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Last Updated :2024/07/25

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Master

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Applied Bioscience

Affiliation (Master)

  • Research Faculty of Agriculture　Fundamental AgriScience Research　Applied Bioscience

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Profile and Settings

Degree

  • Ph.D.（Hokkaido University）

Profile and Settings

  • Contact Point

    tagamiagr.hokudai.ac.jp

  • Name (Japanese)

    Tagami

  • Name (Kana)

    Takayoshi

  • Name

    201601000822435320

Alternate Names

Achievement

Research Interests

  • 動物生化学   Glycosylation   Carbohydrate   X-ray crystallography   carbohydrate-active enzyme   molecular enzymology   

Research Areas

  • Life sciences / Functional biochemistry
  • Life sciences / Applied biochemistry

Research Experience

  • 2016/08 - Today Hokkaido University Research Faculty of Agriculture Assistant professor
  • 2015/04 - 2016/07 Rakuno Gakuen University College of Agriculture, Food and Environment Sciences Lectular
  • 2013/04 - 2015/03 Rakuno Gakuen University College of Agriculture, Food and Environment Sciences Assistant professor

Education

  • 2010/04 - 2013/03  Hokkaido University  Graduate School of Agriculture  Doctoral Course, Division of Agrobiology
  • 2008/04 - 2010/03  Hokkaido University  Graduate School of Agriculture  Master's Course, Division of Agrobiology
  • 2004/04 - 2008/03  Hokkaido University  School of Agriculture  Department of Applied Bioscience

Awards

  • 2023/03 日本農芸化学会 2023年度農芸化学奨励賞
     糖質加水分解酵素の機能構造相関の解明と応用 
    受賞者: 田上貴祥
  • 2021/03 日本農芸化学会 第18回農芸化学研究企画賞
     ブタ血液を原料とする酵素製剤の開発と糖尿病治療薬研究への応用 
    受賞者: 田上 貴祥
  • 2013/02 日本応用糖質科学会北海道支部 奨励賞
     α-グルコシダーゼの長鎖マルトオリゴ糖特異性に関与する構造因子に関する研究 
    受賞者: 田上 貴祥
  • 2011/05 9th Carbohydrate Bioengineering Meeting Best Poster Award
     
    受賞者: Takayoshi Tagami

Published Papers

  • Wataru Saburi, Takayoshi Tagami, Takuya Usui, Jian Yu, Toyoyuki Ose, Min Yao, Haruhide Mori
    Food Bioscience 61 104516 - 104516 2212-4292 2024/10
  • Takashi Tanida, Takayoshi Tagami, Hiroko Sato, Hay Mar Kyaw, Takeshi Fujikawa, Masashi Nagano, Kenji Momozawa, Yojiro Yanagawa, Seiji Katagiri
    Theriogenology 0093-691X 2024/01 [Refereed]
  • Wilaiwan Kaenying, Takayoshi Tagami, Eukote Suwan, Chariwat Pitsanuwong, Sinchai Chomngam, Masayuki Okuyama, Palangpon Kongsaeree, Atsuo Kimura, Prachumporn T. Kongsaeree
    Heliyon 9 (11) e21923 - e21923 2405-8440 2023/11 [Refereed]
  • Weeranuch Lang, Takayoshi Tagami, Yuya Kumagai, Seiya Tanaka, Hye-Jin Kang, Masayuki Okuyama, Wataru Saburi, Haruhide Mori, Tohru Hira, Chaehun Lee, Takuya Isono, Toshifumi Satoh, Hiroshi Hara, Takayuki Kurokawa, Nobuo Sakairi, Yoshiaki Yuguchi, Atsuo Kimura
    Carbohydrate Polymers 319 121185 - 121185 0144-8617 2023/11 [Refereed][Not invited]
  • Tomoya Ota, Wataru Saburi, Takayoshi Tagami, Jian Yu, Shiro Komba, Linda Elizabeth Jewell, Tom Hsiang, Ryozo Imai, Min Yao, Haruhide Mori
    The Journal of biological chemistry 105294 - 105294 2023/09/27 [Refereed]
     
    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.
  • Wataru Saburi, Tomoya Ota, Koji Kato, Takayoshi Tagami, Keitaro Yamashita, Min Yao, Haruhide Mori
    Journal of Applied Glycoscience 70 (2) 43 - 52 1344-7882 2023/05/20 [Refereed]
  • Weeranuch Lang, Takayoshi Tagami, Hye-Jin Kang, Masayuki Okuyama, Nobuo Sakairi, Atsuo Kimura
    Carbohydrate Polymers 307 120629 - 120629 0144-8617 2023/05 [Refereed]
  • Weeranuch Lang, Sarote Sirisansaneeyakul, Takayoshi Tagami, Hye-Jin Kang, Masayuki Okuyama, Nobuo Sakairi, Atsuo Kimura
    Carbohydrate Polymers 305 120565 - 120565 0144-8617 2023/04 [Refereed]
  • Weeranuch Lang, Debashish Mondol, Aphichat Trakooncharoenvit, Takayoshi Tagami, Masayuki Okuyama, Tohru Hira, Nobuo Sakairi, Atsuo Kimura
    Food Hydrocolloids 137 108430 - 108430 0268-005X 2023/04 [Refereed]
  • Wilaiwan Kaenying, Khuanjarat Choengpanya, Takayoshi Tagami, Pakorn Wattana-Amorn, Weeranuch Lang, Masayuki Okuyama, Yaw-Kuen Li, Atsuo Kimura, Prachumporn T. Kongsaeree
    Applied Microbiology and Biotechnology 107 (7-8) 2335 - 2349 0175-7598 2023/03/06 [Refereed]
  • Rikako Shishiuchi, Hyejin Kang, Takayoshi Tagami, Yoshitaka Ueda, Weeranuch Lang, Atsuo Kimura, Masayuki Okuyama
    ACS Omega 7 (50) 47411 - 47423 2470-1343 2022/12/09 [Refereed]
  • Tomohito Iwasaki, Jessica R. Terrill, Kei Kawarai, Yusei Miyata, Takayoshi Tagami, Naoyuki Maeda, Yasuhiro Hasegawa, Takafumi Watanabe, Miranda D. Grounds, Peter G. Arthur
    Acta Histochemica 124 (8) 151959 - 151959 0065-1281 2022/12 [Refereed]
  • Weeranuch Lang, Yuya Kumagai, Shinji Habu, Juri Sadahiro, Takayoshi Tagami, Masayuki Okuyama, Shinichi Kitamura, Nobuo Sakairi, Atsuo Kimura
    Carbohydrate Polymers 291 119562 - 119562 0144-8617 2022/09 [Refereed]
  • Hay Mar Kyaw, Hiroko Sato, Takayoshi Tagami, Yojiro Yanagawa, Masashi Nagano, Seiji Katagiri
    Theriogenology 184 26 - 33 0093-691X 2022/05 [Refereed][Not invited]
     
    Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on Days 2-4 and 13-14 during the estrous cycle. An altered (i.e., loss of the two peaks) profile has been linked to reduced fertility in repeat breeder cows. We previously demonstrated that a form of osteopontin (OPN), with a molecular weight of 29 kDa and found in bull seminal plasma (SP), normalized the EGF profile and restored fertility in repeat breeder cows. OPN has many molecular forms due to post-translational modifications and is abundant in bovine milk. The purpose of the present study was to investigate whether mOPN normalizes the endometrial EGF profile and restores fertility in repeat breeder dairy cows with an altered EGF profile. OPN was separated by one-step anion-exchange column chromatography from the whey of bovine milk. Purified mOPN was verified by Western blotting and peptide mass fingerprinting analyses. The OPN fraction showed three major protein bands of 61, 37 and 31 kDa (peptides I, II, and III, respectively) on SDS-PAGE. All three major bands were identified as OPNs by Western blotting and their tryptic peptide masses were matched at approximately 50, 40, and 10%, respectively, to the bovine OPN amino acid sequence by a peptide mass finger printing analysis. The three bands accounted for approximately 85% of the total protein content and 6-23 mg of OPN was obtained from 1 L of bovine milk. A lyophilized eluate containing 1.3 mg of mOPN (171 cows), 0.5 mL of frozen SP (62 cows), and PBS (84 cows) was infused at estrus into the vagina of repeat breeder cows with an altered EGF profile. Some of the cows treated with mOPN, SP, and PBS (46, 50, and 45 cows, respectively) were inseminated immediately before the infusion and then examined for pregnancy between Days 60 and 65. The rate at which mOPN to normalize the EGF profile (56.1%) was similar to that of SP (58.1%) and higher than that of PBS (23.8%) (P < 0.05). The conception rate after the infusion of mOPN (43.5%) was similar to that of SP (40.0%) and higher than that of PBS (22.2%) (P < 0.05). The present results indicate that the infusion of mOPN into the vagina is a treatment option for repeat breeder cows with an altered EGF profile. Further studies are needed to compare the capacity of the three OPN molecules in milk to normalize the EGF profile, together with their molecular characteristics due to post-translational modifications.
  • Yuya Kumagai, Hideki Kishimura, Weeranuch Lang, Takayoshi Tagami, Masayuki Okuyama, Atsuo Kimura
    Marine Drugs 20 (4) 250 - 250 2022/03/31 [Refereed]
     
    The glycoside hydrolase family 17 β-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of β-(1→3)-glucan, mainly producing laminaribiose, but not of β-(1→3)/β-(1→4)-glucan. The C-terminal-truncated mutants (ΔC466 and ΔC441) showed decreased activity, approximately one-third of that of the WT, and ΔC415 lost almost all activity. An analysis using affinity gel containing laminarin or barley β-glucan revealed a shift in the mobility of the ΔC466, ΔC441, and ΔC415 mutants compared to the WT. Tryptophan residues showed a strong affinity for carbohydrates. Three of four point-mutations of the tryptophan in the C-terminus (W472A, W499A, and W542A) showed a reduction in binding ability to laminarin and barley β-glucan. The C-terminus was predicted to have a β-sandwich structure, and three tryptophan residues (Trp472, Trp499, and Trp542) constituted a putative substrate-binding cave. Linker and substrate-binding functions were assigned to the C-terminus. The N-terminal-truncated mutants also showed decreased activity. The WT formed a trimer, while the N-terminal truncations formed monomers, indicating that the N-terminus contributed to the multimeric form of VvGH17. The results of this study are useful for understanding the structure and the function of GH17 β-1,3-glucanases.
  • Takayoshi Tagami, Minghao Chen, Yuta Furunaga, Asako Kikuchi, Juri Sadahiro, Weeranuch Lang, Masayuki Okuyama, Yoshikazu Tanaka, Tomohito Iwasaki, Min Yao, Atsuo Kimura
    The FEBS Journal 289 (4) 1118 - 1134 1742-464X 2022/02 [Refereed][Not invited]
     
    Glycoside hydrolase family 15 (GH15) inverting enzymes contain two glutamate residues functioning as a general acid catalyst and a general base catalyst, for isomaltose glucohydrolase (IGHase), Glu178 and Glu335, respectively. Generally, a two-catalytic residue-mediated reaction exhibits a typical bell-shaped pH-activity curve. However, IGHase is found to display atypical non-bell-shaped pH-kcat and pH-kcat /Km profiles, theoretically better-fitted to a three-catalytic residue-associated pH-activity curve. We determined the crystal structure of IGHase by the single-wavelength anomalous dispersion method using sulfur atoms and the cocrystal structure of a catalytic base mutant E335A with isomaltose. Although the activity of E335A was undetectable, the electron density observed in its active site pocket did not correspond to an isomaltose but a glycerol and a β-glucose, cryoprotectant, and hydrolysis product. Our structural and biochemical analyses of several mutant enzymes suggest that Tyr48 acts as a second catalytic base catalyst. Y48F mutant displayed almost equivalent specific activity to a catalytic acid mutant E178A. Tyr48, highly conserved in all GH15 members, is fixed by another Tyr residue in many GH15 enzymes; the latter Tyr is replaced by Phe290 in IGHase. The pH profile of F290Y mutant changed to a bell-shaped curve, suggesting that Phe290 is a key residue distinguishing Tyr48 of IGHase from other GH15 members. Furthermore, F290Y is found to accelerate the condensation of isomaltose from glucose by modifying a hydrogen-bonding network between Tyr290-Tyr48-Glu335. The present study indicates that the atypical Phe290 makes Tyr48 of IGHase unique among GH15 enzymes.
  • 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 0175-7598 2022/01/13 [Refereed]
     
    Dextran dextrinase (DDase) catalyzes formation of the polysaccharide dextran from maltodextrin. During the synthesis of dextran, DDase also generates the beneficial material isomaltomegalosaccharide (IMS). The term megalosaccharide is used for a saccharide having DP = 10-100 or 10-200 (DP, degree of polymerization). IMS is a chimeric glucosaccharide comprising α-(1 → 6)- and α-(1 → 4)-linked portions at the nonreducing and reducing ends, respectively, in which the α-(1 → 4)-glucosyl portion originates from maltodextrin of the substrate. In this study, IMS was produced by a practical approach using extracellular DDase (DDext) or cell surface DDase (DDsur) of Gluconobacter oxydans ATCC 11894. DDsur was the original form, so we prepared DDext via secretion from intact cells by incubating with 0.5% G6/G7 (maltohexaose/maltoheptaose); this was followed by generation of IMS from various concentrations of G6/G7 substrate at different temperatures for 96 h. However, IMS synthesis by DDext was limited by insufficient formation of α-(1 → 6)-glucosidic linkages, suggesting that DDase also catalyzes elongation of α-(1 → 4)-glucosyl chain. For production of IMS using DDsur, intact cells bearing DDsur were directly incubated with 20% G6/G7 at 45 °C by optimizing conditions such as cell concentration and agitation efficiency, which resulted in generation of IMS (average DP = 14.7) with 61% α-(1 → 6)-glucosyl content in 51% yield. Increases in substrate concentration and agitation efficiency were found to decrease dextran formation and increase IMS production, which improved the reaction conditions for DDext. Under modified conditions (20% G6/G7, agitation speed of 100 rpm at 45 °C), DDext produced IMS (average DP = 14.5) with 65% α-(1 → 6)-glucosyl content in a good yield of 87%. KEY POINTS: • Beneficial IMS was produced using thermostabilized DDase. • Optimum conditions for reduced dextran formation were successfully determined. • A practical approach was established to provide IMS with a great yield of 87%.
  • Masayuki Okuyama, Ryo Serizawa, Masanari Tanuma, Asako Kikuchi, Juri Sadahiro, Takayoshi Tagami, Weeranuch Lang, Atsuo Kimura
    Journal of Biological Chemistry 296 100398 - 100398 0021-9258 2021/01 [Refereed][Not invited]
     
