研究者情報

マスター

アカウント（マスター）

• 氏名

  佐分利　亘(サブリ　ワタル), サブリ　ワタル

所属（マスター）

• 農学研究院　基盤研究部門　生物機能化学分野

所属（マスター）

• 農学研究院　基盤研究部門　生物機能化学分野

独自項目

syllabus

• 2021, バイオテクノロジー学特論, Advanced Biotechnology, 修士課程, 農学院, バイオテクノロジー、糖質酵素、mRNA分解、植物ウイルスベクター、糖質生合成、酵素改変、木質資源、食品機能、微生物資源、微生物バイオプロセス、技術・データ駆動型
• 2021, バイオテクノロジー学特論演習, Advanced Seminar on Biotechnology, 修士課程, 農学院, バイオテクノロジー，生物利用・物質生産
• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, バイオテクノロジー、糖質酵素、mRNA分解、植物ウイルスベクター、糖質生合成、酵素改変、木質資源、食品機能、微生物資源、微生物バイオプロセス、技術・データ駆動型
• 2021, 食品安全・機能性開発学特論, Advanced Safety and Function of Food, 修士課程, 農学院, 食品安全性、予測微生物、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
• 2021, 食品安全・機能性開発学特論演習, Advanced Seminar on Safety and Function of Food, 修士課程, 農学院, 食品の安全、予測微生物学、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 食品安全性、予測微生物、食料確保と安全性、植物由来機能性食品、微生物由来機能性食品、腸内細菌叢の改変に基づいた機能性食品、食肉由来機能性食品
• 2021, 生物機能化学演習Ⅱ, Seminar on Bioscience and Chemistry II, 学士課程, 農学部, 専門関連英文原書精読
• 2021, 生物機能化学実験Ⅲ, Laboratry Work on Bioscience and Chemistry III, 学士課程, 農学部, 酵素反応、反応速度論、タンパク質精製、電気泳動
• 2021, 生物化学Ⅰ, Biochemistry I, 学士課程, 農学部, 生化学，生体成分，糖質，アミノ酸，タンパク質，酵素，補酵素，ビタミン，脂質，核酸
• 2021, 生物化学Ⅱ, Biochemistry II, 学士課程, 農学部, 代謝，エネルギー，解糖系，トリカルボン酸回路，電子伝達系，酸化的リン酸化，糖新生，ペントースリン酸経路，脂質代謝，光合成，カルビンサイクル，窒素代謝，尿素サイクル
• 2021, 生物化学Ⅲ, Biochemistry III, 学士課程, 農学部, タンパク質，酵素，応用糖質科学，糖質の構造，糖質の機能，糖質の代謝，糖質の合成，糖質の利用

researchmap

プロフィール情報

学位

• 博士(農学)（2006年03月 北海道大学）

プロフィール情報

• 姓

  佐分利, サブリ

• 名

  亘, ワタル

• ID各種

  201201038539083300

業績リスト

研究キーワード

• 蛋白質工学   応用微生物学   糖質合成   酵素化学   応用生物化学   

研究分野

• ライフサイエンス / 応用生物化学

経歴

• 2020年07月 - 現在 北海道大学 大学院農学研究院 准教授
• 2019年04月 - 2020年06月 北海道大学 大学院農学研究院 講師
• 2010年10月 - 2019年03月 北海道大学 大学院農学研究院 助教
• 2006年04月 - 2010年09月 日本食品化工株式会社 研究所 研究員

学歴

• 2001年04月 - 2006年03月   北海道大学   大学院農学研究科
• 1997年04月 - 2001年03月   北海道大学   農学部   応用生命科学科

委員歴

• 2021年03月 - 現在   日本農芸化学会   英文誌編集委員
• 2017年09月 - 現在   応用糖質科学会   Journal of Applied Glycoscience編集委員
• 2011年04月 - 2013年03月   日本農芸化学会   北海道支部会計幹事

受賞

• 2020年09月 日本応用糖質科学会 奨励賞
   

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

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

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

  受賞者: 佐分利 亘
• 2016年09月 日本応用糖質科学会 ポスター賞
   

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

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

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

  受賞者: 佐分利 亘

論文

• 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 2024年10月
• 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年07月11日 

  Abstract Starch degradation in malted barley produces yeast-fermentable sugars. In this study, we compared the amylolytic enzymes and composition of the malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar established in Japan) and Kitanohoshi (the currently used cultivar for beer production). Hokudai 1 malt contained lower activity of amylolytic enzymes than Kitanohoshi malt, although these cultivars contained α-amylase AMY2 and β-amylase Bmy1 as the predominant enzymes. Malt starch hydrolysate of Hokudai 1 contained more limit dextrin and less yeast-fermentable sugars than that of Kitanohoshi. In mixed malt saccharification, a high Hokudai 1 malt ratio increased the limit dextrin levels and decreased the maltotriose and maltose levels. Even though Kitanohoshi malt contained more amylolytic enzymes than Hokudai 1 malt, addition of Kitanohoshi extract containing the amylolytic enzymes did not enhance malt starch degradation of Hokudai 1. Hokudai 1 malt starch was less degradable than Kitanohoshi malt starch.
• 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年04月29日 [査読有り]
   

  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 2023年11月 [査読有り][通常論文]
  
• 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年09月27日 [査読有り]
   

  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年09月21日 [査読有り]
   

  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年07月05日 [査読有り]
   

  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年07月01日 [査読有り]
   

  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年06月23日 [査読有り]
   

  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年03月30日 [査読有り]
   

  α-Glucosidase catalyzes the hydrolysis of α-d-glucosides and transglucosylation. Bacillus sp. AHU2216 α-glucosidase (BspAG13_31A), belonging to the glycoside hydrolase family 13 subfamily 31, specifically cleaves α-(1→4)-glucosidic linkages and shows high disaccharide specificity. We showed previously that the maltose moiety of maltotriose (G3) and maltotetraose (G4), covering subsites +1 and +2 of BspAG13_31A, adopts a less stable conformation than the global minimum energy conformation. This unstable d-glucosyl conformation likely arises from steric hindrance by Asn258 on β→α loop 5 of the catalytic (β/α)8-barrel. In this study, Asn258 mutants of BspAG13_31A were enzymatically and structurally analyzed. N258G/P mutations significantly enhanced trisaccharide specificity. The N258P mutation also enhanced the activity toward sucrose and produced erlose from sucrose through transglucosylation. N258G showed a higher specificity to transglucosylation with p-nitrophenyl α-d-glucopyranoside and maltose than the wild type. E256Q/N258G and E258Q/N258P structures in complex with G3 revealed that the maltose moiety of G3 bound at subsites +1 and +2 adopted a relaxed conformation, whereas a less stable conformation was taken in E256Q. This structural difference suggests that stabilizing the G3 conformation enhances trisaccharide specificity. The E256Q/N258G-G3 complex formed an additional hydrogen bond between Met229 and the d-glucose residue of G3 in subsite +2, and this interaction may enhance transglucosylation.
• 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 2023年03月11日 

  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年12月 [査読有り]
   

  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年10月15日 [査読有り]
   

  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年03月01日 [査読有り]
   

  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年01月24日 [査読有り]
   

  Plants possess many glycoside hydrolase family 1 (GH1) β-glucosidases, which physiologically function in cell wall metabolism and activation of bioactive substances, but most remain uncharacterized. One GH1 isoenzyme AtBGlu42 in Arabidopsis thaliana has been identified to hydrolyze scopolin using the gene deficient plants, but no enzymatic properties were obtained. Its sequence similarity to another functionally characterized enzyme Os1BGlu4 in rice suggests that AtBGlu42 also acts on oligosaccharides. Here, we show that the recombinant AtBGlu42 possesses high kcat/Km not only on scopolin, but also on various β-glucosides, cellooligosaccharides, and laminarioligosaccharides. Of the cellooligosaccharides, cellotriose was the most preferred. The crystal structure, determined at 1.7 Å resolution, suggests that Arg342 gives unfavorable binding to cellooligosaccharides at subsite +3. The mutants R342Y and R342A showed the highest preference on cellotetraose or cellopentaose with increased affinities at subsite +3, indicating that the residues at this position have an important role for chain length specificity.
• 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年01月 [査読有り]
   

  Dextran dextrinase (DDase) catalyzes formation of the polysaccharide dextran from maltodextrin. During the synthesis of dextran, DDase also generates the beneficial material isomaltomegalosaccharide (IMS). The term megalosaccharide is used for a saccharide having DP = 10-100 or 10-200 (DP, degree of polymerization). IMS is a chimeric glucosaccharide comprising α-(1 → 6)- and α-(1 → 4)-linked portions at the nonreducing and reducing ends, respectively, in which the α-(1 → 4)-glucosyl portion originates from maltodextrin of the substrate. In this study, IMS was produced by a practical approach using extracellular DDase (DDext) or cell surface DDase (DDsur) of Gluconobacter oxydans ATCC 11894. DDsur was the original form, so we prepared DDext via secretion from intact cells by incubating with 0.5% G6/G7 (maltohexaose/maltoheptaose); this was followed by generation of IMS from various concentrations of G6/G7 substrate at different temperatures for 96 h. However, IMS synthesis by DDext was limited by insufficient formation of α-(1 → 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of α-(1 → 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 °C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP = 14.7) with 61% α-(1 → 6)-glucosyl content in 51% yield. Increases in substrate concentration and agitation efficiency were found to decrease dextran formation and increase IMS production, which improved the reaction conditions for DDext. Under modified conditions (20% G6/G7, agitation speed of 100 rpm at 45 °C), DDext produced IMS (average DP = 14.5) with 65% α-(1 → 6)-glucosyl content in a good yield of 87%. KEY POINTS: • Beneficial IMS was produced using thermostabilized DDase. • Optimum conditions for reduced dextran formation were successfully determined. • A practical approach was established to provide IMS with a great yield of 87%.
• 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年09月10日 [査読有り]
   

  Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.
• 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年07月14日 [査読有り]
   

  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年02月02日 [査読有り]
  
• 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 2021年 [査読有り]
  
• 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年02月 [査読有り][通常論文]
   

  Trehalose 6-phosphate (Tre6P) is an important intermediate for trehalose biosynthesis. Recent researches have revealed that Tre6P is an endogenous signaling molecule that regulates plant development and stress responses. The necessity of Tre6P in physiological studies is expected to be increasing. To achieve the cost-effective production of Tre6P, a novel approach is required. In this study, we utilized trehalose 6-phosphate phosphorylase (TrePP) from Lactococcus lactis to produce Tre6P. In the reverse phosphorolysis by the TrePP, 91.9 mM Tre6P was produced from 100 mM β-glucose 1-phosphate (β-Glc1P) and 100 mM glucose 6-phosphate (Glc6P). The one-pot reaction of TrePP and maltose phosphorylase (MP) enabled production of 65 mM Tre6P from 100 mM maltose, 100 mM Glc6P, and 20 mM inorganic phosphate. Addition of β-phosphoglucomutase to this reaction produced Glc6P from β-Glc1P and thus reduced requirement of Glc6P as a starting material. Within the range of 20-469 mM inorganic phosphate tested, the 54 mM concentration yielded the highest amount of Tre6P (33 mM). Addition of yeast increased the yield because of its glucose consumption. Finally, from 100 mmol maltose and 60 mmol inorganic phosphate, we successfully achieved production of 37.5 mmol Tre6P in a one-pot reaction (100 mL), and 9.4 g Tre6P dipotassium salt was obtained.
• 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年07月 [査読有り]
   

  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 2019年07月 [査読有り][通常論文]
  
• 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 2019年06月 [査読有り][通常論文]
  
• 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年04月 [査読有り][通常論文]
  
• 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 2019年02月 [査読有り][通常論文]
  
• 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 2018年09月 [査読有り][通常論文]
   

  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 2018年09月01日 [査読有り][通常論文]
   

  Bifidobacteria are a major component of the intestinal microbiota in humans, particularly breast-fed infants. Therefore, elucidation of the mechanisms by which these bacteria colonize the intestine is desired. One approach is transposon mutagenesis, a technique currently attracting much attention because, in combination with next-generation sequencing, it enables exhaustive identification of genes that contribute to microbial fitness. We now describe a transposon mutagenesis system for Bifidobacterium longum subsp. longum 105-A (JCM 31944) based on ISBlo11, a native IS3 family insertion sequence. To build this system, xylose-inducible or constitutive bifidobacterial promoters were tested to drive the expression of full-length or a truncated form at the N terminus of the ISBlo11 transposase. An artificial transposon plasmid, pBFS12, in which ISBlo11 terminal inverted repeats are separated by a 3-bp spacer, was also constructed to mimic the transposition intermediate of IS3 elements. The introduction of this plasmid into a strain expressing transposase resulted in the insertion of the plasmid with an efficiency of >103 CFU/μg DNA. The plasmid targets random 3- to 4-bp sequences, but with a preference for noncoding regions. This mutagenesis system also worked at least in B. longum NCC2705. Characterization of a transposon insertion mutant revealed that a putative α-glucosidase mediates palatinose and trehalose assimilation, demonstrating the suitability of transposon mutagenesis for loss-of-function analysis. We anticipate that this approach will accelerate functional genomic studies of B. longum subsp. longumIMPORTANCE Several hundred species of bacteria colonize the mammalian intestine. However, the genes that enable such bacteria to colonize and thrive in the intestine remain largely unexplored. Transposon mutagenesis, combined with next-generation sequencing, is a promising tool to comprehensively identify these genes but has so far been applied only to a small number of intestinal bacterial species. In this study, a transposon mutagenesis system was established for Bifidobacterium longum subsp. longum, a representative health-promoting Bifidobacterium species. The system enables the identification of genes that promote colonization and survival in the intestine and should help illuminate the physiology of this species.
• 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 2018年07月 [査読有り][通常論文]
  
• 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 2018年01月01日 [査読有り][通常論文]
   

  D-Mannose isomerase (MI) reversibly isomerizes D-mannose to D-fructose, and is attractive for producing D-mannose from inexpensive D-fructose. It belongs to the N-acylglucosamine 2-epimerase (AGE) superfamily along with AGE, cellobiose 2-epimerase (CE), and aldose-ketose isomerase (AKI). In this study, Marinomonas mediterranea Marme_2490, showing low sequence identity with any known enzymes, was found to isomerize D-mannose as its primary substrate. Marme_2490 also isomerized D-lyxose and 4-OH D-mannose derivatives (D-talose and 4-O-monosaccharyl-D-mannose). Its activity for D-lyxose is known in other D-mannose isomerizing enzymes, such as MI and AKI, but we identified, for the first time, its activity for 4-OH D-mannose derivatives. Marme_2490 did not isomerize D-glucose, as known MIs do not, while AKI isomerizes both D-mannose and D-glucose. Thus, Marme_2490 was concluded to be an MI. The initial and equilibrium reaction products were analyzed by NMR to illuminate mechanistic information regarding the Marme_2490 reaction. The analysis of the initial reaction product revealed that β-D-mannose was formed. In the analysis of the equilibrated reaction products in D2O, signals of 2-H of D-mannose and 1-H of D-fructose were clearly detected. This indicates that these protons are not substituted with deuterium from D2O and Marme_2490 catalyzes the intramolecular proton transfer between 1-C and 2-C. The crystal structure of Marme_2490 in a ligand-free form was determined and found that Marme_2490 is formed by an (α/α)6-barrel, which is commonly observed in AGE superfamily enzymes. Despite diverse reaction specificities, the orientations of residues involved in catalysis and substrate binding by Marme_2490 were similar to those in both AKI (Salmonella enterica AKI) and epimerase (Rhodothermus marinus CE). The Marme_2490 structure suggested that the α7→α8 and α11→α12 loops of the catalytic domain participated in the formation of an open substrate-binding site to provide sufficient space to bind 4-OH D-mannose derivatives.
• 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  2017年07月 [査読有り][通常論文]
   

  In plants, cis-jasmone (CJ) is synthesized from a-linolenic acid (LA) via two biosynthetic pathways using jasmonic acid (JA) and iso-12-oxo-phytodienoic acid (iso-OPDA) as key intermediates. However, there have been no reports documenting CJ production by microorganisms. In the present study, the production of fungal-derived CJ by Lasiodiplodia theobromae was observed for the first time, although this production was not observed for Botrytis cinerea, Verticillium longisporum, Fusarium oxysporum, Gibberella fujikuroi, and Cochliobolus heterostrophus. To investigate the biosynthetic pathway of CJ in L. theobromae, administration experiments using [18,18,18-H-2(3), 17,17-H-2(2)] LA (LA-d5), [18,18,18-H-2(3), 17,17-H-2(2)]12-oxo-phytodienoic acid (cis-OPDA-d5), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 8:0 (OPC8-d6), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 6:0 (OPC6-d6), [5', 5', 5'-H-2(3), 4', 4'-H-2(2), 3'-H-2(1)] OPC 4:0 (OPC4-d6), and [11,11-H-2(2), 10,10-H-2(2), 8,8-H-2(2), 2,2-H-2(2)] methyl iso-12-oxo-phytodienoate (iso-MeOPDA-d8) were carried out, revealing that the fungus produced CJ through a single biosynthetic pathway via iso-OPDA. Interestingly, it was suggested that the previously predicted decarboxylation step of 3,7-didehydroJA to afford CJ might not be involved in CJ biosynthesis in L. theobromae.
• 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 2017年 [査読有り][通常論文]
   

  Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce beta-D-glucose 1-phosphate (beta-Glc1P) and D-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40 degrees C, and was stable in the pH range 5.0-8.0 and at up to 30 degrees C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, beta-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, D-mannose 6-phosphate (Man6P). From beta-Glc1P and Man6P, a novel sugar phosphate, alpha-D-Glcp-(1 <-> 1)-alpha-D-Manp6P, was synthesized with 51% yield.
• 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 2016年07月 [査読有り][通常論文]
   

