
• 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.
• 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.
• Functions, structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases

  Wataru Saburi

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 (7) 1294 - 1305 0916-8451 2016/07 [Refereed][Not invited]
   

  Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of -(14)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (/)(6)-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. -Mannoside hydrolase and 4-O--d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in -mannan metabolism, where it epimerizes -d-mannopyranosyl-(14)-d-mannose to -d-mannopyranosyl-(14)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other -mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.
• 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.
• The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat

  Kentaro Sasaki, Chikako Kuwabara, Natsuki Umeki, Mari Fujioka, Wataru Saburi, Hirokazu Matsui, Fumitaka Abe, Ryozo Imai

  JOURNAL OF BIOTECHNOLOGY 228 3 - 7 0168-1656 2016/06 [Refereed][Not invited]
   

  TAD1 (Triticum aestivum defensin 1) is induced during cold acclimation in winter wheat and encodes a plant defensin with antimicrobial activity. In this study, we demonstrated that recombinant TAD1 protein inhibits hyphal growth of the snow mold fungus, Typhula ishikariensis in vitro. Transgenic wheat plants overexpressing TAD1 were created and tested for resistance against T. ishikariensis. Leaf inoculation assays revealed that overexpression of TAD1 confers resistance against the snow mold. In addition, the TAD1-overexpressors showed resistance against Fusarium graminearum, which causes Fusarium head blight, a devastating disease in wheat and barley. These results indicate that TAD1 is a candidate gene to improve resistance against multiple fungal diseases in cereal crops. (C) 2016 Elsevier B.V. All rights reserved.
• 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 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.
• 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.
• 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 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.
• 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 α-glucosidase from Bacillus sp. AHU 2001 with broad substrate specificity

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

  Biochimie Elsevier 108 (108) 140 - 148 6183-1638 2015 [Refereed][Not invited]
   

  α-Glucosidases are ubiquitous enzymes that hydrolyze the α-glucosidic linkage at the non-reducing end of substrates. In this study, we characterized an α-glucosidase (BspAG31A) belonging to glycoside hydrolase family 31 from Bacillus sp. AHU 2001. Recombinant BspAG31A, produced in Escherichia coli, had high hydrolytic activity toward maltooligosaccharides, kojibiose, nigerose, and neotrehalose. This is the first report of an α-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 β → α loop 1 of BspAG31A, 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 BspAG31A showed that Tyr173, situated on the N-terminal domain loop, is associated with the formation of subsite +2. In Y173A, the kcat/Km for maltooligosaccharides slightly decreased with an increasing degree of polymerization compared with wild type. Among the amino acid residues surrounding the substrate binding site, Val543 and Glu545 of BspAG31A were different from the corresponding residues of Bacillus thermoamyloliquefaciens α-glucosidase II, which has higher activity toward isomaltose than BspAG31A. 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.
• 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 alpha-glucosidase HaG from Halomonas sp strain H11

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

  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 70 (Pt 4) 464 - 466 1744-3091 2014/04 [Refereed][Not invited]
   

  The alpha-glucosidase HaG from the halophilic bacterium Halomonas sp. strain H11 catalyzes the hydrolysis of the glucosidic linkage at the nonreducing end of alpha-glucosides, such as maltose and sucrose, to release alpha-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 angstrom resolution was collected and processed. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.2, b = 119.2, c = 177.2 angstrom. 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 beta-1,4-Linked Oligosaccharides Catalyzed by Cellobiose 2-Epimerase, the Sole Enzyme Epimerizing Non-anomeric Hydroxyl Groups of Unmodified Sugars

  Takaaki Fujiwara, Wataru Saburi, Hirokazu Matsui, Haruhide Mori, Min Yao

  JOURNAL OF BIOLOGICAL CHEMISTRY 289 (6) 3405 - 3415 0021-9258 2014/02 [Refereed][Not invited]
   

  Background: The details of the catalytic mechanism of cellobiose 2-epimerase (CE) remains unclear. Results: The crystal structures of Rhodothermus marinus CE in the apo form and complexes with its substrates/products 4-O--d-glucopyranosyl-d-mannnose, epilactose, or cellobiitol (reaction intermediate analog) were elucidated. Conclusion: Epimerization catalyzed by CE proceeds through ring opening, deprotonation/reprotonation, carbon-carbon bond rotation, and ring closure. Significance: This study yielded structural insights into epimerization catalyzed by CE. Cellobiose 2-epimerase (CE) reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified 1,4-linked oligosaccharides, including -1,4-mannobiose, cellobiose, and lactose. CE is responsible for conversion of 1,4-mannobiose to 4-O--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 5-6 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.
• Characterization of a thermophilic 4-O-beta-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 78 (2) 263 - 270 0916-8451 2014/02 [Refereed][Not invited]
   

  4-O-beta-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-beta-D-mannosylD- glucose (Man-Glc) to alpha-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts beta-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-beta-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 degrees C, and it was stable between pH 5.5-8.3 and below 80 degrees 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 beta-D-glucoside and 1,5-anhydro- D-glucitol as acceptor substrates.
• 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

  Tatsuya Sawano, Wataru Saburi, Ken Hamura, Hirokazu Matsui, Haruhide Mori

  FEBS Journal 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. R. albus cellodextrin phosphorylase (RaCDP), classified into glycoside hydrolase family 94, was characterized. Among the cello-oligosaccharides tested, RaCDP had the highest phosphorolytic and synthetic activities towards cellohexaose and cellopentaose, respectively. Sophorose, laminaribiose, β-1,4-xylobiose, β-1,4-mannobiose, and cellobiitol served as acceptors. Site-directed mutational study revealed that Tyr633 is responsible for low affinity to the phosphate group and synthetic activity towards β-1,4-mannobiose. © 2013 FEBS.
• Modulation of Allosteric Regulation by E38K and G101N Mutations in the Potato Tuber ADP-glucose Pyrophosphorylase

  Shinji Wakuta, Yumi Shibata, Yumiko Yoshizaki, Wataru Saburi, Shigeki Hamada, Hiroyuki Ito, Seon-Kap Hwang, Thomas W. Okita, Hirokazu Matsui

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (9) 1854 - 1859 0916-8451 2013/09 [Refereed][Not invited]
   

  The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from alpha-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.
• A Thermophilic Alkalophilic alpha-Amylase from Bacillus sp AAH-31 Shows a Novel Domain Organization among Glycoside Hydrolase Family 13 Enzymes

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

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (9) 1867 - 1873 0916-8451 2013/09 [Refereed][Not invited]
   

  alpha-Amylases (EC 3.2.1.1) hydrolyze internal alpha-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic alpha-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial alpha-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 alpha-amylases. In contrast to known neopullulanase-like alpha-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 degrees C and from pH 6.4 to 11.9. The k(cat) values for soluble starch, gamma-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.
• Identification of Rice beta-Glucosidase with High Hydrolytic Activity towards Salicylic Acid beta-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 77 (5) 934 - 939 0916-8451 2013/05 [Refereed][Not invited]
   

  beta-Glucosidases (EC 3.2.1.21) split beta-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release beta-D-glucose. One of the important functions of plant beta-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonate-related compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid beta-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 1 L of culture medium, 2.1 mg 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 degrees C. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4-9.8 and below 55 degrees C. Native OsTAGG2 and rOsTAGG2P showed 4.5-4.7-fold higher activities towards salicylic acid beta-D-glucoside, an inactive storage-form of salicylic acid, than towards tuberonic acid beta-D-glucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity.
• Crystal structure of Ruminococcus albus cellobiose 2-epimerase: Structural insights into epimerization of unmodified sugar

  Takaaki Fujiwara, Wataru Saburi, Sota Inoue, Haruhide Mori, Hirokazu Matsui, Isao Tanaka, Min Yao

  FEBS LETTERS 587 (7) 840 - 846 0014-5793 2013/04 [Refereed][Not invited]
   