    Glycoside hydrolase family 68 (GH68) enzymes catalyze β-fructosyltransfer from sucrose to another sucrose, so-called transfructosylation. Although regioselectivity of transfructosylation is divergent in GH68 enzymes, there is insufficient information available on the structural factor(s) involved in the selectivity. Here, we found two GH68 enzymes, β-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the β-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and β-(2→6)-transfructosylation (6-TF). We identified His79FFZm and Ala343FFZm and their corresponding Asn84LSZm and Ser345LSZm respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84LSZm and Ser345LSZm (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79FFZm and Ala343FFZm in FFZm (H79N/A343S-FFZm) almost lost 1-TF and acquired 6-TF. H79N/A343S-FFZm exhibited the selectivity like LSZm but did not produce the β-(2→6)-fructoside-linked levan and/or long levanooligosaccharides that LSZm did. We assumed Phe189LSZm to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187FFZm in FFZm to Phe. An H79N/L187F/A343S-FFZm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FFZm (or H79N-FFZm), although without levan formation, suggesting that LSZm has another structural factor for levan production. We also found that FFZm generated a sucrose analog, β-D-fructofuranosyl α-D-mannopyranoside, by β-fructosyltransfer to d-mannose and regarded His79FFZm and Ala343FFZm as key residues for this acceptor specificity. In summary, this study provides insight into the structural factors of regioselectivity and acceptor specificity in transfructosylation of GH68 enzymes.
  • Increased serum malondialdehyde concentration in cows with subclinical ketosis.
    Senoh, T, Oikawa, S, Nakada, K, Tagami, T, Iwasaki, T
    J. Vet. Med. Sci. 81 817 - 820 2019 [Refereed][Not invited]
  • Ma, M, Okuyama, M, Tagami, T, Kikuchi, A, Klahan, P, Kimura, A
    J. Agr. Food Chem. 67 (12) 2280 - 2288 2019 [Refereed][Not invited]
     
    α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of N-glycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)- and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4-glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short- and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (<15 min) synthesized 3II- O-α-glucosyl-maltose and maltotriose, indicating α-1,3-/α-1,4-transferring specificity. A long-term reaction (<24 h) accumulated kojibiose and nigerose using formed glucose as an acceptor substrate. AgdA and AgdB are distinct α-glucosidases. At a high concentration of glucose added exogenously, AgdB largely generated the rare sugars kojibiose and nigerose (exhibiting beneficial physiological functions) with 19% and 24% yields from maltose, respectively.
  • Klahan P, Okuyama M, Jinnai K, Ma M, Kikuchi A, Kumagai Y, Tagami T, Kimura A
    Bioscience, Biotechnology, and Biochemistry 82 (9) 1480 - 1487 2018/09 [Refereed][Not invited]
     
    Herein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligosaccharides. Reaction specificities of wild-type dextranase and its mutant derivatives were examined using dextran and a series of enzymatically prepared p-nitrophenyl α-isomaltooligosaccharides. In experiments with 4-mg·mL-1 dextran, isomaltooligosaccharides with degrees of polymerization (DP) of 3 and 4 were present at the beginning of the reaction, and glucose and isomaltose were produced by the end of the reaction. Increased concentrations of the substrate dextran (40 mg·mL-1) yielded isomaltooligosaccharides with higher DP, and the mutations T558H, W279A/T563N, and W279F/T563N at the -3 and -4 subsites affected hydrolytic activities of the enzyme, likely reflecting decreases in substrate affinity at the -4 subsite. In particular, T558H increased the proportion of isomaltooligosaccharide with DP of 5 in hydrolysates following reactions with 4-mg·mL-1 dextran.Abbreviations CI: cycloisomaltooligosaccharide; CITase: CI glucanotransferase; CITase-Bc: CITase from Bacillus circulans T-3040; DP: degree of polymerization of glucose unit; GH: glycoside hydrolase family; GTF: glucansucrase; HPAEC-PAD: high performance anion-exchange chromatography-pulsed amperometric detection; IG: isomaltooligosaccharide; IGn: IG with DP of n (n, 2‒5); PNP: p-nitrophenol; PNP-Glc: p-nitrophenyl α-glucoside; PNP-IG: p-nitrophenyl isomaltooligosaccharide; PNP-IGn: PNP-IG with DP of n (n, 2‒6); SmDex: dextranase from Streptococcus mutans; SmDexTM: S. mutans ATCC25175 SmDex bearing Gln100‒Ile732.
  • Asako Kikuchi, Masayuki Okuyama, Koji Kato, Shohei Osaki, Min Ma, Yuya Kumagai, Kana Matsunaga, Patcharapa Kiahan, Takayoshi Tagami, Min Yao, Atsuo Kimura
    BIOCHIMIE 142 41 - 50 0300-9084 2017/11 [Refereed][Not invited]
     
    Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, alpha-glucoside hydrolase and agalactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 a-glucoside hydrolase and alpha-galactosidase, were characterized in this study. BT3664 was identified to be an a-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both beta-L-arabinopyranoside and alpha-D-galactopyranoside, and thus we designate BT3661 as a beta-L-arabinopyranosidase/alpha-D-galactosidase, Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 agalactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears G1u361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The k(cat)/K-m value for the hydrolysis of p-nitrophenyl a-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the kcat/Km value for hydrolysis of p-nitrophenyl beta-D-arabinopyranoside. (C) 2017 Published by Elsevier B.V.
  • Min Ma, Masayuki Okuyama, Megumi Sato, Takayoshi Tagami, Patcharapa Klahan, Yuya Kumagai, Haruhide Mori, Atsuo Kimura
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 101 (16) 6399 - 6408 0175-7598 2017/08 [Refereed][Not invited]
     