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

  Okuyama M, Saburi W, Mori H, Kimura A

  Cellular and molecular life sciences : CMLS 73 14 2727 - 2751 2016年07月 [査読有り][通常論文]
   

  alpha-Glucosidases (AGases) and alpha-1,4-glucan lyases (GLases) catalyze the degradation of alpha-glucosidic linkages at the non-reducing ends of substrates to release alpha-glucose and anhydrofructose, respectively. The AGases belong to glycoside hydrolase (GH) families 13 and 31, and the GLases belong to GH31 and share the same structural fold with GH31 AGases. GH13 and GH31 AGases show diverse functions upon the hydrolysis of substrates, having linkage specificities and size preferences, as well as upon transglucosylation, forming specific alpha-glucosidic linkages. The crystal structures of both enzymes were determined using free and ligand-bound forms, which enabled us to understand the important structural elements responsible for the diverse functions. A series of mutational approaches revealed features of the structural elements. In particular, amino-acid residues in plus subsites are of significance, because they regulate transglucosylation, which is used in the production of industrially valuable oligosaccharides. The recently solved three-dimensional structure of GLase from red seaweed revealed the amino-acid residues essential for lyase activity and the strict recognition of the alpha-(1 -> 4)-glucosidic substrate linkage. The former was introduced to the GH31 AGase, and the resultant mutant displayed GLase activity. GH13 and GH31 AGases hydrate anhydrofructose to produce glucose, suggesting that AGases are involved in the catabolic pathway used to salvage unutilized anhydrofructose.
• 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 2016年06月 [査読有り][通常論文]
   

  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 α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis).

  Masuda Y, Okuyama M, Iizuka T, Nakai H, Saburi W, Fukukawa T, Maneesan J, Tagami T, Naraoka T, Mori H, Kimura A

  Bioscience, biotechnology, and biochemistry 80 3 479 - 485 2016年03月 [査読有り][通常論文]
   

  Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an -glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 degrees C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest V-max/K-m value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.
• Structural 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 2016年03月 [査読有り][通常論文]
   

  In Ruminococcus albus, 4-Omicron-beta-D-mannosyl-D-glucose phosphorylase (RaMP1) and beta-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze beta-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-Omicron-beta-D-mannosyl-D-glucose and RaMP2 with/without beta-(1 -> 4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.
• 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 2015年12月 [査読有り][通常論文]
   

  Obesity is one of the major health problems throughout the world. The present study investigated the preventive effect of epilactose - a rare non-digestible disaccharide - on obesity and metabolic disorders in mice fed high-fat (HF) diets. Feeding with HF diets increased body weight gain, fat pad weight and adipocyte size in mice (P<0.01), and these increases were effectively prevented by the use of supplemental epilactose without influencing food intake (P<0.01). Caecal pools of SCFA such as acetic and propionic acids in mice fed epilactose were higher compared with mice not receiving epilactose. Supplemental epilactose increased the expression of uncoupling protein (UCP)-1, which enhances energy expenditure, to 2-fold in the gastrocnemius muscle (P=0.04) and to 1.3-fold in the brown adipose tissue (P=0.02) in mice fed HF diets. Feeding HF diets induced pro-inflammatory macrophage infiltration into white adipose tissue, as indicated by the increased expression of monocyte chemotactic protein-1, TNF-alpha and F4/80, and these increases were attenuated by supplemental epilactose. In differentiated myogenic-like C2C12 cells, propionic acid, but not acetic or n-butyric acids, directly enhanced UCP-1 expression by approximately 2-fold (P<0.01). Taken together, these findings indicate that the epilactose-mediated increase in UCP-1 in the skeletal muscle and brown adipose tissue can enhance whole-body energy expenditure, leading to effective prevention of obesity and metabolic disorders in mice fed HF diets. It is suggested that propionic acid - a bacterial metabolite - acts as a mediator to induce UCP-1 expression in skeletal muscles.
• Identification of rice Os4BGlu13 as a β-glucosidase which hydrolyzes gibberellin A4 1-O-β-d-glucosyl ester, in addition to tuberonic acid glucoside and salicylic acid derivative glucosides.

  Hua Y, Ekkhara W, Sansenya S, Srisomsap C, Roytrakul S, Saburi W, Takeda R, Matsuura H, Mori H, Ketudat Cairns JR

  Archives of biochemistry and biophysics 583 36 - 46 2015年10月 [査読有り][通常論文]
   

  Gibberellin 1-O-beta-D-glucose ester hydrolysis activity has been detected in rice seedling extracts, but no enzyme responsible for this activity has ever been purified and identified. Therefore, gibberellin A4 glucosyl ester (GA(4)-GE) beta-D-glucosidase activity was purified from ten-day rice seedling stems and leaves. The family 1 glycoside hydrolase Os4BGlu13 was identified in the final purification fraction. The Os4BGlu13 cDNA was amplified from rice seedlings and expressed as an N-terminal thioredoxin-tagged fusion protein in Escherichia coll. The purified recombinant Os4BGlu13 protein (rOs4BGlu13) had an optimum pH of 4.5, for hydrolysis of p-nitrophenyl beta-D-glucopyranoside (pNPGlc), which was the best substrate identified, with a k(cat)/K-m of 637 mM(-1) s(-1). rOs4BGlu13 hydrolyzed helicin best among natural glycosides tested (K-cat/K-m, of 74.4 mM(-1) s(-1)). Os4BGlu13 was previously designated tuberonic acid glucoside (TAG) beta-glucosidase (TAGG), and here the k(cat)/K-m of rOsBGlu13 for TAG was 6.68 mM(-1) s(-1), while that for GA4-GE was 3.63 mM(-1) s(-1) and for salicylic acid glucoside (SAG) is 0.88 mM(-1) s(-1). rOs4BGlu13 also hydrolyzed oligosaccharides, with preference for short beta-(1 -> 3)-linked over beta-(1 -> 4)linked glucooligosaccharides. The enzymatic data suggests that Os4BGlu13 may contribute to TAG, SAG, oligosaccharide and GA4-GE hydrolysis in the rice plant, although helicin or a similar compound may be its primary target. (C) 2015 Elsevier Inc. All rights reserved.
• Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-β-d-mannosyl-d-glucose phosphorylase-like protein, UnkA.

  Saburi W, Tanaka Y, Muto H, Inoue S, Odaka R, Nishimoto M, Kitaoka M, Mori H

  Bioscience, biotechnology, and biochemistry 79 6 969 - 977 2015年06月 [査読有り][通常論文]
   

  The aerobic soil bacterium Cellvibrio vulgaris has a beta-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting d-mannose to d-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-beta-d-mannosyl-d-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and beta-(1 -> 4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for beta-(1 -> 4)-mannobiose was 5.5x10(4)-fold higher than it was for d-mannose. Recombinant UnkA phosphorolyzed beta-d-mannosyl-(1 -> 4)-d-glucose and specifically utilized d-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-beta-d-mannosyl-d-glucose phosphorylase.
• 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 2015年03月 [査読有り][通常論文]
   

  Glycoside hydrolase family 13 contains exo-glucosidases specific for alpha-(1 -> 4)- and alpha-(1 -> 6)-linkages including alpha-glucosidase, oligo-1,6-glucosidase, and dextran glucosidase. The alpha-(1 -> 6)-linkage selectivity of Streptococcus mutans dextran glucosidase was altered to alpha-(1 -> 4)-linkage selectivity through site-directed mutations at Val195, Lys275, and Glu371. V195A showed 1300-fold higher k(cat)/K-m for maltose than wild-type, but its k(cat)/K-m for isomaltose remained 2-fold higher than for maltose. K275A and E371A combined with V195A mutation only decreased isomaltase activity. V195A/K275A, V195A/E371A, and V195A/K275A/E371A showed 27-, 26-, and 73-fold higher k(cat)/K-m for maltose than for isomaltose, respectively. Consequently, the three residues are structural elements for recognition of the alpha-(1 -> 6)-glucosidic linkage. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• 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 2015年02月 [査読有り][通常論文]
   

  Streptococcus mutans dextran glucosidase (SmDG) belongs to glycoside hydrolase family 13, and catalyzes both the hydrolysis of substrates such as isomaltooligosaccharides and subsequent transglucosylation to form alpha-(1 -> 6)-glucosidic linkage at the substrate non-reducing ends. Here, we report the 2.4 angstrom resolution crystal structure of glucosyl-enzyme intermediate of SmDG. In the obtained structure, the Trp238 side-chain that constitutes the substrate-binding site turned away from the active pocket, concurrently with conformational changes of the nucleophile and the acid/base residues. Different conformations of Trp238 in each reaction stage indicated its flexibility. Considering the results of kinetic analyses, such flexibility may reflect a requirement for the reaction mechanism of SmDG. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• 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 2015年 [査読有り][通常論文]
   

  Gluconobacter oxydans ATCC 11894 produces dextran dextrinase (DDase, EC 2.4.1.2), which synthesizes dextran from the starch hydrolysate, dextrin and is known to cause ropy beer. G. oxydans ATCC 11894 was believed to possess both a secreted DDase (DDext) and an intracellular DDase (DDint), expressed upon cultivation with dextrin and glucose, respectively. However, genomic Southern blot, peptide mass fingerprinting and reaction product-pattern analyses revealed that both DDext and DDint were identical. The activity in the cell suspension and its liberation from the spheroplast cells indicated that DDint was localized on the cell surface. The localization of DDase was altered during the culture depending on the growth phase. During the early growth stage, DDase was exclusively liberated into the medium (DDext), and the cell-associated form (DDint) appeared after depletion of glucose from the medium. (C) 2014 Elsevier Inc. All rights reserved.
• Biochemical properties and substrate recognition mechanism of GH31 α-glucosidase from Bacillus sp. AHU 2001 with broad substrate specificity.