  Enzymatic epimerization is an important modification for carbohydrates to acquire diverse functions attributable to their stereoisomers. Cellobiose 2-epimerase (CE) catalyzes interconversion between D-glucose and D-mannose residues at the reducing end of beta-1,4-linked oligosaccharides. Here, we solved the structure of Ruminococcus albus CE (RaCE). The structure of RaCE showed strong similarity to those of N-acetyl-D-glucosamine 2-epimerase and aldose-ketose isomerase YihS with a high degree of conservation of residues around the catalytic center, although sequence identity between them is low. Based on structural comparison, we found that His184 is required for RaCE activity as the third histidine added to two essential histidines in other sugar epimerases/isomerases. This finding was confirmed by mutagenesis, suggesting a new catalytic mechanism for CE involving three histidines. Structured summary of protein interactions: RaCE and X-ray crystallography (View interaction) (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
• Identification and Characterization of Cellobiose 2-Epimerases from Various Aerobes

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

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (1) 189 - 193 0916-8451 2013/01 [Refereed][Not invited]
   

  Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of beta-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 beta-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.
• Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis

  Ken Hamura, Wataru Saburi, Hirokazu Matsui, Haruhide Mori

  Carbohydrate Research 379 21 - 25 1873-426X 2013 [Refereed][Not invited]
   

  Cellobiose phosphorylase (EC 2.4.1.20, CBP) catalyzes the reversible phosphorolysis of cellobiose to α-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 kcat/Km 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-β-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-β-D-glucopyranosyl-D-mannose and 4-O-β-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-β-D-glucopyranosyl-D-mannose and 4-O-β-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively. © 2013 Elsevier Ltd.
• COLD SHOCK DOMAIN PROTEIN 3 is involved in salt and drought stress tolerance in Arabidopsis

  Myung-Hee Kim, Shunya Sato, Kentaro Sasaki, Wataru Saburi, Hirokazu Matsui, Ryozo Imai

  FEBS OPEN BIO 3 438 - 442 2211-5463 2013 [Refereed][Not invited]
   

  Cold shock proteins (CSPs) of bacteria are produced in response to cold and function as RNA chaperones that are essential for cold adaptation. Arabidopsis thaliana COLD SHOCK DOMAIN PROTEIN 3 (AtCSP3) shares a domain with bacterial CSPs and is involved in acquisition of freezing tolerance. Our previous study revealed that many of the genes that are down regulated in an AtCSP3 knockout mutant (atcsp3-2) are functionally associated with responses to salt and drought as well as cold. Here, we examined the involvement of AtCSP3 in salt and drought stress tolerance. We found that AtCSP3 is induced during salt and drought stresses, and is regulated by ABA. Pi knockout mutant of AtCSP3 (atcsp3-2) showed lower survival rates after salt and drought stress treatments. Conversely, the AtCSP3-overexpressing plants displayed higher survival rates after treatment with these stresses. Most of the genes that were down regulated in the atcsp3-2 mutant were found to be inducible upon salt and drought stresses, and upregulated in the AtCSP3-overexpressors. Together, our data demonstrates that AtCSP3 is involved in the regulation of salt and drought stress tolerance in Arabidopsis. (C) 2013 The Authors. Published by Elsevier B.V. oil behalf of Federation of European Biochemical Societies. All rights reserved.
• Metabolic Mechanism of Mannan in a Ruminal Bacterium, Ruminococcus albus, Involving Two Mannoside Phosphorylases and Cellobiose 2-Epimerase DISCOVERY OF A NEW CARBOHYDRATE PHOSPHORYLASE, beta-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE

  Ryosuke Kawahara, Wataru Saburi, Rei Odaka, Hidenori Taguchi, Shigeaki Ito, Haruhide Mori, Hirokazu Matsui

  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 (CE; EC 5.1.3.11), which converts cellobiose to 4-O-beta-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, beta-1,4-mannobiose is epimerized to 4-O-beta-D-mannosyl-D-glucose (Man-Glc) by CE, and Man-Glc is phosphorolyzed to alpha-D-mannosyl 1-phosphate (Man1P) and D-glucose by Man-Glc phosphorylase (MP; EC 2.4.1.281). 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 toward D-glucose and 6-deoxy-D-glucose in the presence of Man1P, whereas 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: beta-linked glucobioses, maltose, N, N'-diacetylchitobiose, and beta-1,4-mannooligosaccharides. Particularly, beta-1,4-mannooligosaccharides served as significantly better acceptor substrates for RaMP2 than D-glucose. In the phosphorolytic reactions, RaMP2 had weak activity toward beta-1,4-mannobiose but efficiently degraded beta-1,4-mannooligosaccharides longer than beta-1,4-mannobiose. Consequently, RaMP2 is thought to catalyze the phosphorolysis of beta-1,4-mannooligosaccharides longer than beta-1,4-mannobiose to produce Man1P and beta-1,4-mannobiose.
• 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.
• Immobilization of a Thermostable Cellobiose 2-Epimerase from Rhodothermus marinus JCM9785 and Continuous Production of Epilactose

  Hiroki Sato, Wataru Saburi, Teruyo Ojima, Hidenori Taguchi, Haruhide Mori, Hirokazu Matsui

  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 (8) 1584 - 1587 0916-8451 2012/08 [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 degrees C at a space velocity of 8h(-1). The epilactose concentration of the resulting eluate was 30 g/L, and this was maintained for 13d.
• 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 7 76 (7) 1378 - 1383 0916-8451 2012/07 [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.
• 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.
• alpha-Glucosylated 6-gingerol: chemoenzymatic synthesis using alpha-glucosidase from Halomonas sp H11, and its physical properties

  Teruyo Ojima, Kenta Aizawa, Wataru Saburi, Takeshi Yamamoto

  CARBOHYDRATE RESEARCH 354 59 - 64 0008-6215 2012/06 [Refereed][Not invited]
   

  6-Gingerol [(S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one] is a biologically active compound and is abundant in the rhizomes of ginger (Zingiber officinale). It has some beneficial functions in healthcare, but its use is limited because of its insolubility in water and its heat-instability. To improve these physical properties, the glucosylation of 6-gingerol was investigated using alpha-glucosidases (EC. 3.2.1.20) from Aspergillus niger, Aspergillus nidulans ABPU1, Acremonium strictum, Halomonas sp. H11, and Saccharomyces cerevisiae, and cyclodextrin glucanotransferases (CGTase, EC. 2.4.1.19) from Bacillus coagulans, Bacillus sp. No. 38-2, Bacillus clarkii 7364, and Geobacillus stearothermophilus. Among these, only alpha-glucosidase from Halomonas sp. H11 (HaG) transferred a glucosyl moiety to 6-gingerol, and produced glucosylated compounds. The chemical structure of the reaction product, determined by nuclear magnetic resonance spectroscopy and mass spectrometry, was (S)-5-(O-alpha-D-glucopyranosyl)-1-(4-hydroxy-3-methoxyphenyl) decan-3-one (5-alpha-Glc-gingerol). Notably, the regioisomer formed by glucosylation of the phenolic OH was not observed at all, indicating that HaG specifically transferred the glucose moiety to the 5-OH of the beta-hydroxy keto group in 6-gingerol. Almost 60% of the original 6-gingerol was converted into 5-alpha-Glc-gingerol by the reaction. In contrast to 6-gingerol, 5-alpha-Glc-gingerol, in the form of an orange powder prepared by freeze-drying, was water-soluble and stable at room temperature. It was also more stable than 6-gingerol under acidic conditions and to heat. (C) 2012 Elsevier Ltd. All rights reserved.
• 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 27 287 (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 [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).
• Characterization of Halomonas sp Strain H11 alpha-Glucosidase Activated by Monovalent Cations and Its Application for Efficient Synthesis of alpha-D-Glucosylglycerol

  Teruyo Ojima, Wataru Saburi, Takeshi Yamamoto, Toshiaki Kudo

  APPLIED AND ENVIRONMENTAL MICROBIOLOGY 6 78 (6) 1836 - 1845 0099-2240 2012/03 [Refereed][Not invited]
   