    Aspergillus niger alpha-glucosidase (ANG), a member of glycoside hydrolase family 31, catalyzes hydrolysis of alpha-glucosidic linkages at the non-reducing end. In the presence of high concentrations of maltose, the enzyme also catalyzes the formation of alpha-(1 -> 6)-glucosyl products by transglucosylation and it is used for production of the industrially useful panose and isomaltooligosaccharides. The initial transglucosylation by wild-type ANG in the presence of 100 mM maltose [Glc(alpha 1-4)Glc] yields both alpha-(1 -> 6)- and alpha-(1 -> 4)-glucosidic linkages, the latter constituting similar to 25% of the total transfer reaction product. The maltotriose [Glc(alpha 1-4)Glc(alpha 1-4)Glc], alpha-(1 -> 4)-glucosyl product disappears quickly, whereas the alpha-(1 -> 6)-glucosyl products panose [Glc(alpha 1-6)Glc(alpha 1-4)Glc], isomaltose [Glc(alpha 1-6)Glc], and isomaltotriose [Glc(alpha 1-6)Glc(alpha 1-6)Glc] accumulate. To modify the transglucosylation properties of ANG, residue Asn694, which was predicted to be involved in formation of the plus subsites of ANG, was replaced with Ala, Leu, Phe, and Trp. Except for N694A, the mutations enhanced the initial velocity of the alpha-(1 -> 4)-transfer reaction to produce maltotriose, which was then degraded at a rate similar to that by wild-type ANG. With increasing reaction time, N694F and N694W mutations led to the accumulation of larger amounts of isomaltose and isomaltotriose than achieved with the wild-type enzyme. In the final stage of the reaction, the major product was panose (N694A and N694L) or isomaltose (N694F and N694W).
  • Okuyama M, Matsunaga K, Watanabe K, Yamashita K, Tagami T, Kikuchi A, Ma M, Klahan P, Mori H, Yao M, Kimura A
    The FEBS Journal 284 (5) 766 - 783 2017/02 [Refereed][Not invited]
     
    The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from α-glycosidases can give poor yields of desired products because they require generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using β-galactosyl azide and α-galactosyl transfer from α-galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, β-galactosyl azide and acceptor. A three-dimensional structure of the mutant enzyme in complex with the transglycosylation product, β-lactosyl α-d-galactoside, was solved to elucidate the ligand-binding aspects of the α-galactosidase. Subtle differences at the β→α loops 1, 2 and 3 of the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside recognition is conserved among GH27, 31, 36 and 97 α-galactosidases. DATABASE: The atomic coordinates (code: 5E1Q) have been deposited in the Protein Data Bank.
  • Michi YAMADA, Yuki WATANABE, Hanako YOSHIDA, Syunoa MITANI, Takayuki HARA, Takayoshi TAGAMI, Tomohito IWASAKI, Mikio SUGANO, Kazushige TAKEHANA, Shinji SUGIYAMA, Tetsuya EBIHARA, Yoh-ichi KOYAMA, Koji YAMADA, Hiroki NAKATSUJI
    Nihon Yoton Gakkaishi 54 (3) 130 - 141 0913-882X 2017 [Refereed][Not invited]
  • Masayuki Okuyama, Masashi Miyamoto, Ichiro Matsuo, Shogo Iwamoto, Ryo Serizawa, Masanari Tanuma, Min Ma, Patcharapa Klahan, Yuya Kumagai, Takayoshi Tagami, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 81 (8) 1503 - 1511 0916-8451 2017 [Refereed][Not invited]
     
    The recombinant catalytic alpha-subunit of N-glycan processing glucosidase II from Schizosaccharomyces pombe (SpGII alpha) was produced in Escherichia coli. The recombinant SpGII alpha exhibited quite low stability, with a reduction in activity to <40% after 2-days preservation at 4 degrees C, but the presence of 10% (v/v) glycerol prevented this loss of activity. SpGII alpha, a member of the glycoside hydrolase family 31 (GH31), displayed the typical substrate specificity of GH31 alpha-glucosidases. The enzyme hydrolyzed not only alpha-(1 -> 3)-but also alpha-(1 -> 2)-, alpha-(1 -> 4)-, and alpha-(1 -> 6)-glucosidic linkages, and p-nitrophenyl alpha-glucoside. SpGII alpha displayed most catalytic properties of glucosidase II. Hydrolytic activity of the terminal alpha-glucosidic residue of Glc(2)Man(3)-Dansyl was faster than that of Glc(1)Man(3)-Dansyl. This catalytic alpha-subunit also removed terminal glucose residues from native N-glycans (Glc(2)Man(9)GlcNAc(2) and Glc(1)Man(9)GlcNAc(2)) although the activity was low.
  • Yang-Hsin Hsu, Takayoshi Tagami, Kana Matsunaga, Masayuki Okuyama, Takashi Suzuki, Naonobu Noda, Masahiko Suzuki, Hanako Shimura
    PLANT JOURNAL 89 (2) 325 - 337 0960-7412 2017/01 [Refereed][Not invited]
     
    Because structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue-flowered and accumulates delphinidin 3-O-p-coumaroylrutinoside5-O-malonylglucoside-3'5'-O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve-flowered, and LC-MS analyses showed that AL accumulated delphinidin 3-O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.
  • Julan Liao, Masayuki Okuyama, Keigo Ishihara, Yoshinori Yamori, Shigeo Iki, Takayoshi Tagami, Haruhide Mori, Seiya Chiba, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 (9) 1747 - 1752 0916-8451 2016/09 [Refereed][Not invited]
     
    The recombinant AglB produced by Pichia pastoris exhibited substrate inhibition behavior for the hydrolysis of p-nitrophenyl -galactoside, whereas it hydrolyzed the natural substrates, including galactomanno-oligosaccharides and raffinose family oligosaccharides, according to the Michaelian kinetics. These contrasting kinetic behaviors can be attributed to the difference in the dissociation constant of second substrate from the enzyme and/or to the ability of the leaving group of the substrates. The enzyme displays the grater k(cat)/K-m values for hydrolysis of the branched -galactoside in galactomanno-oligosaccharides than that of raffinose and stachyose. A sequence comparison suggested that AglB had a shallow active-site pocket, and it can allow to hydrolyze the branched -galactosides, but not linear raffinose family oligosaccharides.
  • Takayoshi Tagami, Eri Miyano, Juri Sadahiro, Masayuki Okuyama, Tomohito Iwasaki, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 (32) 16438 - 16447 0021-9258 2016/08 [Refereed][Not invited]
     