  Saburi W, Okuyama M, Kumagai Y, Kimura A, Mori H

  Biochimie 108 108 140 - 148 Elsevier 2015年 [査読有り][通常論文]
   

  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 2014年05月 [査読有り][通常論文]
   

  The hydrolytic activity of a thermophilic alkalophilic alpha-amylase from Bacillus sp. AAH-31 (AmyL) toward soluble starch was enhanced through optimization of amino acid (aa) residues situated near the substrate binding site. Twenty-four selected aa residues were replaced with Ala, and Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of AmyL's aa sequence with related enzymes. Y426A, H431A,I509A, and K549A showed notably higher activity than the wild type at 162-254% of wildtype activity. Tyr426, His431, and Ile509 were predicted to be located near subsite -2, while Lys549 was near subsite +2. Ser, Ala, Ala, and Met were found to be the best aa residues for the positions of Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single mutations at distant positions were effective in enhancing catalytic activity. The double-mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of the selected single mutations, showed 340%, 252%, and 271% of wild type activity, respectively. Triple and quadruple-mutant enzymes of the selected mutations did not show higher activity than the best double-mutant, Y426S/K549M. (C) 2014 Elsevier B.V. All rights reserved.
• Crystallization and preliminary X-ray crystallographic analysis of α-glucosidase HaG from Halomonas sp. strain H11.

  Shen X, Saburi W, Gai ZQ, Komoda K, Yu J, Ojima-Kato T, Kido Y, Matsui H, Mori H, Yao M

  Acta crystallographica. Section F, Structural biology communications 70 Pt 4 464 - 466 2014年04月 [査読有り][通常論文]
   

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

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

  The Journal of biological chemistry 289 6 3405 - 3415 2014年02月 [査読有り][通常論文]
   

  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-β-D-mannosyl-D-glucose phosphorylase from Rhodothermus marinus.

  Jaito N, Saburi W, Odaka R, Kido Y, Hamura K, Nishimoto M, Kitaoka M, Matsui H, Mori H

  Bioscience, biotechnology, and biochemistry 78 2 263 - 270 2014年02月 [査読有り][通常論文]
   

  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 2013年11月 [査読有り][通常論文]
   

  Dextran glucosidase from Streptococcus mutans (SmDG) catalyzes the hydrolysis of an alpha-1,6-glucosidic linkage at the non-reducing end of isomaltooligosaccharides and dextran. This enzyme has an Asp-194 catalytic nucleophile and two catalytically unrelated Cys residues, Cys-129 and Cys-532. Cys-free SmDG was constructed by replacement with Ser (C129S/C532S (2CS), the activity of which was the same as that of the wild type, SmDG). The nucleophile mutant of 2CS was generated by substitution of Asp-194 with Cys (D194C-2CS). The hydrolytic activity of D194C-2CS was 8.1 x 10(-4) % of 2CS. KI-associated oxidation of D194C-2CS increased the activity up to 0.27% of 2CS, which was 330 times higher than D194C-2CS. Peptide-mapping mass analysis of the oxidized D194C-2CS (Ox-D194C-2CS) revealed that Cys-194 was converted into cysteine sulfinate. Ox-D194C-2CS and 2CS shared the same properties (optimum pH, pI, and substrate specificity), whereas Ox-D194C-2CS had much higher transglucosylation activity than 2CS. This is the first study indicating that a more acidic nucleophile (-SOO-) enhances transglycosylation. The introduction of cysteine sulfinate as a catalytic nucleophile could be a novel approach to enhance transglycosylation.
• Characterization of Ruminococcus albus cellodextrin phosphorylase and identification of a key phenylalanine residue for acceptor specificity and affinity to the phosphate group.

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

  The FEBS journal 280 18 4463 - 4473 2013年09月 [査読有り][通常論文]
   

  Ruminococcus albus has the ability to intracellularly degrade cello-oligosaccharides primarily via phosphorolysis. In this study, the enzymatic characteristics of R. albus cellodextrin phosphorylase (RaCDP), which is a member of glycoside hydrolase family 94, was investigated. RaCDP catalyzes the phosphorolysis of cellotriose through an ordered 'bi bi' mechanism in which cellotriose binds to RaCDP before inorganic phosphate, and then cellobiose and glucose 1-phosphate (Glc1P) are released in that order. Among the cello-oligosaccharides tested, RaCDP had the highest phosphorolytic and synthetic activities towards cellohexaose and cellopentaose, respectively. RaCDP successively transferred glucosyl residues from Glc1P to the growing cello-oligosaccharide chain, and insoluble cello-oligosaccharides comprising a mean of eight residues were produced. Sophorose, laminaribiose, β-1,4-xylobiose, β-1,4-mannobiose and cellobiitol served as acceptors for RaCDP. RaCDP had very low affinity for phosphate groups in both the phosphorolysis and synthesis directions. A sequence comparison revealed that RaCDP has Gln at position 646 where His is normally conserved in the phosphate binding sites of related enzymes. A Q646H mutant showed approximately twofold lower apparent Km values for inorganic phosphate and Glc1P than the wild-type. RaCDP has Phe at position 633 corresponding to Tyr and Val in the +1 subsites of cellobiose phosphorylase and N,N′-diacetylchitobiose phosphorylase, respectively. A F633Y mutant showed higher preference for cellobiose over β-1,4-mannobiose as an acceptor substrate in the synthetic reaction than the wild-type. Furthermore, the F633Y mutant showed 75- and 1100-fold lower apparent Km values for inorganic phosphate and Glc1P, respectively, in phosphorolysis and synthesis of cellotriose. 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 2013年09月 [査読有り][通常論文]
   

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

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

  Bioscience, biotechnology, and biochemistry 77 9 1867 - 1873 2013年09月 [査読有り][通常論文]
   

  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 β-glucosidase with high hydrolytic activity towards salicylic acid β-D-glucoside.

  Himeno N, Saburi W, Wakuta S, Takeda R, Matsuura H, Nabeta K, Sansenya S, Ketudat Cairns JR, Mori H, Imai R, Matsui H

  Bioscience, biotechnology, and biochemistry 77 5 934 - 939 2013年05月 [査読有り][通常論文]
   

  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 2013年04月 [査読有り][通常論文]
   

  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 2013年01月 [査読有り][通常論文]
   

  Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of 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 2013年 [査読有り][通常論文]
   

  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 2013年 [査読有り][通常論文]
   

  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, β-1,4-MANNOOLIGOSACCHARIDE PHOSPHORYLASE.

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

  The Journal of biological chemistry 287 50 42389 - 42399 50 2012年12月 [査読有り][通常論文]
   

  Ruminococcus albus is a typical ruminal bacterium digesting cellulose and hemicellulose. Cellobiose 2-epimerase (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 2012年09月 [査読有り][通常論文]
   

  Bacteroides thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family 66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482) in Escherichia coli and determined that recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization > 4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase 66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases.
• 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 2012年08月 [査読有り][通常論文]
   

  Cellobiose 2-epimerase (CE) efficiently forms epilactose which has several beneficial biological functions. A thermostable CE from Rhodothermus marinus was immobilized on Duolite A568 and packed into a column. Lactose (100 g/L) was supplied to the reactor, kept at 50 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 α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31.

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

  Bioscience, biotechnology, and biochemistry 76 7 1378 - 1383 7 2012年07月 [査読有り][通常論文]
   

  α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS–PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimu...
• 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 2012年06月 [査読有り][通常論文]
   

  A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val(837)-Met(932) for PsDex and Tyr(404)-Tyr(492) for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala(39)-Ser(1304)) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp(189), Asp(340), Glu(412), and Asp(1254) of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1,500 to 1/40,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using alpha-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a beta- or alpha-isomaltotetraosyl azide, respectively, strongly indicating Asp(340) and Glu(412) as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from alpha-isomaltotetraosyl fluoride in the presence of NaN3.
• α-Glucosylated 6-gingerol: chemoenzymatic synthesis using α-glucosidase from Halomonas sp. H11, and its physical properties.

  Ojima T, Aizawa K, Saburi W, Yamamoto T

  Carbohydrate research 354 59 - 64 2012年06月 [査読有り][通常論文]
   

  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 α-glucosidase-catalyzed conversion of 1,5-anhydrofructose.