  An alpha-glucosidase (HaG) with the following unique properties was isolated from Halomonas sp. strain H11: (i) high transglucosylation activity, (ii) activation by monovalent cations, and (iii) very narrow substrate specificity. The molecular mass of the purified HaG was estimated to be 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). HaG showed high hydrolytic activities toward maltose, sucrose, and p-nitrophenyl alpha-D-glucoside (pNPG) but to almost no other disaccharides or malto-oligosaccharides higher than trisaccharides. HaG showed optimum activity to maltose at 30 degrees C and pH 6.5. Monovalent cations such as K+, Rb+, Cs+, and NH4+ increased the enzymatic activity to 2- to 9-fold of the original activity. These ions shifted the activity-pH profile to the alkaline side. The optimum temperature rose to 40 degrees C in the presence of 10 mM NH4+, although temperature stability was not affected. The apparent K-m and k(cat) values for maltose and pNPG were significantly improved by monovalent cations. Surprisingly, k(cat)/K-m for pNPG increased 372- to 969-fold in their presence. HaG used some alcohols as acceptor substrates in transglucosylation and was useful for efficient synthesis of alpha-D-glucosylglycerol. The efficiency of the production level was superior to that of the previously reported enzyme Aspergillus niger alpha-glucosidase in terms of small amounts of by-products. Sequence analysis of HaG revealed that it was classified in glycoside hydrolase family 13. Its amino acid sequence showed high identities, 60%, 58%, 57%, and 56%, to Xanthomonas campestris WU-9701 alpha-glucosidase, Xanthomonas campestris pv. raphani 756C oligo-1,6-glucosidase, Pseudomonas stutzeri DSM 4166 oligo-1,6-glucosidase, and Agrobacterium tumefaciens F2 alpha-glucosidase, respectively.
• 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 [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.
• OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling

  Shinji Wakuta, Erika Suzuki, Wataru Saburi, Hideyuki Matsuura, Kensuke Nabeta, Ryozo Imai, Hirokazu Matsui

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 409 (4) 634 - 639 0006-291X 2011/06 [Refereed][Not invited]
   

  The synthesis of JA-IIe was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice. (C) 2011 Elsevier Inc. All rights reserved.
• Ingestion of epilactose, a non-digestible saccharide, improves postgastrectomy osteopenia and anemia in rats through the promotion of intestinal mineral absorption. - Comparative analyses of two non-digestible saccharides, epilactose and fructooligosaccha

  Taguchi Hidenori, Suzuki Takuya, Nishimukai Megumi, Yokoshima Satoru, Ojima Teruyo, Yamamoto Takeshi, Saburi Wataru, Hara Hiroshi, Kaneda Isamu, Onodera Shuichi, Shiomi Norio, Matsui Hirokazu

  Journal of Applied Glycoscience Supplement The Japanese Society of Applied Glycoscience 2011 (0) 74 - 74 2011 [Refereed][Not invited]
  
• Ingestion of Epilactose, a Non-digestible Disaccharide, Improves Postgastrectomy Osteopenia and Anemia in Rats through the Promotion of Intestinal Calcium and Iron Absorption

  Takuya Suzuki, Megumi Nishimukai, Aki Shinoki, Hidenori Taguchi, Satoru Fukiya, Atsushi Yokota, Wataru Saburi, Takeshi Yamamoto, Hiroshi Hara, Hirokazu Matsui

  JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 58 (19) 10787 - 10792 0021-8561 2010/10 [Refereed][Not invited]
   

  Gastrectomy often results in osteopenia and anemia because of calcium (Ca) and iron (Fe) malabsorption. Here, we investigated the effects of feeding epilactose, a non-digestible disaccharide, on gastrectomy-induced osteopenia, anemia, and Ca and Fe malabsorption in male Sprague Dawley rats. Totally gastrectomized or sham-operated rats were fed the control or epilactose (50 g/kg) diets for 30 days. Gastrectomy severely decreased intestinal Ca and Fe absorption, femoral bone strength, Ca content, hemoglobin concentration, and hematocrit. These decreases were partly or totally restored by feeding epilactose. Feeding epilactose increased the cecal tissue weight and the soluble Ca concentration and short-chain fatty acid pools of the cecal contents. Collectively, the increases in cecal mucosal area and/or soluble Ca concentration of the cecal contents, resulting from short-chain fatty acid production by intestinal microbes, are thought to be responsible for the epilactose-mediated promotion of Ca and Fe absorption in the gastrectomized rats.
• Ingestion of epilactose, a non-digestible saccharide, prevents gastrectomy-induced calcium malabsorption, iron malabsorption, osteopenia, and anemia in rats.

  Suzuki Takuya, Nishimukai Megumi, Taguchi Hidenori, Hamada Shigeki, Saburi Wataru, Yamamoto Takeshi, Ito Susumu, Hara Hiroshi, Matsui Hirokazu

  Journal of Applied Glycoscience Supplement 日本応用糖質科学会 2010 90 - 90 2010
• 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
• 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 [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.
• Gene cloning and enzymatic characteristics of a novel gamma-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364.

  Nakagawa Y, Saburi W, Takada M, Hatada Y, Horikoshi K

  Biochimica et biophysica acta 1784 (12) 2004 - 2011 0006-3002 2008/12 [Refereed][Not invited]
  
• 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.
• 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 0022-2836 2008/05 [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.
• 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 (Pt 9) 774 - 776 1744-3091 2007/09 [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.
• 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 1570-9639 2006/04 [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.
• Enzymatic synthesis of alkyl α-2-deoxyglucosides by alkyl alcohol resistant α-glucosidase from Aspergillus niger

  Young-Min Kim, Masayuki Okuyama, Haruhide Mori, Hiroyuki Nakai, Wataru Saburi, Seiya Chiba, Atsuo Kimura

  Tetrahedron Asymmetry 16 (2) 403 - 409 0957-4166 2005/01/24 [Refereed][Not invited]
   

  Aspergillus niger α-glucosidase (ANGase) was used for an efficient syntheses of alkyl α-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 α-2-deoxyglucosidic bond and the two-dimensional NMR (HMBC) spectra showed to be made up of 2-deoxyglucosyl and alkyl moieties. © 2004 Elsevier Ltd. All rights reserved.
• 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]
  
• Isolation and characterization of cDNA encoding P-19.5 protein accumulated preferentially at early stage of carrot somatic embryogenesis

  Takuma Sano, Mamoru Nishimoto, Wataru Saburi, Atsuo Kimura, Hiroshi Yasuda, Masahiro Uchibatake, Takuji Ohwada, Hiroshi Masuda

  Plant Science 167 (6) 1211 - 1217 0168-9452 2004/12 [Refereed][Not invited]
   

  We found P-19.5 protein that preferentially accumulated in cell clusters and globular embryos at the early stage of carrot somatic embryogenesis. P-19.5 protein was isolated from 9-day-old cell clusters with SDS-PAGE, and then determined the amino acid sequences of a N-terminal peptide and some internal peptides. Using each primer for these peptides, the full-length sequence of cDNA encoding P-19.5 protein was determined by analyzing 3′-RACE and 5′-RACE products. We regard P-19.5 protein as an allergen-like protein because the P-19.5 protein sequence predicted from cDNA was homologous to celery major allergen (75%), carrot major allergen (70-72%), Pimpinella brachycarpa pathogenesis-related protein (71%), and parsley pathogenesis-related protein (63%). We found concomitantly two separate cDNAs (P-16 and P-19.5 H cDNAs) during analyzing P-19.5 cDNA: P-16 cDNA was cloned and isolated from P-16 protein that was recognized against P-19.5 protein antiserum, and P-19.5 H cDNA that had homology (80.5% amino acid identity) with P-19.5 cDNA was detected during analyzing P-19.5 cDNA. Northern blotting revealed that P-19.5 and P-19.5 H transcripts were highly expressed at early stages of cell cluster and globular embryos, whereas P-16 transcript was invariably expressed at all stages from cell clusters to plantlets. © 2004 Elsevier Ireland Ltd. All rights reserved.

MISC

• 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
• 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
• [Review] Structures and Functions of Cellobiose 2-Epimerase and Related Enzymes

  Saburi Wataru  Bulletin of Applied Glycoscience  11-  (1)  22  -34  2021  [Refereed]
   

  セロビオース2-エピメラーゼ(CE)は,β-(1→4)-二糖の還元末端Glc残基をMan残基にエピメリ化する.β-マンナンの代謝においてManβ-4ManからManβ-4Glcを生成し,加リン酸分解へと導くとされる.CEの利用によるラクトースからのエピラクトースの生産技術が整備され,エピラクトースにプレバイオティクス効果など有益な生理機能が見出された.CEの構造は,触媒部位も含めてアシルグルコサミン2-エピメラーゼ(AGE)やマンノースイソメラーゼ(MI)などと類似し,AGEスーパーファミリーを形成する.(α/α)₆バレルからなる触媒ドメインの8番目と12番目のα-ヘリックス上のHisが一般酸・塩基触媒として基質の2-Hの授受に働くcis-エンジオレート中間体を経由したエピメリ化機構が提唱された.一方,MIなどイソメラーゼでは,8番目のα-ヘリックス上のHisが一般酸・塩基触媒として基質の1-Cと2-Cの間でのプロトンの分子内転移を行う機構が考えられた.AGEスーパーファミリーからManをエピメリ化するマンノース2-エピメラーゼが発見され,GlcからのManの生産への応用が期待された.