    The actinobacterium Kribbella flavida NBRC 14399(T) produces cyclobis-(1 -> 6)-alpha-nigerosyl (CNN), a cyclic glucotetraose with alternate alpha-(1 -> 6)- and alpha-(1 -> 3)-glucosidic linkages, from starch in the culture medium. We identified gene clusters associated with the production and intracellular catabolism of CNN in the K. flavida genome. One cluster encodes 6-alpha-glucosyl-transferase and 3-alpha-isomaltosyltransferase, which are known to coproduce CNN from starch. The other cluster contains four genes annotated as a transcriptional regulator, sugar transporter, glycoside hydrolase family (GH) 31 protein (Kfla1895), and GH15 protein (Kfla1896). Kfla1895 hydrolyzed the alpha-(1 -> 3)-glucosidic linkages of CNN and produced isomaltose via a possible linear tetrasaccharide. The initial rate of hydrolysis of CNN (11.6 s(-1)) was much higher than that of panose (0.242 s(-1)), and hydrolysis of isomaltotriose and nigerose was extremely low. Because Kfla1895 has a strong preference for the alpha-(1 -> 3)-isomaltosyl moiety and effectively hydrolyzes the alpha-(1 -> 3)-glucosidic linkage, it should be termed 1,3-alpha-isomaltosidase. Kfla1896 effectively hydrolyzed isomaltose with liberation of beta-glucose, but displayed low or no activity toward CNN and the general GH15 enzyme substrates such as maltose, soluble starch, or dextran. The k(cat)/K-m for isomaltose (4.81 +/- 0.18 s(-1) mM(-1)) was 6.9- and 19-fold higher than those for panose and isomaltotriose, respectively. These results indicate that Kfla1896 is a new GH15 enzyme with high substrate specificity for isomaltose, suggesting the enzyme should be designated an isomaltose glucohydrolase. This is the first report to identify a starch-utilization pathway that proceeds via CNN.
  • Yasushi Masuda, Masayuki Okuyama, Takahisa Iizuka, Hiroyuki Nakai, Wataru Saburi, Taro Fukukawa, Janjira Maneesan, Takayoshi Tagami, Tetsushi Naraoka, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 80 (3) 479 - 485 0916-8451 2016/03 [Refereed][Not invited]
     
    Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an -glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 degrees C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest V-max/K-m value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.
  • Yuya Kumagai, Keitaro Yamashita, Takayoshi Tagami, Misugi Uraji, Kun Wan, Masayuki Okuyama, Min Yao, Atsuo Kimura, Tadashi Hatanaka
    FEBS JOURNAL 282 (20) 4001 - 4014 1742-464X 2015/10 [Refereed][Not invited]
     
    Endo--1,4-mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide (GGM5; 6(III),6(IV)--d-galactosyl mannopentaose) to GGM3 and M2, whereas TfMan hydrolyzed GGM5 to GGM4 and M1. To determine the region involved in the substrate specificity, we constructed chimeric enzymes of StMan and TfMan and evaluated their substrate specificities. Moreover, the crystal structure of the catalytic domain of StMan (StMandC) and the complex structure of the inactive mutant StE273AdC with M6 were solved at resolutions of 1.60 and 1.50 angstrom, respectively. Structural comparisons of StMandC and the catalytic domain of TfMan lead to the identification of a subsite around -1 in StMandC that could accommodate a galactose branch. These findings demonstrate that the two loops (loop7 and loop8) are responsible for substrate recognition in GH5 actinomycete mannanases. In particular, Trp281 in loop7 of StMan, which is located in a narrow and deep cleft, plays an important role in its affinity toward linear substrates. Asp310 in loop8 of StMan specifically bound to the galactosyl unit in the -1 subsite.
  • Takayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 290 (3) 1796 - 1803 0021-9258 2015/01 [Refereed][Not invited]
     
    The alpha-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower K-m and higher k(cat)/K-m for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligo-saccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of alpha-glycosidases. The clear correlation obtained between log K-i of the acarviosyl-maltooligosaccharides and log(K-m/k(cat)) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the K-m for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates.
  • Janjira Maneesan, Hideyuki Matsuura, Takayoshi Tagami, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 (12) 2064 - 2068 0916-8451 2014/12 [Refereed][Not invited]
     
    alpha-1,4-Glucan lyases [glycoside hydrolase family (GH) 31] catalyze an elimination reaction to form 1,5-anhydro-d-fructose (AF), while GH31 alpha-glucosidases normally catalyze a hydrolytic reaction. We determined that a small amount of AF was produced by GH31 Aspergillus niger alpha-glucosidase from maltooligosaccharides by elimination reaction, likely via an oxocarbenium ion intermediate.
  • Kyung-Mo Song, Masayuki Okuyama, Mariko Nishimura, Takayoshi Tagami, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (8) 1759 - 1765 0916-8451 2013/08 [Refereed][Not invited]
     
    The specificity for the alpha-1,4- and alpha-1,6-glucosidic linkages varies among glycoside hydrolase family 31 alpha-glucosidases. This difference in substrate specificity has been considered to be due to the difference in an aromatic residue on beta ->alpha loop 1 in the catalytic domain with a (beta/alpha)(8) barrel fold; i.e., the enzymes having Tyr and Trp on beta ->alpha loop 1 were respectively described as alpha-1,4-specific and alpha-1,6-specific alpha-glucosidases. Schwanniomyees oceidentalis alpha-glucosidase, however, prefers the alpha-1,4-glucosidic linkage, although the enzyme possesses Trp324 at the corresponding position. The mutation of Trp324 to Tyr decreased the ability for hydrolysis of the alpha-1,6-glucosidic linkage and formation of the alpha-1,6-glucosidic linkage in transglycosylation, indicating Trp324 to be closely associated with alpha-1,6 specificity, even if the enzyme preferred the alpha-1,4-glucosidic linkage. The mutant enzyme was found to catalyze the production of the branched oligosaccharide, 2,4-di-O-(alpha-D-glucopyranosyl)-D-glucopyranose, more efficiently than the wild-type enzyme.
  • Takayoshi Tagami, Keitaro Yamashita, Masayuki Okuyama, Haruhide Mori, Min Yao, Atsuo Kimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 (26) 19296 - 19303 0021-9258 2013/06 [Refereed][Not invited]
     
    Sugar beet alpha-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher k(cat)/K-m values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific alpha-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 angstrom resolution. The active site pocket of SBG is formed by a (beta/alpha)(8) barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (beta/alpha)(8) barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.
  • Takayoshi Tagami, Yoshiyuki Tanaka, Haruhide Mori, Masayuki Okuyama, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 77 (2) 312 - 319 0916-8451 2013/02 [Refereed][Not invited]
     
    Acarbose is a pseudo-tetrasaccharide and one of the most effective inhibitors of glycoside hydrolases. Its derivatives, acarviosyl-maltooligosaccharides, which have longer maltooligosaccharide parts than the maltose unit of acarbose, were synthesized using a disproportionating enzyme partially purified from adzuki cotyledons. The enzyme was identified as a typical type-1 disproportionating enzyme (DPE1) by primary structure analysis. It produced six compounds from 100 mm acarbose and 7.5% (w/v) of maltotetraose-rich syrup. The masses of the six products were confirmed to accord with acarviosyl-maltooligosaccharides with the degrees of polymerization = 5-10 (AC5-AC10) by electrospray ionization mass spectrometry. H-1 and C-13 NMR spectra indicated that AC5-AC10 were alpha-acarviosyl-(1 -> 4)maltooligosaccharide, which have maltotriose-maltooctaose respectively in the maltooligosaccharide part. A predominance of AC7 in the products at the early stage of the reaction indicated that DPE1 catalyzes the transfer of the acarviosyl-glucose moiety from acarbose to the acceptors. ACn can be useful tools as new inhibitors of glycoside hydrolases.
  • Takayoshi Tagami, Masayuki Okuyama, Hiroyuki Nakai, Young-Min Kim, Haruhide Mori, Kazunori Taguchi, Birte Svensson, Atsuo Kimura
    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1834 (1) 329 - 335 1570-9639 2013/01 [Refereed][Not invited]
     
    Glycoside hydrolase family 31 alpha-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger alpha-glucosidase (ANG) is specific for short-chain substrates with the highest k(cat)/K-m for maltotriose, while sugar beet alpha-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k(cat)/K-m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K-m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K-m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AG5. (C) 2012 Elsevier B.V. All rights reserved.
  • Lukana Ngiwsara, Gaku Iwai, Takayoshi Tagami, Natsuko Sato, Hiroyuki Nakai, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 76 (10) 1967 - 1974 0916-8451 2012/10 [Refereed][Not invited]
     
    Honeybees, Apis mellifera, possess three alpha-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-M preferred sucrose to maltose as substrate and formed only alpha-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the alpha-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) alpha-glucosidases and related enzymes.