  Kim YM, Saburi W, Yu S, Nakai H, Maneesan J, Kang MS, Chiba S, Kim D, Okuyama M, Mori H, Kimura A

  The Journal of biological chemistry 287 27 22441 - 22444 27 2012年06月 [査読有り][通常論文]
   

  alpha-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form alpha-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for alpha-glucosidases. alpha-Glucosidase-catalyzed hydration produced alpha-glucose from AF, which plateaued with time. Combined reaction with alpha-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger alpha-glucosidase (31AG), which is stable in organic solvent, produced ethyl alpha-glucoside from AF in 80% ethanol. The findings indicate that alpha-glucosidases catalyze trans-addition. This is the first report of alpha-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.
• 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 2012年04月 [査読有り][通常論文]
   

  Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose to produce alpha-D-glucopyranosyl phosphate (Glc1P) and D-glucose. It is an essential enzyme for the metabolism of cello-oligosaccharides in a ruminal bacterium, Ruminococcus albus. In this study, recombinant R. albus CBP (RaCBP) produced in Escherichia coli was characterized. It showed highest activity at pH 6.2 at 50 degrees C, and was stable in a pH range of 5.5-8.8 and at below 40 degrees C. It phosphorolyzed only cellobiose efficiently, and the reaction proceeded through a random-ordered bi hi mechanism, by which inorganic phosphate and cellobiose bind in random order and D-glucose is released before Glc1P. In the synthetic reaction, RaCBP showed highest activity to D-glucose, followed by 6-deoxy-D-glucose. D-Mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, 1,5-anhydro-D-glucitol, and gentiobiose also served as acceptors, although the activities for them were much lower than for D-glucose. D-Glucose acted as a competitive-uncompetitive inhibitor of the reverse synthetic reaction, which bound not only the Glc1P site (competitive) but also the ternary enzyme-Glc1P-D-glucose complex (uncompetitive).
• Characterization of Halomonas sp. strain H11 α-glucosidase activated by monovalent cations and its application for efficient synthesis of α-D-glucosylglycerol.

  Ojima T, Saburi W, Yamamoto T, Kudo T

  Applied and environmental microbiology 78 6 1836 - 1845 6 2012年03月 [査読有り][通常論文]
   

  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 2011年11月 [査読有り][通常論文]
   

  Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of beta-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinas DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 degrees C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 degrees C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.
• 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 2011年06月 [査読有り][通常論文]
   

  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.
• 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

  Journal of Applied Glycoscience Supplement 2011 0 74 - 74 The Japanese Society of Applied Glycoscience 2011年 [査読有り][通常論文]
  
• 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 2010年10月 [査読有り][通常論文]
   

  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.
• 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

  Journal of Applied Glycoscience Supplement 2010 90 - 90 日本応用糖質科学会 2010年
• 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

  Journal of Applied Glycoscience Supplement 2009 34 - 34 日本応用糖質科学会 2009年
• 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 2008年12月 [査読有り][通常論文]
   

  Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an alpha-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms of substrate chain length specificity. SMDG efficiently hydrolyzes both short- and long-chain substrates, while O16G acts on only short- chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short beta -> alpha loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced by a smaller amino acid, alanine, asparagine or proline; and (ii) short beta -> alpha loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates, as implied by structural analysis.
• 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 2008年12月 [査読有り][通常論文]
  
• 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年09月 [査読有り][通常論文]
   

  α-Glucosidase (EC 3.2.1.20), an exo-glycosylase to hydrolyze α-glucosidic linkage, is characterized by the variety of substrate specificity. Enzyme also catalyzes the transglucosylation, on which industrial interests focus due to the production of valuable glucooligosaccharides. α-Glucosidase is a physiologically important enzyme in most of organisms (microorganisms, insects, plants and animals including human). Therefore, there are many types of α-glucosidases to display unique functions, in which we are interested. This report describes the recently analyzed unique functions of α-glucosidases by mainly focusing on honeybee α-glucosidase isoenzymes, dextran glucosidase, multiple forms of rice α-glucosidases, and Escherichia coli α-xylosidase. © 2008 Woodhead Publishing Limited. All rights reserved.
• Substrate recognition mechanism of alpha-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans

  Hironori Hondoh, Wataru Saburi, Haruhide Mori, Masayuki Okuyama, Toshitaka Nakada, Yoshiki Matsuura, Atsuo Kimura

  JOURNAL OF MOLECULAR BIOLOGY 378 4 913 - 922 2008年05月 [査読有り][通常論文]
   

  We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the alpha-1,6-glucosidic linkage of isomaltooli-gosaccharides from their non-reducing ends to produce alpha-glucose. By using the mutant of catalytic acid Glu236 -> Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 angstrom resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (beta/alpha)(8)-barrel in domain A, which is common to the alpha-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites -1 to +2. The environment of the glucose residue at subsite -1 is similar to the environment of this residue in the alpha-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite -1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile alpha-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile alpha-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the alpha-amylase structure from Bacillus subtilis. The comparison with the alpha-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of alpha-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes. (c) 2008 Elsevier Ltd. All rights reserved.
• 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 2007年09月 [査読有り][通常論文]
   

  Dextran glucosidase from Streptococcus mutans is an exo-hydrolase that acts on the nonreducing terminal alpha-1,6-glucosidic linkage of oligosaccharides and dextran with a high degree of transglucosylation. Based on amino-acid sequence similarity, this enzyme is classified into glycoside hydrolase family 13. Recombinant dextran glucosidase was purified and crystallized by the hanging-drop vapour-diffusion technique using polyethylene glycol 6000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 72.72, b = 86.47, c = 104.30 angstrom. A native data set was collected to 2.2 angstrom resolution from a single crystal.
• Structural elements in dextran glucosidase responsible for high specificity to long chain substrate.

  Saburi W, Mori H, Saito S, Okuyama M, Kimura A

  Biochimica et biophysica acta 1764 4 688 - 698 2006年04月 [査読有り][通常論文]
   

  Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta -> alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar k(cat)/k(m) value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta -> alpha loop 4 of B. subtilis oligo- 1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta ->alpha loop 4 were important for high activity to long-chain substrates. (c) 2006 Elsevier B.V. All rights reserved.
• Enzymatic synthesis of alkyl α-2-deoxylglucosides by alkyl alcohol resistant α-glucosidase from Aspergillus niger

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

  Tetrahedron Asymmetry 16 2 403 - 409 2005年01月24日 [査読有り][通常論文]
   

  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年 [査読有り][通常論文]
  
• 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 2004年12月 [査読有り][通常論文]
   

  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年
• 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年
• β1-3/1-6グルカンの部分構造をもつオリゴ糖の化学合成

  太田智也, 佐分利亘, 今場司朗, 森春英 日本農芸化学会大会講演要旨集(Web) 2023 2023年
• 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年
• 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年
• Microdochium nivale由来GH55ラミナリナーゼによるラミナリン分解機構の解明

  太田智也, 佐分利亘, 今場司朗, JEWELL Linda, HSIANG Tom, 今井亮三, 森春英 日本農芸化学会大会講演要旨集(Web) 2022 2022年
• 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年
• 【総説：―受賞論文―】 セロビオース2-エピメラーゼと類縁酵素群の構造と機能に関する研究

  佐分利 亘 応用糖質科学：日本応用糖質科学会誌 11 (1) 22 -34 2021年 [査読有り]
   

  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年
• 植物病原性真菌Microdochium nivale由来菌体外β-グルコシダーゼの機能解析

  太田智也, 佐分利亘, JEWELL Linda, HSIANG Tom, 今井亮三, 森春英 日本農芸化学会大会講演要旨集(Web) 2020 2020年
• 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年
• 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年
• 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年08月20日 [査読無し][通常論文]
  
• 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年
• 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年
• 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年01月 

  CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.
• グリコシル転移酵素を活用した穀物澱粉改変技術の開発

  佐分利 亘 年報 34 216 -221 2018年
• 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 (応用糖質科学シンポジウム)

  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年05月 [査読無し][通常論文]
   

  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年03月05日 [査読無し][通常論文]
  
• 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年10月07日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年
• 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年11月14日 [査読無し][通常論文]
  
• P055 Trehalose-induced Systemic Resistance(TSR)の分子基盤(ポスター発表,植物化学調節学会第50回大会)

  手塚 大介, 和久田 真司, 加藤 英樹, 松浦 英幸, 佐分 利亘, 森 春英, 松井 博和, 今井 亮三 植物化学調節学会研究発表記録集 50 (0) 73 -73 2015年10月01日 [査読無し][通常論文]
  
• 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年10月01日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• UDP-Glc非依存性配糖体化酵素による新規なジャスモン酸の配糖体化と その生理的意義

  竹松 知紀, 瀬戸 義哉, 宮澤 吉郎, 和久田 真司, 荻原 毅, 佐分利 亘, 森 春英, 高橋 公咲, 松浦 英幸 天然有機化合物討論会講演要旨集 57 Oral14 2015年 

  【目 的】

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

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

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

  【研究方法および結果】

  1. UDP-Glc非依存性配糖化酵素の発見

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

  2. イネ由来のUDP-Glc非依存性配糖化酵素の同定

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

  (View PDFfor the rest of the abstract.)