• 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.
• Development of modulation technology of cereal starch with transglycosylases

  佐分利 亘  年報  34-  216  -221  2018
• Structures and Functions of Cellobiose 2-Epimerase and β-Mannoside Phosphorylases Involved in β-Mannan Degradation

  佐分利 亘, 加藤 公児, 姚 閔, 松井 博和, 森 春英  応用糖質科学 = Bulletin of applied glycoscience : 日本応用糖質科学会誌  7-  (2)  69  -75  2017/05  [Not refereed][Not invited]
   

  β-マンナンは主としてマンノシル基がβ-(1→4)-結合した主鎖を持つ多糖であり，植物細胞壁のヘミセルロースの構成多糖や貯蔵多糖として存在する。Ruminococcus albusやBacteroides fragilisなどの細菌は，β-マンナン分解物のβ-(1→4)-マンノビオース(Manβ1-4Man)をセロビオース2-エピメラーゼ(CE)によりエピメリ化し，生じたManβ1-4Glcを4-O-β-D-マンノシル-D-グルコースホスホリラーゼ(MGP)により加リン酸分解することで代謝する(CE-MGP経路)。R. albusは，長鎖のβ-(1→4)-マンノオリゴ糖を好むβ-1,4-マンノオリゴ糖ホスホリラーゼ(MOP)も有する。CEはN-アセチルグルコサミン2-エピメラーゼやアルドースケトースイソメラーゼなどの単糖異性化酵素と構造的類似性を持ち，MGPやMOPは主としてマンノシドホスホリラーゼからなる糖質加水分解酵素ファミリー130に分類される。本稿では，これら酵素の機能と構造について概説する。
• 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  2016/08/20  [Not refereed][Not invited]
  
• 穀物澱粉を原料とした新規な複合糖質の開発

  佐分利亘  飯島藤十郎記念食品科学振興財団年報  31-  48‐53  -53  2016/08  [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]
  
• 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]
  
• 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]
  
• コムギの低温誘導性ディフェンシンは雪腐病および赤かび病抵抗性を向上させる

  佐々木健太郎, 宇梶慎子, 梅木菜月, 藤岡真里, 佐分利亘, 松井博和, 安倍史高, 今井亮三  農研機構生物機能利用研究部門成果情報(Web)  2016-  2016
• オリゴ糖異性化酵素および関連酵素の分子基盤と応用

  佐分利亘  日本農芸化学会北海道支部講演会講演要旨  2015-  2  2015/11/14  [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]
  
• 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]
  
• 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

  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  5-  (3)  42  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]
  
• 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  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).

• 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]
  
• 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]
  
• 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]
  
• オリゴ糖異性化酵素の分子基盤と有用オリゴ糖合成への応用

  佐分利亘  バイオサイエンスとインダストリー  72-  (5)  385  -389  2014/09/01  [Not refereed][Not invited]
  
• Ba-8 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(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))

  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 : 日本応用糖質科学会誌  4-  (3)  B40  2014/08/20  [Not refereed][Not invited]
  
• 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]
  
• ルーメン細菌に見られたヘミセルロースの新しい分解機構 オリゴ糖異性化酵素と加リン酸分解酵素による糖代謝

  佐分利亘  化学と生物  52-  (1)  13  -14  2014/01/01  [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.
• Acidophilic β-Galactosidase from Aspergillus niger AHU7120 with Lactose Hydrolytic Activity Under Simulated Gastric Conditions

  Saburi Wataru, Ueno Hiroshi M, Matui Hirokazu  Journal of applied glycoscience  61-  (2)  53  -57  2014  [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-  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]
  
• Ap-12 セロビオース2-エピメラーゼの異性化反応機構の解明(セルラーゼ,一般講演,日本応用糖質科学会平成25年度大会(第62回))

  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 : 日本応用糖質科学会誌  3-  (3)  B30  2013/08/20  [Not refereed][Not invited]
  
• Ca-1 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(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))

  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 : 日本応用糖質科学会誌  3-  (3)  B37  2013/08/20  [Not refereed][Not invited]
  
• Ca-4 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(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))

  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 : 日本応用糖質科学会誌  3-  (3)  B38  2013/08/20  [Not refereed][Not invited]
  
• 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]
  
• 低温馴化により誘導される植物ディフェンシンTAD1はコムギの雪腐病抵抗性に関与する

  梅木菜月, 梅木菜月, 小野瑞穂, 小野瑞穂, 藤岡真理, 植原愛, 宇梶慎子, 安倍史高, 佐々木健太郎, 佐分利亘, 松井博和, 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  31st-  184  2013/08/20  [Not refereed][Not invited]
  
• 2つのRuminococcus albus NE1由来マンノシルグルコースホスホリラーゼアイソザイムの生化学的機能解析

  佐分利亘  飯島藤十郎記念食品科学振興財団年報  28-  299  2013/08  [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。
• 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  -141  2013/05  [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。
• 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]
  
• シロイヌナズナにおけるCYP94B2の機能解析

  天野就基, 北岡直樹, 川出洋, 谷口沙希, 佐分利亘, 松井博和, 松浦英幸  日本農芸化学会大会講演要旨集(Web)  2013-  4A44A10 (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]
  
• 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]
  
• イネの傷害時のSAGをdonorとしたTAに対する糖転移反応

  竹松知紀, 宮澤吉郎, 瀬戸義哉, 和久田真司, 佐分利亘, 鍋田憲助, 松井博和, 松浦英幸  日本農芸化学会北海道支部講演会講演要旨  2012-  15  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]
  
• Bp1-11 シロイヌナズナADP-glucose pyrophosphorylase遺伝子の発現制御因子の解析(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))

  石塚 佐都子, 和久田 真司, 佐分 利亘, 今井 亮三, 松井 博和  応用糖質科学 : 日本応用糖質科学会誌  2-  (3)  B38  2012/08/20  [Not refereed][Not invited]
  
• Bp1-12 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(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))

  尾高 伶, 佐分 利亘, 川原 良介, 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)  B38  2012/08/20  [Not refereed][Not invited]
  
• Bp1-13 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(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))

  澤野 達也, 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)  B38  2012/08/20  [Not refereed][Not invited]
  
• Ba2-8 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(バイオマス関連酵素(キチナーゼ・キトサナーゼ他),一般講演,日本応用糖質科学会平成24年度大会(第61回))

  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 : 日本応用糖質科学会誌  2-  (3)  B53  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]
  
• シロイヌナズナADP‐glucose pyrophosphorylase遺伝子の発現制御因子の解析

  石塚佐都子, 和久田真司, 佐分利亘, 今井亮三, 松井博和  応用糖質科学  2-  (3)  (38)  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]
  
• イネのストレス応答とトレハロース代謝

  藪内威志, 島周平, 佐分利亘, 松井博和, 今井亮三  日本農芸化学会大会講演要旨集(Web)  2012-  2A30A06 (WEB ONLY)  2012/03/05  [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]
  
• シロイヌナズナ由来シトクロムP450 CYP94C1の機能解析

  谷口沙希, 和久田真司, 北岡直樹, 佐分利亘, 川出洋, 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)  2012-  3A30P18 (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]
  
• 嫌気性ルーメン細菌Ruminococcus albus由来セロビオース 2‐エピメラーゼの立体構造解析

  藤原孝彰, 佐分利亘, 松井博和, 姚閔, 田中勲  PFシンポジウム要旨集  29th-  30  2012  [Not refereed][Not invited]
  
• エピラクトースの酵素合成と機能

  佐分 利亘, 田口 秀典, 鈴木 卓弥  応用糖質科学 : 日本応用糖質科学会誌 = Bulletin of applied glycoscience  1-  (4)  291  -295  2011/11/20  [Not refereed][Not invited]
   