MISC

Presentations

  • α-グルコシダーゼの一般酸塩基触媒残基変異酵素を用いたグルコシルエステルの合成  [Not invited]
    藤井大河, 田上貴祥, 木村淳夫, 奥山正幸
    令和4年度日本農芸化学会 北海道・東北支部 合同支部会  2022/09
  • ブタ血清 maltase-glucoamylase の保存方法および粗精製方法の検討  [Not invited]
    枇杷千尋, 田上貴祥, 奥山正幸
    令和4年度日本農芸化学会 北海道・東北支部 合同支部会  2022/09
  • デキストラン代謝遺伝子群にコードされた solute-binding protein の機能および結晶構造の解析  [Not invited]
    宮園駿介, 田上貴祥, 奥山正幸
    令和4年度日本農芸化学会 北海道・東北支部 合同支部会  2022/09
  • GH55 β-1,3-グルカナーゼのエンド様式の作用と構造  [Not invited]
    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
    日本応用糖質科学会2022年度大会  2022/09 
    ポスター賞受賞
  • Kribbella flavida由来GH31 α-1,3-イソマルトシダーゼの基質特異性と結晶構造の解析  [Not invited]
    楊恵佳, 田上貴祥, 木村淳夫, 奥山正幸
    日本応用糖質科学会2022年度大会  2022/09
  • Bifidobacterium adolescentis由来α-ガラクトシダーゼの基質特異性  [Not invited]
    大江 剛平, 奥山 正幸, 田上 貴祥, 中川 雄登, 菊池 麻子, 木村 淳夫
    令和3年度日本応用糖質科学会北海道支部講演会  2022/01
  • デキストラン代謝関連遺伝子群にコードされたsolute-binding proteinの特異性解析  [Not invited]
    宮園駿介, 田上貴祥, 奥山正幸, 木村淳夫
    令和3年度日本応用糖質科学会北海道支部講演会  2022/01
  • 多糖資化遺伝子群に含まれる2種のSolute-binding proteinの機能構造解析  [Not invited]
    宮園駿介, 田上貴祥, 奥山正幸, 木村淳夫
    日本応用糖質科学会令和3年度大会  2021/09
  • Bifidobacterium adolescentis由来α−ガラクトシダーゼの機能解析  [Not invited]
    大江剛平, 奥山正幸, 田上貴祥, 木村淳夫
    日本応用糖質科学会令和3年度大会  2021/09
  • 新奇α-L-glucosidaseの機能および結晶構造の解析  [Not invited]
    猪内 里花子, 田上 貴祥, 奥山 正幸, 木村 淳夫
    日本応用糖質科学会令和3年度大会  2021/09
  • Isomaltose glucohydrolase に特徴的なPhe290の機能解析  [Not invited]
    田上 貴祥, 古永 雄太, 奥山 正幸, 木村 淳夫
    日本農芸化学会2021年度大会  2021/03
  • α-グルコシダーゼによるα-マンノシル基転移反応の受容体特異性の解析  [Not invited]
    藤井大河, 床波 篤, 田上貴祥, 奥山正幸, 木村淳夫
    令和2年度日本応用糖質科学会北海道支部会  2021/01
  • 糖質加水分解酵素ファミリー97 α-ガラクトシダーゼの糖転移反応の解析  [Not invited]
    岡本菜央, 菊池麻子, 田上貴祥, 奥山正幸, 木村淳夫
    令和2年度日本応用糖質科学会北海道支部会  2021/01
  • タマネギのフルクトオリゴ糖分解に関わる酵素の性質  [Not invited]
    乕田春佳, 上野敬司, 髙木惇生, 奥山正幸, 田上貴祥, 木村淳夫, 義平大樹, 小野寺秀一
    令和2年度日本応用糖質科学会北海道支部会  2021/01
  • 糸状菌 α-グルコシダーゼ B への部位特異的飽和変異導入が生成物特異性に与える影響  [Not invited]
    床波 篤, Min Ma, 奥山 正幸, 田上 貴祥, 木村 淳夫
    2020年度日本農芸化学会北海道支部/第50回日本栄養・食糧学会北海道支部合同学術講演会  2020/12
  • 組換え大腸菌を用いたラフィノースの細胞工学的生産  [Not invited]
    松井亨太, 奥山正幸, 田上貴祥, 木村淳夫
    2020年度日本農芸化学会北海道支部/第50回日本栄養・食糧学会北海道支部合同学術講演会  2020/12
  • GH13 に発見された α-1,3-グルコシダーゼの基質特異性を支配する構造因子  [Not invited]
    中山 英里香, 田上 貴祥, 奥山 正幸, 木村 淳夫
    2020年度日本農芸化学会北海道支部/第50回日本栄養・食糧学会北海道支部合同学術講演会  2020/12
  • 組換えオステオポンチンによるリピートブリーダー牛の子宮内膜上皮成長因子（EGF）濃度正常化と受胎性回復効果の検証  [Not invited]
    佐藤 弘子, Hay Mar Kyaw, 栁川 洋二郎, 永野 昌志, 田上 貴祥, 片桐 成二
    第61回日本卵子学会  2020/10
  • Capacity of milk osteopontin to normalize endometrial epidermal growth factor profile in repeat breeder dairy cows  [Not invited]
    Hay Mar Kyaw, 栁川洋二郎, 田上貴祥, 永野昌志, 片桐成二
    第163回 日本獣医学会学術集会  2020/09
  • New treatment option to improve fertility by intravaginal infusion of recombinant osteopontin in repeat breeder dairy cows  [Not invited]
    佐藤弘子, 田上貴祥, 栁川洋二郎, 永野昌志, 片桐成二
    8th Sapporo Summer Symposium for One Health  2020/09
  • Effect of milk osteopontin on the normalization of endometrial epidermal growth factor profile in repeat breeder dairy cows  [Not invited]
    Hay Mar Kyaw, Takayoshi Tagami, Yojiro Yanagawa, Masashi Nagano, Seiji Katagiri
    8th Sapporo Summer Symposium for One Health  2020/09
  • 細菌 trehalose 6-phosphate hydrolaseの活性中心に結合するリン酸イオンおよび基質との複合体構造の分子解析  [Not invited]
    猪内 里花子, Klahan Patcharapa, 田上 貴祥, 田口陽大, 佐分利 亘, 森 春英, 奥山 正幸, 木村 淳夫
    日本応用糖質科学会令和2年度大会  2020/09
  • GH97 α-グルコシドヒドロラーゼが示す多様な基質特異性: 新奇な基質認識を与える2酵素と分子機構  [Not invited]
    菊池 麻子, 田上 貴祥, 奥山 正幸, 木村 淳夫
    日本応用糖質科学会令和2年度大会  2020/09
  • α-グルコシダーゼを用いた α-マンノシルオリゴ糖の合成  [Not invited]
    前山和輝, 田上貴祥, 奥山正幸, 木村淳夫
    日本農芸化学会2020年度大会  2020/03
  • ブタ血清由来マルターゼ-グルコアミラーゼを構成する2 つのサブユニットは独立に機能する  [Not invited]
    渡邊 憲, 田上貴祥, 奥山正幸, 木村淳夫
    日本農芸化学会2020年度大会  2020/03
  • ニゲロース特異的な Solute-binding protein の発見  [Not invited]
    田上貴祥, 壷井芙美, 佐藤宏樹, 奥山正幸, 佐分利亘, 森 春英, 木村淳夫
    日本農芸化学会2020年度大会  2020/03
  • 放線菌由来 GH13_30 α-グルコシダーゼの機能解析  [Not invited]