• 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年10月01日 [査読無し][通常論文]
  
• 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年10月01日 [査読無し][通常論文]
  
• 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年10月01日 [査読無し][通常論文]
  
• 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年10月01日 [査読無し][通常論文]
  
• 19. 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(口頭発表,植物化学調節学会第49回大会)

  高橋 直希, 川又 彩, 手塚 大介, 佐分利 亘, 松浦 英幸, 森 春英, 今井 亮三 植物化学調節学会研究発表記録集 49 (0) 37 -37 2014年10月01日 [査読無し][通常論文]
  
• 43. 傷害時のイネにおけるサリチル酸グルコシドを糖ドナーとするツベロン酸への糖転移反応(口頭発表,植物化学調節学会第49回大会)

  竹松 知紀, 瀬戸 義哉, 宮澤 吉郎, 和久田 真司, 佐分利 亘, 森 春英, 高橋 公咲, 松浦 英幸 植物化学調節学会研究発表記録集 49 (0) 61 -61 2014年10月01日 [査読無し][通常論文]
  
• 52. 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(口頭発表,植物化学調節学会第49回大会)

  手塚 大介, 坂井 志帆, 和久田 真司, 加藤 英樹, 松浦 英幸, 佐分利 亘, 森 春英, 松井 博和, 今井 亮三 植物化学調節学会研究発表記録集 49 (0) 70 -70 2014年10月01日 [査読無し][通常論文]
  
• 62. 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(口頭発表,植物化学調節学会第49回大会)

  武田 遼介, 佐分利 亘, 姫野 奈美, 和久田 真司, 松浦 英幸, 今井 亮三, 松井 博和, 森 春英 植物化学調節学会研究発表記録集 49 (0) 80 -80 2014年10月01日 [査読無し][通常論文]
  
• 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年09月22日 [査読無し][通常論文]
  
• 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年09月01日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年04月30日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年01月01日 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
   

  Acidophilic β-galactosidase is a useful enzyme as digestive supplement used to alleviate symptoms of lactose intolerance. Aspergilli are the source of several acidophilic β-galactosidases that retain enzymatic activity under gastric conditions. In this study, we investigated the extracellular acidophilic β-galactosidase activity of six Aspergillus niger strains, AHU7104, AHU7120, AHU7217, AHU7294, AHU7295 and AHU7296; A. niger AHU7120 was selected as an enzyme source. β-Galactosidase from A. niger AHU7120 (AnBGal) was purified from culture supernatant. Its N-terminal sequence was identical to that of An01g12150, which belongs to the glycoside hydrolase family 35, from A. niger CBS 513.88. The DNA sequence of AnBGal was identical to An01g12150. Recombinant AnBGal (rAnBGal) harboring yeast α-factor signal sequence was expressed in Pichia pastoris, and 21.9 mg of purified rAnBGal with 129 U/mg of enzyme activity was isolated from 200 mL of culture supernatant. Native and recombinant AnBGal enzymes showed similar pH optima, pH stability, and kinetics for p-nitrophenyl β-D-galactopyranoside and lactose; rAnBGal showed slightly lower thermal stability than the native enzyme. Lactose in milk was rapidly degraded by rAnBGal at higher pH values (range, 2.0‒3.5), consistent with the pH optimum of AnBGal. We estimated that 3.5 μM AnBGal may degrade ≥ 66% of lactose before gastric half-emptying of ingested milk. These data indicate that AnBGal may help alleviate symptoms of lactose intolerance.
• 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年 [査読無し][通常論文]
   

  Spectrophotometric quantification method of carbohydrates is useful for processing multiple samples. In this study, we established colorimetric quantification for 4-O-β-D-mannosyl-D-glucose (Man-Glc) and β-(1→4)-mannobiose (Man2). For quantification of Man-Glc, phosphorolysis of Man-Glc catalyzed by 4-O-β-D-mannosyl-D-glucose phosphorylase (MGP) was coupled with quantification of D-glucose by the glucose oxidase-peroxidase method. In addition to MGP, cellobiose 2-epimerase (CE) was added for quantification of Man2. In both quantifications, a good linear relationship was obtained between A505 and the sample concentration (0-0.5 mM). The A505 values obtained at various concentrations of Man2 and Man-Glc were almost identical to those with equivalent D-glucose concentrations. Kinetic parameters of Ruminococcus albus and Rhodothermus marinus CEs for the epimerization of Man2 were determined using the quantification method for Man-Glc. Both enzymes showed 5-15-fold higher kcat/Km values than those for cellobiose and lactose, which supports the prediction that these enzymes utilize Man2 as a substrate in the β-mannan metabolic pathway.
• 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年 [査読無し][通常論文]
  
• 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年11月27日 [査読無し][通常論文]
  
• 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年11月27日 [査読無し][通常論文]
  
• 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年11月27日 [査読無し][通常論文]
  
• 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年11月27日 [査読無し][通常論文]
  
• 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年11月27日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
  
• 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年05月20日 [査読無し][通常論文]
   

  セロビオース2-エピメラーゼ(CE)は,セロビオースやラクトースなどβ-1,4結合からなるオリゴ糖の還元末端のグルコース残基をマンノース残基に可逆的に異性化する。CEをラクトースに作用させて得られるエピラクトース(4-O-β-D-ガラクトシル-D-マンノース)は優れた腸内細菌叢改善効果を有し,新たな食品素材として有望である。本研究では,CEを用いたエピラクトースの実用的合成法の確立を目的とした。Ruminococcus albus由来CEのアミノ酸配列を基に配列類似性検索を行い,大規模培養が可能な好気性細菌よりCEホモログを探索した。見出されたCEのうちRhodothermus marinus由来酵素(RmCE)は耐熱性に優れ,エピラクトースの工業的製造に適した特性を備えていた。固定化RmCEによる連続反応では,遊離酵素よりも少ない酵素量でエピラクトースを合成可能なことが示された。反応液を濃縮してラクトースを結晶化,除去した後,樹脂分画もしくは結晶化を行うことでCE反応液からエピラクトースを約90%の純度に精製した。
• 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年05月 [査読無し][通常論文]
   

  セロビオース2-エピメラーゼ(CE)は，セロビオースやラクトースなどβ-1，4結合からなるオリゴ糖の還元末端のグルコース残基をマンノース残基に可逆的に異性化する。CEをラクトースに作用させて得られるエピラクトース(4-O-β-D-ガラクトシル-D-マンノース)は優れた腸内細菌叢改善効果を有し，新たな食品素材として有望である。本研究では，CEを用いたエピラクトースの実用的合成法の確立を目的とした。Ruminococcus albus由来CEのアミノ酸配列を基に配列類似性検索を行い，大規模培養が可能な好気性細菌よりCEホモログを探索した。見出されたCEのうちRhodothermus marinus由来酵素(RmCE)は耐熱性に優れ，エピラクトースの工業的製造に適した特性を備えていた。固定化RmCEによる連続反応では，遊離酵素よりも少ない酵素量でエピラクトースを合成可能なことが示された。反応液を濃縮してラクトースを結晶化，除去した後，樹脂分画もしくは結晶化を行うことでCE反応液からエピラクトースを約90%の純度に精製した。
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年11月01日 [査読無し][通常論文]
  
• 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年11月01日 [査読無し][通常論文]
  
• 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年11月01日 [査読無し][通常論文]
  
• 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年11月01日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年08月20日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年11月20日 [査読無し][通常論文]
   

  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年11月20日 [査読無し][通常論文]
   

  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年11月20日 [査読無し][通常論文]
   

  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年11月 [査読無し][通常論文]
   

  Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of beta-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinas DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 degrees C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 degrees C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.
• 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年09月 [査読無し][通常論文]
   

  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年08月25日 [査読無し][通常論文]
  
• 2Dp12 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(酵素学・酵素工学,一般講演)

  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 0 (63) 138 -138 2011年08月25日 [査読無し][通常論文]
  
• 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年08月 [査読無し][通常論文]
   

  Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl2 increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl2 also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca2+, were considered to stabilize SmDG. On the other hand, CaCl2 had no significant effect on catalytic reaction. The enhanced stability by Ca2+ was probably related to calcium binding in the beta -> alpha loop 1 of the (beta/alpha)(8) barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年12月31日 [査読無し][通常論文]
  
• α-グルコシダーゼとシクロデキストリン生成酵素の共反応による非還元性末端にα-1,3結合およびα-1,6結合したグルコシル基を有する二つのα-1,4-グルカンの生成

  佐分利 亘, 佐分利 上村, 飯塚 貴久, 山本 健, 高田 正保 Journal of applied glycoscience 57 (4) 231 -237 2010年10月20日 [査読無し][通常論文]
   

