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Ota Tomoya

Research Faculty of Agriculture　Fundamental AgriScience Research　Bioscience and ChemistryAssistant Professor

Researcher basic information

■ Degree
  • 博士（農学）, 北海道大学, Mar. 2024
■ URL
researchmap URLホームページURL■ Various IDs
J-Global ID■ Research Keywords and Fields
Research Keyword
  • 糖質関連酵素
  • 酵素化学
  • 有機合成化学
  • 応用生物化学
Research Field
  • Life Science, Applied biochemistry
■ Educational Organization

Career

■ Career
Career
  • Apr. 2024 - Present
    Hokkaido University, Graduate School of Agriculture Research Faculty of Agriculture, Assistant professor
  • Apr. 2021 - Mar. 2024
    北海道大学アンビシャス博士人材フェローシップ（SDGs）
Educational Background
  • Apr. 2019 - Mar. 2024, Hokkaido University, Graduate School of Agriculture
  • Apr. 2015 - Mar. 2019, Hokkaido University, School of Agriculture, Department of Bioscience and Chemistry
Committee Memberships
  • May 2025 - Present
    日本農芸化学会, 北海道支部会計幹事
  • May 2024 - Feb. 2026
    公益社団法人日本農芸化学会, 2025年度大会実行委員会委員

Research activity information

■ Awards
  • Dec. 2023, 日本農芸化学会北海道支部, 学生会員奨励賞
  • Aug. 2023, 日本農芸化学会北海道支部, 優秀発表賞
  • Sep. 2022, 日本応用糖質科学会, ポスター賞
■ Papers
  • Mechanism for synthesis of isomaltooligosaccharides from maltooligosaccharides by GH15 α-glucan 4(6)-α-glucosyltransferase.
    Tianyi Qin; Wataru Saburi; Momo Sawada Otsubo; Haruki Oshita; Kenta Kanai; Tomoya Ota; Birte Svensson; Haruhide Mori
    The FEBS journal, 16 Dec. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, Isomaltosaccharides, composed of α-(1 → 6)-d-glucosyl residues, exhibit diverse beneficial properties depending on the degree of polymerization (DP) and attract great interest across multiple industries. The anomer-retaining transglucosidase, dextran dextrinase (DDase)-which synthesizes the α-(1 → 6)-d-glucose polymer dextran from α-(1 → 4)-glucan maltooligosaccharides (MOSs)-shows structural similarity to anomer-inverting α-glucohydrolases belonging to glycoside hydrolase family 15 (GH15). Here, we show a new GH15 transglucosidase, α-glucan 4(6)-α-glucosyltransferase, from Tepidibacillus decaturensis (Td46GT) and its mechanism for converting MOS into isomaltooligosaccharide (IMO). Td46GT catalyzes DDase-like d-glucosyl-transfer reactions: α-(1 → 4)-transglucosylation to MOS and α-(1 → 6)-transglucosylation to short-chain MOS of DP 2-3 and IMO. Unlike DDase, it does not produce dextran. Kinetic analyses of the two-substrate reactions revealed that the acceptors determined formed linkages. Relative acceptor-substrate specificity constants (RASCs) indicated that maltotriose and maltose were the best acceptors for α-(1 → 4)- and α-(1 → 6)-transglucosylations, respectively. The subsite affinities calculated from the RASCs were consistent with those obtained from kcat/Km values for single-substrate reactions. Time-dependent changes in the MOS and IMO concentrations during the reaction were quantitatively simulated using RASCs and kcat/Km values. Structure prediction suggested Td46GT possesses a substrate-binding site similar to DDase, and site-directed mutagenesis identified Phe418 and Tyr612 as critical residues at subsite +2 for MOS and IMO binding. Our results suggested that Td46GT possesses distinct MOS- and IMO-binding subsites in a single active pocket, which are shared by both acceptor and donor substrates, and that the binding manner of the acceptor determines the product specificity of transglucosylation.
  • 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, Nov. 2023, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, 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 nonreducing 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-1 mM-1) were much lower than to laminarin (5920 s-1 mM-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. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases.
  • 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, 87, 10, 1111, 1121, 21 Sep. 2023, [Peer-reviewed], [Lead author], [International Magazine]
    English, Scientific journal, 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 for 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 the 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.
  • 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, 23 Jun. 2023, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
    English, Scientific journal, 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 (Basel, Switzerland), 28, 7, 30 Mar. 2023, [Peer-reviewed], [International Magazine]
    English, Scientific journal, 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.
    Wataru Saburi; Tomoya Ota; Koji Kato; Takayoshi Tagami; Keitaro Yamashita; Min Yao; Haruhide Mori
    Journal of applied glycoscience, 70, 2, 43, 52, 2023, [Peer-reviewed], [Domestic magazines]
    English, Scientific journal, 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.
■ Other Activities and Achievements
■ Syllabus
  • 生物機能化学実験Ⅲ, 2024年, 学士課程, 農学部
  • 生物学実験Ⅰ, 2024年, 学士課程, 農学部
  • 化学概論, 2024年, 学士課程, 農学部
  • 生物機能化学演習Ⅱ, 2024年, 学士課程, 農学部
■ Affiliated academic society
  • 日本応用糖質科学会
  • 日本農芸化学会