  エピラクトース(4-O-β-D-ガラクトシル-D-マンノース)は,加熱牛乳中に微量存在する希少糖である。ラクトースのアルカリ異性化やβ-ガラクトシダーゼによる糖転移反応により本糖質を合成可能であることが示されていたが,効率の低さや基質のコストの高さから,生理機能解析を実施できるほど大量の高純度エピラクトースを合成することは困難であった。しかし,セロビオース2-エピメラーゼ(CE)を用いることによりラクトースからエピラクトースを効率的に合成可能であることが見出され,当該酵素反応液からのエピラクトースの精製法が開発された。このことからエピラクトースの生理機能解析が比較的容易となり,様々な有益な生理機能が明らかにされてきた。本稿では,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効率的合成方法ならびに生理機能について概説する。
• オリゴ糖研究の最前線 その1 機能性オリゴ糖の研究 エピラクトースの酵素合成と機能

  佐分利亘, 田口秀典, 鈴木卓弥  応用糖質科学  1-  (4)  291  -295  2011/11/20  [Not refereed][Not invited]
   

  エピラクトース(4-O-β-D-ガラクトシル-D-マンノース)は,加熱牛乳中に微量存在する希少糖である。ラクトースのアルカリ異性化やβ-ガラクトシダーゼによる糖転移反応により本糖質を合成可能であることが示されていたが,効率の低さや基質のコストの高さから,生理機能解析を実施できるほど大量の高純度エピラクトースを合成することは困難であった。しかし,セロビオース2-エピメラーゼ(CE)を用いることによりラクトースからエピラクトースを効率的に合成可能であることが見出され,当該酵素反応液からのエピラクトースの精製法が開発された。このことからエピラクトースの生理機能解析が比較的容易となり,様々な有益な生理機能が明らかにされてきた。本稿では,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効率的合成方法ならびに生理機能について概説する。
• エピラクトースの酵素合成と機能

  佐分 利亘, 田口 秀典, 鈴木 卓弥  応用糖質科学 : 日本応用糖質科学会誌 = Bulletin of applied glycoscience  1-  (4)  291  -295  2011/11/20  [Not refereed][Not invited]
   

  エピラクトース(4-O-β-D-ガラクトシル-D-マンノース)は,加熱牛乳中に微量存在する希少糖である。ラクトースのアルカリ異性化やβ-ガラクトシダーゼによる糖転移反応により本糖質を合成可能であることが示されていたが,効率の低さや基質のコストの高さから,生理機能解析を実施できるほど大量の高純度エピラクトースを合成することは困難であった。しかし,セロビオース2-エピメラーゼ(CE)を用いることによりラクトースからエピラクトースを効率的に合成可能であることが見出され,当該酵素反応液からのエピラクトースの精製法が開発された。このことからエピラクトースの生理機能解析が比較的容易となり,様々な有益な生理機能が明らかにされてきた。本稿では,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効率的合成方法ならびに生理機能について概説する。
• 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.
• Physiological and Biochemical Characterization of Three Nucleoside Diphosphate Kinase Isozymes from Rice (Oryza sativa L.)

  Akihiko Kihara, Wataru Saburi, Shinji Wakuta, Myung-Hee Kim, Shigeki Hamada, Hiroyuki Ito, Ryozo Imai, Hirokazu Matsui  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  75-  (9)  1740  -1745  2011/09  [Not refereed][Not invited]
   

  Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the gamma-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. In this study, we examined the subcellular localization, tissue-specific gene expression, and enzymatic characteristics of three rice NDPK isozymes (OsNDPK1-OsNDPK3). Sequence comparison of the three OsNDPKs suggested differential subcellular localization. Transient expression of green fluorescence protein-fused proteins in onion cells indicated that OsNDPK2 and OsNDPK3 are localized to plastid and mitochondria respectively, while OsNDPK1 is localized to the cytosol. Expression analysis indicated that all the OsNDPKs are expressed in the leaf, leaf sheath, and immature seeds, except for OsNDPK1, in the leaf sheath. Recombinant OsNDPK2 and OsNDPK3 showed lower optimum pH and higher stability under acidic pH than OsNDPK1. In ATP formation, all the OsNDPKs displayed lower K-m values for the second substrate, ADP, than for the first substrate, NTP, and showed lowest and highest K-m values for GTP and CTP respectively.
• 好塩細菌Halomonas sp.H11株より単離した新規α‐グルコシダーゼの諸性質

  小島晃代, 佐分利亘, 山本健, 工藤俊章  日本生物工学会大会講演要旨集  63rd-  138  -138  2011/08/25  [Not refereed][Not invited]
  
• 2Dp12 Properties of novel alpha-glucosidase isolated from halophilic bacterium Halomonas sp. H11 strain

  Ojima Teruyo, Saburi Wataru, Yamamoto Takeshi, Kudo Toshiaki  日本生物工学会大会講演要旨集  0-  (63)  138  -138  2011/08/25  [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.
• Ochromonas danica由来β‐1,3‐グルカンホスホリラーゼのクローニングと機能解析

  磯野直人, 柳原和典, 山本豊, 十万真奈, 佐分利亘, 山本健, 小林裕子, 小林一成, 久松眞  応用糖質科学  1-  (3)  (57)  2011/07/20  [Not refereed][Not invited]
  
• 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]
  
• 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, 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]
  
• 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]
  
• 難消化性二糖エピラクトースによるカルシウム吸収・鉄吸収,骨形成,貧血の回復作用に関する研究―胃切除ラットにおけるフラクトオリゴ糖との比較検討―

  田口秀典, 鈴木卓弥, 西向めぐみ, 横嶋悟, 小島晃代, 山本健, 佐分利亘, 原博, 金田勇, 小野寺秀一, 塩見徳夫, 松井博和  応用糖質科学  1-  (3)  (43)  2011/07/20  [Not refereed][Not invited]
  
• 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]
  
• イネ培養細胞におけるスクロース誘導性タンパク質の解析

  アンストーン ワスサン, 和久田真司, 濱田茂樹, 伊藤浩之, 佐分利亘, 今井亮三, 松井博和  日本農芸化学会大会講演要旨集  2011-  13  2011/03/05  [Not refereed][Not invited]
  
• イネNucleoside diphosphate kinaseの生理的諸性質の解析

  木原明彦, 和久田真司, 金明姫, 濱田茂樹, 佐分利亘, 伊藤浩之, 今井亮三, 松井博和  日本農芸化学会大会講演要旨集  2011-  14  2011/03/05  [Not refereed][Not invited]
  
• セロビオース2‐エピメラーゼによるエピラクトースの酵素合成と生理機能

  松井博和, 田口秀典, 佐分利亘  日本農芸化学会大会講演要旨集  2011-  SHI34  2011/03/05  [Not refereed][Not invited]
  
• イネの病傷害ストレス応答におけるOsJAR1およびOsJAR2の機能

  鈴木絵里香, 和久田真司, 佐分利亘, 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  2011-  5  2011  [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]
  
• ヒトにおける新規糖質・メガロシュガーの消化性と生体利用性

  高見昌之, 田辺賢一, 中村禎子, 佐分利亘, 奥恒行  日本食物繊維学会誌  14-  (2)  141  2010/12/31  [Not refereed][Not invited]
  
• Production of Two α-1,4-Glucans Having Glucosyl Residues Linked by α-1,3-and α-1,6-Linkages at the Non-reducing End by the Coupled Reaction of α-Glucosidase and Cyclodextrin Glucanotransferase

  SABURI Wataru, SABURI KAMIMURA Yukari, IIZUKA Takahisa, YAMAMOTO Takeshi, TAKADA Masayasu  Journal of applied glycoscience  57-  (4)  231  -237  2010/10/20  [Not refereed][Not invited]
   