  イソマルトオリゴ糖やニゲロオリゴ糖などのオリゴ糖は，α-amylaseやβ-amylaseによりmaltoseやmaltotrioseまで澱粉を加水分解し，α-glucosidaseによる糖転移反応を行うことにより製造される．このため，当該シラップは，2-4糖の比較的低分子糖を主成分とする．一方，高分子糖は低甘味であり飲食物のコク味やボディー感増強効果がある他，低浸透圧のため小腸への刺激が少なくエネルギー給源として優れる．本研究では，シクロデキストリン生成酵素(CGTase)とα-glucosidaseを枝切り酵素存在下で同時に澱粉へ作用させることにより，β-amylase耐性グルカンが得られることを見出した．β-amylaseによる作用をほとんど受けないことから本グルカンは非還元性末端あるいはその近傍にα-1,4結合以外の結合が導入されたと考えられた．本グルカンの重合度は，α-glucosidaseの鎖長特異性に依存し，高分子基質に高活性を示す <i>Acremonium strictum</i> 酵素(ASG)を使用した場合は長鎖(重合度6-10)の，低分子基質へ高い特異性を有する <i>Aspergillus niger</i> 酵素(ANG)を使用した場合は短鎖(重合度4-6)の糖が主成分であった(Fig. 2)．得られたグルカンのβ-amylaseによる分解がα-amylaseにより促進されたことから還元性末端部は主にα-1,4-結合からなると考えられた．メチル化分析により，ASGおよびANGを用いて得られたグルカン(それぞれBRGIおよびBRGII)はそれぞれα-1,3結合およびα-1,6結合が非還元性末端あるいはその近傍に導入されたことが明らかとなった．BRGI含有シラップを調製し，澱粉加水分解物からなるコーンシラップと室温保存1カ月での外観を比較したところ，BRGI含有シラップはコーンシラップと異なり清澄性を保ち，高い耐老化性を示した(Fig. 8).
• 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年08月 [査読無し][通常論文]
   

  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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年05月01日 [査読無し][通常論文]
  
• Bacillus clarkii 7364 株が生産する γ-Cyclodextrin に高い特異性を有する二つの酵素

  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 Journal of applied glycoscience 57 (2) 121 -129 2010年04月20日 [査読無し][通常論文]
   

  好アルカリ性細菌<i>Bacillus clarkii</i> 7364株が菌体外に分泌するcyclodextrin glucanotransferase (CGTase)は，澱粉を基質としたとき，&gamma;-cyclodextrin (&gamma;-CD)を優先的に生成する&gamma;-CGTaseである．また，本酵素(Cgt)をコードする遺伝子の約200 bases下流には，cyclodextrinase (Cda)をコードする遺伝子が存在する．本研究では，CgtおよびCdaの機能解析を行った．Cgtは至適pH 10.0の好アルカリ酵素であった．Cgtを1%(w/v)可溶性澱粉に作用させると，CD組成は&alpha;-CD<0.1%，&beta;-CD 3.5%および&gamma;-CD 27.5%となった．Cgtおよび他起源のCGTaseのアミノ酸配列を比較したところ，Cgtではサブサイト+3，+2，-3および-7においてアミノ酸の欠失および相違が認められた．サブサイト+2に位置するAla223を三つの塩基性アミノ酸(His，LysおよびArg)に置換した変異体は，いずれも中性pHでの&gamma;-CD合成比活性が向上した．これは，Ala223の塩基性アミノ酸への置換により，プロトン化した側鎖のアミノ基が基質と相互作用を示すことによるものと推察される．一方，Cdaは&gamma;-CDのみに高い加水分解活性を示すcyclodextrinaseであった．さらに，Cdaはマルトトリオース単位で糖転移させる活性を保持していた．Cdaおよび他起源のCD分解酵素のアミノ酸配列を比較したところ，Cdaでは他起源のCD分解酵素が保持するNドメインが欠失し，C末端側が100アミノ酸以上長かった．CD分解酵素のNドメインは二量体形成に寄与するが，CdaはNドメインを欠失しているにもかかわらず，12量体を形成していた．これらの特徴は他起源のCD分解酵素では認められず，Cdaは新奇なCD分解酵素であることが示唆される．
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• Streptococcus mutans Dextran Glucosidase 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 Journal of applied glycoscience 56 (2) 111 -117 2009年04月20日 [査読無し][通常論文]
   

  <i>Streptococcus mutans</i>由来のdextran glucosidase(DGase)は，デキストランやイソマルトオリゴ糖の非還元末端の&alpha;-1,6結合を加水分解し，&alpha;-glucoseを遊離する保持型酵素である．またDGaseは，一次構造に基づきGH family 13に分類されることや，高い糖転移活性を示すことも明らかとなっている．本研究では，&alpha;-1,6結合に特異的に作用するDGaseの基質認識機構をX線結晶構造解析により解明した．X線結晶構造解析の結果，DGaseは(&beta;/&alpha;)₈バレル構造を持ち，その活性部位はポケット状になっていることが明らかとなった．イソマルトトリオース複合体の構造解析より，三つのサブサイト(-1～+2)が確認された．サブサイト-1において基質は多くの水素結合により認識されており，その様式はGH family 13の他の酵素と類似していた．加えてAsp60とArg398が基質のグルコース残基のO4原子と水素結合を形成しており，これらの結合が基質の非還元末端の認識に重要であると考えられる．サブサイト+1では，Lys275とGlu371が基質と水素結合しており，基質のコンフォメーションを制御していた．加えて切断される&alpha;-1,6結合の6位のメチレン基とVal195の間に疎水的相互作用が確認され，これらの残基が基質認識に重要であることが示唆された．
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• デキストリンデキストラナーゼ遺伝子の異種宿主発現および組換え酵素の解析

  貞廣 樹里, 佐分利 亘, 森 春英, 奥山 正幸, 岡田 嚴太郎, 木村 淳夫 Journal of Applied Glycoscience Supplement 2008 (0) 100 -100 2008年 [査読無し][通常論文]
  
• γ-CDに高い特異性を有するBacillus clarkii 7364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 Journal of Applied Glycoscience Supplement 2008 (0) 98 -98 2008年 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年08月30日 [査読無し][通常論文]
  
• 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年08月30日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年07月20日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• Purification, Characterization, and Sequence Analysis of Two α-Amylase Isoforms from Azuki Bean, Vigna angularis, Showing Different Affinity towards β-Cyclodextrin Sepharose(Biochemistry & Molecular Biology)

  MAR San San, MORI Haruhide, LEE Jin-Ha, FUKUDA Kenji, SABURI Wataru, FUKUHARA Arinobu, OKUYAMA Masayuki, CHIBA Seiya, KIMURA Atsuo Bioscience, biotechnology, and biochemistry 67 (5) 1080 -93 2003年05月 [査読無し][通常論文]
   

  Two α-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a β-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl_2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17) : k_<cat>, 71.8 and 55.5 s^<-1>, K_m, 0.113 and 0.097 mg/ml ; for blocked 4-nitr...
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  
• 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年03月05日 [査読無し][通常論文]
  

所属学協会

• 日本応用糖質科学会   日本農芸化学会   

共同研究・競争的資金等の研究課題

• 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

  研究期間 : 2021年07月 -2024年03月 

  代表者 : 今井 亮三, 佐分利 亘
• 糖質の効率的変換に有用な糖質異性化酵素の構造基盤の解明と機能強化

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2021年04月 -2024年03月 

  代表者 : 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．
• D-マンノ－ス骨格を持つ糖鎖による免疫・炎症反応制御とその分子基盤の解析

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2019年04月 -2022年03月 

  代表者 : 戸田 雅子, 佐分利 亘, 比能 洋, 新谷 尚弘

   

  真菌類や植物には結合様式の異なる様々なα型とβ型のマンナン（D-マンノ－スを含む多糖類）が含まれる。マンナンは「免疫系に対する機能を持つ食品成分」と注目され、その機能性の科学的解析が求められている。本研究はマンノ－ス骨格を持つ分子の免疫学的な意義を明らかすることを目的とする。また、日本の国菌と言われる「麹菌」を用いてマンノシル化アレルゲンを発現する麹菌を構築し、抗アレルギ－作用を持つマンナン分子の作製を目指す。本年度はまずβ型マンノオリゴ糖の調製を行い、その免疫機能性を解析した。その結果、β-Man-(1→4)-Manやβ-Man-(1→4)-Glc骨格を持つオリゴ糖の中で、β-1,4-マンノビオースがマウス骨髄樹状細胞を高レベルで活性化することが明らかになった。また、α型マンナンをβ-1,4-マンノビオースや他の多糖類と共に樹状細胞を刺激すると、樹状細胞による抗炎症サイトカイン産生が増強されることを見いだした。αマンノオリゴ糖に関しては、α-1,2およびα-1,6結合への伸長方向が任意に制御可能な保護基の組み合わせによるαマンノオリゴ糖の合成ルート構築を実施した。この目的を満たす共通中間体の最適化を行い、中間体を使用することによりそれぞれのα-1,2およびα-1,6結合型の直鎖オリゴ糖をベンジルグリコシド体として調製するルート構築に成功した。マンノシル化アレルゲンを発現する麹菌に関しては、オボアルブミン（OVA：モデルアレルゲン）の cDNAを麹菌グルコアミラーゼglaAの触媒ドメインをコードするDNAの下流に連結し、GlaA-OVA融合タンパク質として麹菌で発現させた。2日間液体培養した培養上清500 µL分をSDS-PAGEに供し、Coomassie Brilliant Blue R-250染色したところ、OVAの発現が確認できたが発現量は低かった。
• 糖質の多様化高機能化に向けた酵素法アプローチの新展開

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 森 春英, 佐分利 亘

   