    佐分利亘, 田上貴祥, 壷井芙美, 奥山正幸, 木村淳夫, 森 春英
    日本農芸化学会2020年度大会  2020/03
  • 治療試験用オステオポンチンの調製と野外試験の現状  [Invited]
    田上 貴祥
    第112回 日本繁殖生物学会  2019/09
  • デキストランデキストリナーゼのSer923の機能解析  [Not invited]
    嶌村有季乃, 田上貴祥, 奥山正幸, 木村淳夫
    日本応用糖質科学会令和元年度大会  2019/09
  • β-Fructofuranosidaseの糖転移作用：飽和変異導入による改変  [Not invited]
    髙木惇生, 奥山正幸, 田上貴祥, 木村淳夫
    日本応用糖質科学会令和元年度大会  2019/09
  • 組み換えオステオポンチンによる牛子宮内膜上皮成長因子濃度の正常化効果の検証  [Not invited]
    佐藤 弘子, Hay Mar KYAW, 栁川 洋二郎, 永野 昌志, 田上 貴祥, 片桐 成二
    第112回 日本繁殖生物学会  2019/09
  • Bacteroides thetaiotaomicron由来GH97酵素：5つの機能未知パラログの解析  [Not invited]
    菊池麻子, 中川雄登, 奥山正幸, 田上貴祥, 木村淳夫
    日本農芸化学会2019年度大会  2019/03
  • Bacteroides thetaiotaomicron由来α-galactosidase(BtGal97a)の天然基質に関する研究  [Not invited]
    中川雄登, 松永夏奈, 菊池麻子, 奥山正幸, 田上貴祥, 木村淳夫
    日本農芸化学会2019年度大会  2019/03
  • ブタ血清由来マルターゼ-グルコアミラーゼを形成する酵素ユニットの機能  [Not invited]
    渡邊 憲, 田上貴祥, 奥山正幸, 木村淳夫
    日本農芸化学会2019年度大会  2019/03
  • ブタ血清由来α-グルコシダーゼの cDNA クローニングと組換え酵素の機能解析  [Not invited]
    渡邊憲, 田上貴祥, 奥山正幸, 木村淳夫
    公益社団法人 日本農芸化学会 東北・北海道合同支部大会 (東北支部第 153 回大会)  2018/09
  • 改変 β-fructofuranosidase による 6-ケストースの合成および精製  [Not invited]
    髙木惇生, 芹沢領, 奥山正幸, 田上貴祥, 上野敬司, 小野寺秀一, 木村淳夫
    公益社団法人 日本農芸化学会 東北・北海道合同支部大会 (東北支部第 153 回大会)  2018/09
  • Structural and functional investigation of starch-metabolizing enzymes  [Invited]
    Takayoshi Tagami
    13th International Symposium of the Protein Society of Thailand  2018/08
  • α-galactosidaseに想定される新奇な基質認識機構  [Not invited]
    菊池 麻子, 田上 貴祥, 奥山 正幸, 木村 淳夫
    日本農芸化学会2018年度大会  2018
  • デキストラン デキストリナーゼの多糖合成に関与するアミノ酸の同定  [Not invited]
    佐々木 優希, 熊谷 裕也, 貞廣 樹里, ラング ビーラヌッチ, 田上 貴祥, 奥山 正幸, 木村 淳夫
    日本農芸化学会2018年度大会  2018
  • Amino acid residues to govern substrate recognition of trehalose-6-phosphate hydrolase from Streptococcus mutans NBRC13955  [Not invited]
    Patcharapa KLAHAN, Masayuki OKUYAMA, Takayoshi TAGAMI, Atsuo KIMURA
    日本農芸化学会2018年度大会  2018
  • Enzymatic characterization of trehalose-6-phosphate hydrolase from Streptococcus mutants NBRC13955  [Not invited]
    P. Klahan, M. Okuyama, T. Tagami, A. Kimura
    12th Carbohydrate Bioengineering Meeting  2017/04
  • a-Glucosidase newly found in Aspergillus niger displays high specificity to a-(1→3)-glucosidic linkage  [Not invited]
    M. Ma, M. Okuyama, T. Tagami, A. Kimura
    12th Carbohydrate Bioengineering Meeting  2017/04
  • Characterization of thermostable α-glucosidase from Geobacillus caldoxylosilyticus NBRC 107762, which displays remarkable transglucosylation  [Not invited]
    Min MA, Masayuki OKUYAMA, Takayoshi TAGAMI, Atsuo KIMURA
    日本農芸化学会2017年度大会  2017
  • シングルアンカー型イソマルトメガロ糖におけるケルセチン-3-O-β-グルコシド可溶化に寄与する糖質構造の決定  [Not invited]
    土生 慎二, ラング ビーラヌッチ, 熊谷 裕也, 貞廣 樹里, 植草 聡太, 田上 貴祥, 奥山 正幸, 原 博, 木村 淳夫
    日本農芸化学会2017年度大会  2017
  • Vibrio由来glycoside hydrolase family 17の機能未知なC末端領域は酵素活性の発現に必須である  [Not invited]
    熊谷 祐也, 田上 貴祥, 奥山 正幸, 木村 淳夫
    日本農芸化学会2017年度大会  2017
  • 新奇な基質認識機構を有するα-galactosidaseに関する研究  [Not invited]
    菊池麻子, 奥山正幸, 田上貴祥, 木村淳夫
    平成２９年度 日本農芸化学会 北海道支部第１回講演会  2017
  • GH15 isomaltose glucohydrolaseの基質認識に関わる構造因子の同定  [Not invited]
    田上貴祥, 陳明皓, 奥山正幸, 岩_智仁, 田中良和, 姚閔, 木村淳夫
    日本応用糖質科学会平成29年度大会  2017
  • Structure element to regulate transglucosidation specificity of Aspergillus niger α-glucosidase  [Not invited]
    M. Ma, M. Okuyama, M. Sato, T. Tagami, H. Mori, A. Kimura
    6th Symposium on the Alpha-Amylase Family  2016/09
  • Reaction mechanism of glycosidases by studying 1,5-anhydro-d-fructose (AF) production  [Not invited]
    J. Maneesan, H. Matsuura, T. Tagami, H. Mori, A. Kimura
    28th International carbohydrate symposium  2016/07
  • Purification and characterization of a-1,3-glucosidase from Aspergillus niger  [Not invited]
    M. Ma, T. Tagami, M. Okuyama, A. Kimura
    28th International carbohydrate symposium  2016/07