  The isomalto- and nigero-oligosaccharides are usually produced from starch by the combination of α-glucosidase and starch degrading enzymes, such as α- and β-amylases. In this study, a new reaction system for the production from starch of two α-1,4 glucans having α-1,6- and α-1,3-linked glucosyl residues at or near the non-reducing end was established. These glucans were efficiently produced by the coupled reaction of α-glucosidase and cyclodextrin glucanotransferase (CGTase) with negligible cyclodextrin production. The produced glucans underwent very little hydrolyzation by β-amylase but α-amylase clearly enhanced the digestion of glucan. This indicated that glucosidic linkage other than α-1,4-linkage was introduced at or near the non-reducing end of the glucan and the reducing end part of the glucan was mainly composed of α-1,4-linkages. The glucosidic linkage introduced was dependent on the specificity of α-glucosidase for glucosidic linkage. α-Glucosidases from <i>Aspergillus niger</i> (ANG) and <i>Acremonium strictum</i> (ASG) produced α-1,6- and α-1,3-glucosidic linkages, respectively. The chain length distribution also varied according to the specificity of α-glucosidases for substrate chain length. The major DP of the glucans produced by ANG and ASG were 4-6 and 6-10, respectively. The glucan produced by the coupled reaction was highly resistant to retrogradation. The syrup including this glucan maintained transparency following storage at room temperature for 1 month. In contrast, control syrup including the starch hydrolysate lost transparency despite the lower content of long-chain glucan. This indicates that the glucosyl residue linked by α-1,3-linkage present at or near the non-reducing end of glucan strongly inhibits aggregation of glucan and provides retrogradation tolerance.
• Practical Preparation of Epilactose Produced with Cellobiose 2-Epimerase from Ruminococcus albus NE1

  Wataru Saburi, Takeshi Yamamoto, Hidenori Taguchi, Shigeki Hamada, Hirokazu Matsui  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY  74-  (8)  1736  -1737  2010/08  [Not refereed][Not invited]
   

  A practical purification method for a non-digestible disaccharide, epilactose (4-O-beta-galactosyl-D-mannose), was established. Epilactose was synthesized from lactose with cellobiose 2-epimerase and purified by the following procedure: (i) removal of lactose by crystallization, (ii) hydrolysis of lactose by beta-galactosidase, (iii) digestion of monosaccharides by yeast, and (iv) column chromatography with Na-form cation exchange resin. Epilactose of 91.1% purity was recovered at 42.5% yield.
• 各種二重変異米澱粉の特性の評価

  安東竜一, 佐分利亘, 高田正保, 中村保典, 藤田直子  J Appl Glycosci  57-  (Suppl.)  31  -22  2010/07/20  [Not refereed][Not invited]
  
• 難消化性二糖エピラクトースによる小腸カルシウム吸収促進作用機序に関する研究

  鈴木卓弥, 西向めぐみ, 武知真希, 田口秀典, 濱田茂樹, 佐分利亘, 山本健, 伊藤進, 原博, 松井博和  J Appl Glycosci  57-  (Suppl.)  48  -91  2010/07/20  [Not refereed][Not invited]
  
• 難消化性二糖エピラクトースによるカルシウム吸収・鉄吸収,骨形成,貧血の回復作用に関する研究~胃切除ラットでの評価

  鈴木卓弥, 西向めぐみ, 田口秀典, 濱田茂樹, 佐分利亘, 山本健, 伊藤進, 原博, 松井博和  J Appl Glycosci  57-  (Suppl.)  48  2010/07/20  [Not refereed][Not invited]
  
• ヒトおよびラット小腸粘膜ならびにヒト唾液消化酵素を用いた新規糖質・メガロ糖の消化性の検討

  高見昌之, 田辺賢一, 中村禎子, 佐分利亘, 奥恒行  日本栄養・食糧学会大会講演要旨集  64th-  84  2010/05/01  [Not refereed][Not invited]
  
• Characterization of Two γ-Cyclodextrin-specific Enzymes from Bacillus clarkii 7364

  NAKAGAWA Yoshinori, SABURI Wataru, YAMAMOTO Takeshi, TAKADA Masayasu, OGAWA Koichi, YAMAMOTO Mikio, HATADA Yuji, NAKAMURA Nobuyuki, HORIKOSHI Koki  Journal of applied glycoscience  57-  (2)  121  -129  2010/04/20  [Not refereed][Not invited]
   

  The alkaliphilic soil bacterium <i>Bacillus clarkii</i> 7364 was found to produce cyclodextrin glucanotransferase (CGTase), an enzyme which converts starch into &gamma;-cyclodextrin (&gamma;-CD) with high specificity. The bacterium also intracellularly produced cyclodextrinase (CDase). The gene encoding the CDase (Cda) was located about 200 bases downstream of the gene encoding the CGTase (Cgt). Comparison of the amino acid sequence of Cgt with those of other CGTases revealed that several amino acids which contribute to substrate binding are absent or different at subsites +3, +2, -3 and -7 in Cgt. The replacement of Ala223 at subsite +2 by three basic amino acids, histidine, lysine and arginine, strongly enhanced &gamma;-CD-forming activity in the neutral pH range, although the optimum pH for the activity (pH 10.0) and CD production specificity remained the same. The interaction between the protonated amino group in the side chain of these basic amino acids and the linear substrate is thought to play an important role in the enhancement of the activity. Cda had extremely high hydrolytic specificity for &gamma;-CD. Cda also displayed a transglycosylation activity, where a maltotriose moiety could be transferred, unlike other CD-degrading enzymes. In Cda, the N-domain normally found in other CD-degrading enzymes which functions in the dimerization, thus contributing to the preference for CDs, was deleted and instead, a long extra sequence was present in the C-terminus. Despite the lack of the N-domain, Cda showed a dodecameric structure. These unique features indicate that Cda is a novel CD-degrading enzyme.
• CGTaseとα‐glucosidaseの共反応による新規分岐糖生成反応

  佐分利亘, 上村由香里, 飯塚貴久, 山本健, 高田正保  日本農芸化学会大会講演要旨集  2010-  23  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]
  
• 新規糖質・メガロ糖の消化性とヒトにおける利用性の検討

  高見昌之, 田辺賢一, 中村禎子, 佐分利亘, 奥恒行  日本栄養・食糧学会九州・沖縄支部大会講演要旨集  2010-  17  2010  [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(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]
  
• Bacillus clarkii7364株が生産するγ‐cyclodextrinに高い特異性を有する2つの酵素

  中川佳紀, 佐分利亘, 山本健, 高田正保, 小川浩一, 山本幹男, 秦田勇二, 中村信之, 掘越弘毅  J Appl Glycosci  56-  (Suppl.)  61  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, &alpha;-glucosidase, oligo-1,6-glucosidase and dextran glucosidase, which hydrolyze the non-reducing end glucosidic linkages of maltooligo- and/or isomaltoolligosaccharides, are categorized as &alpha;-glucoside hydrolase. Despite a high similarity in the sequence and overall structure of those family enzymes, GH 13 &alpha;-glucoside hydrolases show a wide range of substrate specificity. Until now, three crystal structures of &alpha;-glucoside hydrolase, dextran glucosidase from <i>Streptococcus mutans</i> (DGase), oligo-1,6-glucosidase from <i>Bacillus cereus</i> (O16G), and &alpha;-glucosidase from <i>Geobacillus</i> sp. HTA-462 (GSJ) have been determined. In this study, we have performed the structural comparison of these &alpha;-glucoside hydrolases. Their overall structures are generally similar, and consist of three major domains A, B and C as found in many &alpha;-amylase family enzymes. The significant structural differences in these enzymes are mainly found in loop regions. GSJ has a shorter &beta;&rarr;&alpha; 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 &alpha;-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 &alpha;-amylases since the key residues in substrate binding are conserved in both primary and tertiary structures.
• 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]
  
• 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]
  
• γ‐CDに高い特異性を有するBacillus clarkii7364由来cyclodextrinaseの諸性質

  中川佳紀, 佐分利亘, 高田正保, 秦田勇二, 掘越弘毅  J Appl Glycosci  55-  (Suppl.)  49  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]
  
• 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]
  
• Heterologous expression of dextrin dextranase gene and analysis of recombinant enzyme

  Sadahiro Juri, Saburi Wataru, Mori Haruhide, Okuyama Masayuki, Okada Gentaro, Kimura Atsuo  Journal of Applied Glycoscience Supplement  2008-  (0)  100  -100  2008  [Not refereed][Not invited]
  
• Enzymatic characteristics of a novel γ-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364

  Nakagawa Yoshinori, Saburi Wataru, Takada Masayasu, Hatada Yuji, Horikoshi Koki  Journal of Applied Glycoscience Supplement  2008-  (0)  98  -98  2008  [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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• 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]
  
• Purification, characterization, and sequence analysis of two alpha-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards beta-cyclodextrin sepharose.