  糖質は構成糖と結合様式により顕著な多様性を示す．食品機能性素材やバイオ素材等，素材の潜在的宝箱であり，機能発見と応用利用に向けて，多様性に対応可能な効率的合成手法の確立が必須である．本申請研究は，合成酵素と転移酵素利用を利用した糖質多様化に向けた合成手法の確立を目的としている． 合成酵素利用による糖質合成に関しては，ショ糖からの糖ヌクレオチド合成酵素の高機能化に向けた変異酵素等の解析，および安定的な利用条件について検討を行った．糖供与体基質としてショ糖に依存しない糖ヌクレオチド合成酵素について，利用条件の検討を進めた．本酵素単独での反応による新規オリゴ糖数種の合成も行い，構造決定により新規化合物であることを確認した．反応条件検討により収率向上を図った． 糖転移酵素関連では，TS酵素（N末端側を伸長型酵素）は伸長部分を含めて主要3ドメインにより構成され，各ドメイン単独のタンパク質，ならびに各ドメイン欠失タンパク質を調製・機能解析を行い，各ドメインが有する機能を実験的に明らかにした．マルトオリゴ糖からイソマルトオリゴ糖形成に至る逐次的反応を明らかにした． 糖質の合成に関して，植物において代謝制御物質として機能するトレハロース６-リン酸を，酵素により合成する反応系を確立した．本反応では，マルトオリゴ糖を唯一の炭素源として，3酵素を含むワンポット反応により目的の糖リン酸を合成するものであり，簡便かつ有効な合成系である．
• 微生物が持つ新しい糖質異性化酵素群を活用した糖質変換技術の開発と応用

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 佐分利 亘

   

  多様な構造を持つ糖質には様々な優良な機能が知られている。自然界に豊富に存在する糖質は極めて限定的で，豊富な糖質を希少糖質へと変換する技術を開発することで、新しい糖質の応用が拓けると考えられる．本研究では，単糖変換を触媒する異性化酵素であり，グルコースからマンノースへの一段階異性化反応を触媒するマンノース2-エピメラーゼ (ME) について機能・構造を解明すること，ホモログの機能解析を通じて新たな単糖変換酵素を取得することを目的とした。本年度は，MEに特徴的な長いループ構造に注目して3つの細菌に由来するMEホモログの機能を大腸菌組換え酵素を用いて解析し，いずれのホモログにもME活性を認めた。このことから，MEに特徴的な長いループ構造に着目することで精度良くMEを取得できることが示された．また，興味深いことに，これらの基質を検討する中で，ME酵素がキシロースやリキソースなどの単糖にも作用し，エピメラーゼ活性のみならずイソメラーゼ活性も微弱ながら示すことが明らかになった．Runella slithformis由来MEの高活性化変異酵素の取得に先立ち、変異酵素のハイスループットスクリーニング系を検討した。すなわち，96穴プレートを活用した小規模培養系による組換え酵素生産条件，溶菌試薬による酵素抽出条件と無細胞抽出液の酵素活性の評価方法を確立した。単糖にも有意に高いエピメリ化活性を持つMelioribacter roseus由来セロビオース2-エピメラーゼ (CE) について，本酵素に見られたMEに対応する長いループに注目し，この削除変異酵素を調製して基質特異性を検討した．当該変異酵素は野生型酵素より明らかに低い単糖に対する特異性を示した．ループの短縮化に伴う単糖特異性の低下から，本ループ構造がM. roseus由来CEの単糖への高い活性に重要なことが明らかになった。
• 植物の病害抵抗性を支配するシグナル物資の活性化の分子基盤

  日本学術振興会：科学研究費助成事業 基盤研究(Ｃ)

  研究期間 : 2015年10月 -2018年03月 

  代表者 : 松井 博和, 佐分利 亘

   

  植物ではシグナル物質の多くは配糖化により不活性化され，加水分解により活性化される．本研究ではツベロン酸(TA)配糖体 を加水分解するイネβ-グルコシダーゼTAGG2がサリチル酸グルコシド (SAG) にも高い活性を持つことに注目し，SAG分解に重要なアミノ酸残基を特定し，アポプラストへの局在を明らかにした．TAGG2過剰発現イネではTA量の増加は見られたが，SA量の明確な増加は見られなかった．いもち菌接種試験では，TAGG過剰発現体において野生型よりも病斑形成が促進されることが明らかになった．シロイヌナズナホモログはSAGよりもラミナリオリゴ糖などオリゴ糖基質によく作用した．
• オリゴ糖・多糖・配糖体の酵素合成の分子基盤的研究

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 森 春英, 佐分利 亘

   

  糖質は，構成単糖と結合様式と重合度によって顕著な多様性を持つ分子群である．有用化合物が多数存在すると予想されるが，機能性の解析には潤沢な化合物が必要であり，効率的な大量合成系確立が必須である．本研究では，天然に豊富に存在する糖質からの転換として，合成酵素と転移酵素を利用した高度な糖質合成系確立を実施した．合成酵素利用では，ショ糖を出発物質として合成酵素の基質（UDP-Glcなどの糖ヌクレオチド）を供給するワンポット反応により二糖の合成系を確立した．新規転移酵素はマルトオリゴ糖に作用し，グルコース単位での転移反応を触媒する酵素群を見いだした．
• オリゴ糖異性化酵素とその類縁酵素の構造基盤の解明

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2014年04月 -2016年03月 

  代表者 : 佐分利 亘

   

  セロビオース2-エピメラーゼ (CE) は，β1-4二糖の還元末端グルコース残基をマンノース残基に異性化する。本酵素は触媒ドメインおよび触媒部位構造を他の単糖異性化酵素と共有する。本研究では，これら酵素の構造機能相関を解析した。Rhodothermus marinus由来CEの基質結合部位残基への変異により二糖特異性に重要なアミノ酸残基を決定した。また，本酵素に僅かなイソメラーゼ活性を認め，Cardicellulosiruptor saccharolyticus由来酵素との比較からイソメラーゼ活性に重要な構造を見出した。機能未知タンパク質の解析から、新規なマンノース異性化酵素を見出した。
• 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)

  研究期間 : 2014年04月 -2016年03月 

  代表者 : 佐分利 亘
• 植物ホルモンの新規な代謝酵素の同定と機能解析

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2012年04月 -2015年03月 

  代表者 : 松井 博和, 佐分 利亘, 松浦 英幸

   

  ジャスモン酸 (JA) は病傷害ストレス応答，生長などに関与する植物ホルモンである．本研究ではJAシグナルの不活性化に働くと推定したCYP94D1，CYP94D2およびILL6の機能解析を行った．CYP94D1については遺伝子発現量の調整により表現型の変化が認められた．すなわち，遺伝子欠損株ではJAへの感受性の低下，過剰発現体ではJAの水酸化物の誘導体ツベロン酸グルコシドの蓄積が見られた．
• 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

  研究期間 : 2014年04月 -2015年03月 

  代表者 : 佐分利 亘
• ２つの新規な糖質ホスホリラーゼの分子解析

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2012年04月 -2014年03月 

  代表者 : 佐分利 亘

   

  反芻動物の第一胃ルーメンに共生する偏性嫌気性細菌Ruminococcus albusは二つのマンノシルグルコースホスホリラーゼ (I型酵素とII型酵素) を持つ。基質特異性を検討したところ，I型酵素はマンノシルグルコースに特異的な酵素であり，II型酵素はマンノオリゴ糖に高い活性を示す酵素であることが判明した．合成反応では，I型酵素のみ6-OHグルコース誘導体をアクセプタ基質とした．アミノ酸配列の比較から推定したアミノ酸残基の変異酵素の解析から，I型酵素のAsp129が触媒残基と考えられた．また，I型酵素のIle212がグルコース6位誘導体への合成活性に重要な残基であることが明らかになった．
• 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

  研究期間 : 2013年08月 -2014年03月 

  代表者 : 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)

  研究期間 : 2012年04月 -2014年03月 

  代表者 : 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)

  研究期間 : 2012年08月 -2013年03月 

  代表者 : 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)

  研究期間 : 2011年09月 -2012年03月 

  代表者 : 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)

  研究期間 : 2011年04月 -2012年03月 

  代表者 : 佐分利 亘

産業財産権

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

  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年05月13日

  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年03月18日

  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年07月17日

  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年09月19日

  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年08月08日

  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年01月16日

  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年11月22日

  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年03月22日

  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年03月21日

  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年01月10日

  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年09月21日

  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年07月12日

  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年11月17日

  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年11月04日

  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年04月28日

  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年08月19日

  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年06月25日

  佐分利 亘, 森 春英, 奥山 正幸, 木村 淳夫, 山本 健, 小川 浩一  日本食品化工株式会社  201103044027154701
• 特開2010-095701:新規分岐グルカン並びにその製造方法および用途  2010年04月30日

  佐分利, 亘, 上 村, 由香里, 大 畑, 祐一郎, 山 本, 健, 高 田 正  日本食品化工株式会社  201003099272270506
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  佐分利, 亘, 上 村, 由香里, 大 畑, 祐一郎, 山 本, 健, 高 田 正  日本食品化工株式会社  201103001378520711
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  佐分利 亘, 森 春英, 奥山 正幸, 木村 淳夫, 山本 健, 小川 浩一  日本食品化工株式会社  200903003995134491