  San San Mar, Haruhide Mori, Jin-Ha Lee, Kenji Fukuda, Wataru Saburi, Arinobu Fukuhara, Masayuki Okuyama, Seiya Chiba, Atsuo Kimura  Bioscience, biotechnology, and biochemistry  67-  (5)  1080  -93  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.
• 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]
  
• 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]
  

Association Memberships

• THE JAPANESE SOCIETY OF APPLIED GLYCOSCIENCE   JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY   

Research Projects

• ゲノムデザインと自由な書き換えによる高収量コムギの創出

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2021/07 -2024/03 

  Author : 今井 亮三, 佐分利 亘
• Molecular basis and functional modulation of carbohydrate epimerases and isomerases useful for carbohydrate conversion

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2021/04 -2024/03 

  Author : 佐分利 亘

   

  糖質の高度利用には，希少糖質の効率合成法が必要である．本研究では，微生物の多様な糖質代謝を支える異性化酵素の中でも，セロビオース2-エピメラーゼやマンノースイソメラーゼなど様々な糖質異性化酵素を含む酵素群に注目し，反応特異性を制御する機構の解明と高活性変異酵素の開発を目的とした．セロビオース2-エピメラーぜの中にはエピメラーゼ活性のみを示す酵素 (1機能CE) とエピメラーゼ活性に加えてイソメラーゼ活性を示す酵素 (2機能CE) が存在するが，この特異性の違いを説明する構造は明らかではなかった．そこで，1機能CEと2機能CEの間で構造領域を入れ換えた一連のキメラ酵素を作成した．具体的には，(α/α)6バレルの触媒ドメインを構成するαヘリックス1と2，3と4，5と6，7と8，9と10，11と12について，1機能酵素の構造を2機能酵素に移植した．これら6つのキメラ酵素のうち，最初の3つの変異酵素は活性型酵素として得られた．この機能について解析を進めている． マンノースエピメラーゼ (ME酵素) については高活性変異酵素の取得のため，ハイスループットスクリーニング系を検討した．本スクリーニング系では，マンノーストランスポーター遺伝子を欠失させることでマンノース資化性を失われた大腸菌を用い，この細胞表層にME酵素を発現させることでマンノース資化性の回復，また，活性に応じた生育速度の増加により高活性変異酵素をスクリーニングすることを計画した．マンノーストランスポーターを構成する3タンパク質全ての遺伝子を欠失させることでマンノース資化性を完全に失わせることができた．この大腸菌変異株をホストとし，氷核タンパク質のNドメインを付加したME酵素を発現させると，ME酵素は外膜画分に生産され，マンノース資化性の回復が確認された．この系を利用して高活性変異酵素をスクリーニングする予定である．
• Analysis of immunological properties of mannose-based molecules and its action mechanisms.

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

  Date (from‐to) : 2019/04 -2022/03 

  Author : Toda Masako

   

  The aim of this study was to identify immunological function of mannose-based molecules and its action mechanisms. Among α-mannooligosaccharides, long chain molecules such as α-Man-(1→6)-Man4 inhibited lipopolysaccharide (LPS)-stimulated activation of bone marrow derived murine dendritic cells (BMDC). In contrast, among β-mannooligosaccharides, β-Man-(1→4)-Man activated BMDC via engagement of toll-like receptor 4. We also found that α-mannan from yeast promotes anti-inflammatory responses (e.g., IL-10 production) in BMDC when it was stimulated with LPS. Metabolome analysis showed that α-mannan altered levels of many metabolites in BMDC, e.g., an increased level of lactic acid, the final product of glycolysis, and a reduced level of succinic acid, an inflammatory metabolite of TCA cycle. The results suggest that α-mannan induces reprogram of cellular metabolism and thereby promotes anti-inflammatory response in BMDC.
• Utilization of enzymes for production of a variety of carbohydrates

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

  Date (from‐to) : 2018/04 -2021/03 

  Author : MORI Haruhide

   

  To develop the enzymatic production of carbohydrates, some enzymes were investigated in this study. As glycosyltransferases using sugar nucleotides as glycosyl donor, SS-enzyme and GS-enzyme were used. New disaccharides were produced using the SS-enzyme, in one-pot reaction with sucrose. The SS-enzyme was engineered for phosphorylated products. A new type of GS-enzyme was found. As transglycosylases, enzymes producing useful oligosaccharides and cyclic oligosaccharides were analyzed. Functions of the domains of the multi-domain structure, residues involved in the acceptor binding and determining their product preference were clarified.
• Carbohydrate conversion by novel bacterial carbohydrate epimerases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2018/04 -2021/03 

  Author : Saburi Wataru

   

  Carbohydrates with various structures have a variety of excellent functions, but kind of carbohydrates, which are abundantly present, is extremely limited. Therefore, it is necessary to establish the enzymatic synthesis technology using abundant carbohydrate resources for the utilization of useful rare carbohydrates. In this study, we clarified the functions of mannose 2-epimerase (ME) homologs from several strains, such as Dyadobacter fermentans, in addition to the originally discovered enzyme from Runella slithyformis. In addition, a cellobiose 2-epimerase with broad specificity that acts on monosaccharides such as glucose and galactose in addition to β1-4 disaccharides was discovered through functional analysis of ME homologs.
• Molecular mechanism of activation of signal compounds regulating resistance against pathogen in plants

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2015/10 -2018/03 

  Author : Hirokazu Matsui

   

  In plants, physiologically active compounds are inactivated by glycosylation and activated by hydrolysis of glycosides. In this study, rice beta-glucosidase TAGG2, which has high activity towards both tuberonic acid (TA) beta-glucoside and salicylic acid beta-glucoside (SAG), was biochemically and physiologically characterized. Important amino acid residues involved in recognition of SAG were determined through site-directed mutagenesis, and localization of TAGG2 to apoplast was confirmed using transgenic rice expressing a fusion protein of GFP and TAGG2. In transgenic rice expressing TAGG2, over-accumulation of TA was observed but SA level was not changed significantly. Blast infection analysis suggested overexpression of the TAGG2 gene negatively regulated the resistance against pathogens. Homologous enzymes in Arabidopsis had high activity towards oligosaccharide substrates such as laminarioligosaccharide rather than SAG.
• Enzymatic synthesis of oligosaccharides, polysaccharides, and glycosides

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

  Date (from‐to) : 2015/04 -2018/03 

  Author : Mori Haruhide

   

  Carbohydrate includes a wide variety of molecules because of the diversity of constituent monosaccharides, linkages, and degrees of polymerization. They are highly expected to contain functional compounds useful for human life. Therefore, various synthetic methods are required for synthesis of various saccharides. In this study, enzymatic conversion from highly abundant carbohydrates in nature was established with two types of enzymes: glycoside synthase and glycosyl transferase. In the method with synthase, sugar nucleotides, substrates for glycoside synthases, were provided from sucrose, and disaccharides were efficiently produced through the one-pot reactions. A new group of glycosyl transferases were found. The enzymes in it acted on maltooligosaccharides and catalyzed glucosyl transfer reactions.
• Molecular basis for oligosaccharide epimerases and their related enzymes.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2014/04 -2016/03 

  Author : SABURI Wataru

   

  Cellobiose 2-epimerase (CE) epimerizes the reducing end glucose residue of beta1-4 disaccharides to mannose residue. It shares the catalytic domain and site structures with other monosaccharide isomerases/epimerases. In this study, structure-function relationship of these enzymes was analyzed. Important amino acid residues for high selectivity for disaccharides in Rhodothermus marines CE were determined through site-directed mutation. Slight isomerization activity was observed in R. marines CE, and important structure for isomerase activity was determined based on the comparison of the structures between R. marines and Cardicellulosiruptor saccharolyticus CEs. A novel enzyme acting on mannose was found from function unknown proteins.
• オリゴ糖異性化酵素とその類縁酵素の構造基盤の解明

  文部科学省：科学研究費補助金 若手研究(B)

  Date (from‐to) : 2014/04 -2016/03 

  Author : 佐分利 亘
• Identification and functional analysis of novel metabolizing enzymes of plant hormones

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

  Date (from‐to) : 2012/04 -2015/03 

  Author : MATSUI Hirokazu, SABURI Wataru, MATSUURA Hideyuki

   

  Jasmonate (JA) is a plant hormone regulating abiotic and biotic stress responses and growth. In this study, we analyzed the physiological and biochemical functions of CYP94D1, CYP94D2, and ILL6 proteins, which were predicted to inactivate the JA signal. In the analysis of mutant plants with higher and lower expression levels of CYP94D1 than wild type plant, we observed phenotypic differences. The transgenic plants lucking CYP94D1 showed higher resistance to external JA. The overexpressing plants of CYP94D1 accumulate higher level of tuberonic acid glucoside than wild type.
• オリゴ糖異性化酵素ならびに類縁酵素群の機能解明と有用糖質の効率合成への応用展開

  野田産業科学研究所：奨励研究助成

  Date (from‐to) : 2014/04 -2015/03 

  Author : 佐分利 亘
• Molecular analysis of two novel carbohydrate phosphorylases

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)

  Date (from‐to) : 2012/04 -2014/03 

  Author : SABURI Wataru

   

  A ruminal anaerobic baceteium, Ruminococcus albus, has two mannosylglucose phosphorylase isozymes (Type-I and Type-II), phosphorolyzing mannosylglucose to alpha-mannose 1-phosphate and glucose. Anyalysis of substrate specificity revealed that Type-I was specific to mannosylglucose, but Type-II had higher activity to mannooligosaccharide than mannosylglucose. These enzymes showed different acceptor substrate specificity in the synthetic reaction: Type-I enzyme showed synthetic activity to 6-OH glucose derivatives unlike in contrast to Type-II, which was completely inert to these substrates. Asp129 of Type-I enzyme was determined to be catalytic amino acid residue based on sequence comparison and site-directed mutagenesis analysis. Ile212 of Type-I enzyme was important for recognition of 6-OH glucose derivatives as acceptor substrates in the synthetic reaction (reverse reaction).
• プレバイオティクスとして機能するエピラクトースの実用的合成法の開発

  科学技術振興機構：A-STEP 研究成果最適展開支援プログラム 第1回【FS】 探索タイプ

  Date (from‐to) : 2013/08 -2014/03 

  Author : 佐分利 亘
• 2つの新規な糖質ホスホリラーゼの分子解析

  文部科学省：科学研究費補助金 若手研究(B)

  Date (from‐to) : 2012/04 -2014/03 

  Author : 佐分利 亘
• プレバイオティクスの革新的酵素合成技術の開発

  ノーステック財団：研究開発助成事業」(フードイノベーション創造支援事業 研究シーズ発掘補助金)

  Date (from‐to) : 2012/08 -2013/03 

  Author : 佐分利 亘
• バイオリアクターを利用したエピラクトースの効率的合成法の開発

  ノーステック財団：「研究開発助成事業」(若手研究人材育成事業 Talent補助金)

  Date (from‐to) : 2011/09 -2012/03 

  Author : 佐分利 亘
• ルーメン細菌によるヘミセルロース分解の分子機構の解析

  公益財団法人秋山記念生命科学振興財団：研究助成 (奨励助成)

  Date (from‐to) : 2011/04 -2012/03 

  Author : 佐分利 亘

Industrial Property Rights

• 特許第5982087号:内臓脂肪蓄積予防または蓄積改善剤  2016/08/05

  松井 博和, 田口 秀典, 濱田 茂樹, 宮下 和夫, 伊藤 進, 松浦 正男, 木口 雅夫, 横谷 亮, 山本 健, 佐分利 亘  日本食品化工株式会社, 国立大学法人北海道大学, 株式会社化合物安全性研究所  201603010684373826
• 特許第5933321号:新規なα−グルコシダーゼとその製造法並びに用途  2016/05/13

  小島 晃代, 相沢 健太, 和田 幸樹, 佐分利 亘, 山本 健, 工藤 俊章  日本食品化工株式会社, 国立大学法人 長崎大学  201603003156993778
• 特許第5903230号:エピラクトースを含有する動物用飼料組成物を含有する液状の動物用配合飼料の用途  2016/03/18

  本間 満, 上西 寛司, 中島 肇, 藤田 孝, 阿部 健太郎, 松井 博和, 佐分利 亘, 田口 秀典  雪印種苗株式会社  201603010521225250
• 特許第5778888号:分岐糖類を含有する風味改善剤および製剤用マスキング剤  2015/07/17

  佐分利, 亘, 上 村, 由香里, 山 本, 健, 高 田 正  日本食品化工株式会社  201503008415363456
• 特許第5615584号:高純度エピラクトースの製造方法  2014/09/19

  佐分利 亘, 山本 健  日本食品化工株式会社  201403023507906210
• 特許第5590615号:β1,3−グルカンの製造方法  2014/08/08

  磯野 直人, 山本 豊, 佐分利 亘  国立大学法人三重大学  201403049059820502
• 特開2014-005447:澱粉分解物および飲食品用味質改善剤ならびにその用途  2014/01/16

  飯塚 貴久, 影嶋 富美, 佐分利 亘, 柿野 あけみ, 山本 健, 高田 正保  日本食品化工株式会社  201403045972049700
• 特許第5414926号:澱粉分解物および飲食品用味質改善剤ならびにその用途  2013/11/22

  飯塚 貴久, 影嶋 富美, 佐分利 亘, 柿野 あけみ, 山本 健, 高田 正保  日本食品化工株式会社  201403040019728479
• 特許第5224572号:デキストラン生成酵素遺伝子、デキストラン生成酵素およびその製造方法、デキストランの製造方法  2013/03/22

  木村 淳夫, 森 春英, 奥山 正幸, 佐分利 亘, 千葉 誠哉, 岡田 嚴太郎, 山本 健, 山本 幹男  国立大学法人北海道大学, 日本食品化工株式会社  201303089862201646
• 特開2013-051904:エピラクトースを含有する動物用飼料組成物、その組成物を含有する動物用配合飼料、及びその用途  2013/03/21

  本間 満, 上西 寛司, 中島 肇, 藤田 孝, 阿部 健太郎, 松井 博和, 佐分利 亘, 田口 秀典  雪印メグミルク株式会社, 雪印種苗株式会社  201303002682696657
• 特開2013-005793:新規なα−グルコシダーゼとその製造法並びに用途  2013/01/10

  小島 晃代, 相沢 健太, 和田 幸樹, 佐分利 亘, 山本 健, 工藤 俊章  日本食品化工株式会社, 国立大学法人 長崎大学  201303038329804874
• 特許第5092049号:セロビオース2−エピメラーゼおよびその用途  2012/09/21

  佐分利 亘, 小島 晃代, 山本 健, 松井 博和  日本食品化工株式会社, 国立大学法人北海道大学  201303045638740941
• 特開2012-130332:セロビオース2−エピメラーゼおよびその用途  2012/07/12

  佐分利 亘, 小島 晃代, 山本 健, 松井 博和  日本食品化工株式会社, 国立大学法人北海道大学  201203064122628468
• 特開2011-231035:内臓脂肪蓄積予防または蓄積改善剤  2011/11/17

  松井 博和, 田口 秀典, 濱田 茂樹, 宮下 和夫, 伊藤 進, 松浦 正男, 木口 雅夫, 横谷 亮, 山本 健, 佐分利 亘  日本食品化工株式会社, 国立大学法人北海道大学, 株式会社化合物安全性研究所  201103061666598914
• 特開2011-217701:高純度エピラクトースおよびその製造方法  2011/11/04

  佐分利 亘, 山本 健  日本食品化工株式会社  201103029212146538
• 特開2011-083248:分岐糖類を含有する風味改善剤および製剤用マスキング剤  2011/04/28

  佐分利, 亘, 上 村, 由香里, 山 本, 健, 高 田 正  日本食品化工株式会社  201103014968847206
• WO2010-092997:β1,3−グルカンの製造方法  2010/08/19

  磯野 直人, 山本 豊, 佐分利 亘  国立大学法人三重大学  201203046386496901
• 特許第4537733号:アノマー保持型糖加水分解酵素変異体及びその製造方法  2010/06/25

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