  • 環状四糖[cyclo-(1→6)-(α-nigerosyl-nigerosyl)]の分解を担う2種の糖質加水分解酵素の発見  [Not invited]
    田上貴祥, 貞廣樹里, 奥山正幸, 木村淳夫, 岩崎智仁
    日本農芸化学会2016年度大会  2016
  • Kribbella flavida由来isomaltosyltransferaseの機能解析  [Not invited]
    宮野江梨, 田上貴祥, 奥山正幸, 木村淳夫
    日本農芸化学会2016年度大会  2016
  • Efficient production and characterization of α-1,3-glucosidase from Aspergillus niger  [Not invited]
    Min MA, Takayoshi TAGAMI, Masayuki OKUYAMA, Atsuo KIMURA
    日本農芸化学会2016年度大会  2016
  • ブタロース肉とヒレ肉におけるトリポリリン酸加水分解活性の比較  [Not invited]
    岩崎智仁, 大塚創太, 田上貴祥, 舩津保浩, 石下真人
    日本畜産学会第121回大会  2016
  • GH97 α-galactosidase求核触媒変異酵素が触媒する糖転移反応の反応機構  [Not invited]
    松永 夏奈, 奥山 正幸, 渡辺 健一, 田上 貴祥, 山下 恵太郎, 姚 閔, 森 春英, 木村 淳夫
    日本応用糖質科学会平成28年度大会  2016
  • 長鎖阻害剤の利用による植物a-グルコシダーゼの機能構造相関の解明  [Invited]
    田上貴祥, 山下恵太郎, 奥山正幸, 森春英, 姚 閔, 木村淳夫
    平成27年度 応用糖質科学シンポジウム  2015/09
  • Structural and biochemical studies of sugar beet a-glucosidase exhibiting high specificity for long-chain substrates  [Not invited]
    T. Tagami, K. Yamashita, M. Okuyama, H. Mori, M. Yao, A. Kimura
    11th Carbohydrate Bioengineering Meeting  2015/05
  • A transglycosylation of catalytic nucleophile mutant of GH97 a-galactosidase with an external nucleophile  [Not invited]
    M. Okuyama, K. Matsunaga, K. Watanabe, T. Tagami, K. Yamashita, H. Mori, M. Yao, A. Kimura
    11th Carbohydrate Bioengineering Meeting  2015/05
  • テンサイa-グルコシダーゼの高重合度基質特異性はらせん構造の長鎖基質に適したサブサイト構造に起因する  [Not invited]
    田上貴祥, 山下恵太郎, 奥山正幸, 森 春英, 姚 閔, 木村淳夫
    日本農芸化学会2015年度大会  2015
  • 食肉タンパク質の溶解性と熱ゲル化特性に及ぼすアミノ酸添加の影響  [Not invited]
    船津保浩, 岩_智仁, 田上貴祥
    FOOMA JAPAN 2015  2015
  • Production of 1,5-anhydro-d-fructose by a-glucosidase belong to glycoside hydrolase family 13 and 31  [Not invited]
    Janjira Maneesan, Hideyuki Matsuura, Takayoshi Tagami, Momoko Kobayashi, Masayuki Okuyama, Haruhide Mori, Atsuo Kimura
    日本農芸化学会2014年度大会  2014
  • 筋損傷部位における酸化タンパク質領域の可視化  [Not invited]
    岩_智仁, 石崎恵梨, 田上貴祥, 美名口順, 竹花一成, Miranda Grounds, Peter Arthur
    日本畜産学会第118回大会  2014
  • 放線菌Kribbella flavidaによるオリゴ糖合成の検討  [Not invited]
    田上貴祥, 奥山正幸, 岩_智仁, 木村淳夫
    日本農芸化学会平成26年度北海道支部・東北支部合同支部会  2014
  • Enzymatic synthesis of acarviosyl-maltooligosaccharides and their inhibitory effects on a-glucosidase  [Not invited]
    T. Tagami, Y. Tanaka, H. Mori, M. Okuyama, A. Kimura
    26th International Carbohydrate Symposium  2012/07
  • Structural insights into specificity for long-chain substrates of sugar beet a-glucosidase  [Not invited]
    T. Tagami, K. Yamashita, M. Okuyama, H. Mori, M. Yao, A. Kimura
    Plant and Seaweed Polysaccharides Symposium 2012  2012/07
  • Acarviosyl-maltooligosaccharideの酵素合成とテンサイa-glucosidaseに対する基質アナログとしての利用  [Not invited]
    田上貴祥, 山下恵太郎, 田中良幸, 奥山正幸, 森 春英, 姚 閔, 木村淳夫
    日本応用糖質科学会平成24年度大会  2012
  • Aspergillus niger由来a-glucosidaseの基質特異性に関与するアミノ酸残基の解析  [Not invited]
    佐藤恵美, 田上貴祥, 奥山正幸, 森春英, 木村淳夫
    日本農芸化学会北海道支部平成24年度支部講演会  2012
  • Structural element determining the diverse specificities for chain-length of substrate in glycoside hydrolase family 31 a-glucosidases  [Not invited]
    T. Tagami, M. Okuyama, H. Mori, A. Kimura
    9th Carbohydrate Bioengineering Meeting  2011/05
  • GH 31 a-glucosidase における基質の鎖長認識機構の解明  [Not invited]
    田上貴祥, 奥山正幸, 森 春英, 木村淳夫
    日本農芸化学会北海道支部平成23年度夏期シンポジウム  2011
  • GH family 31に属するAspergillus niger 由来a-glucosidaseの鎖長特異性の改変  [Not invited]
    田上貴祥, 奥山正幸, 森 春英, 木村淳夫
    日本応用糖質科学会平成23年度大会  2011
  • Dextran glucosidase (DexB) ウォーターパス改変体の機能と構造  [Not invited]
    小林桃子, 山下恵太郎, 田上貴祥, 本同宏成, 森 春英, 奥山正幸, 姚閔, 木村敦夫
    日本応用糖質科学会平成23年度大会  2011
  • Shifting specificity of Aspergillus niger a-glucosidase from short-chain substrates to long-chain substrates  [Not invited]
    T. Tagami, T. Nishimura, M. Okuyama, H. Mori, A. Kimura
    Plant Polysaccharide and Applied Glycoscience Workshop 2010  2010/07
  • GH family 31 a-グルコシダーゼにおける基質の鎖長認識機構の解明  [Not invited]
    田上貴祥, 西村崇志, 奥山正幸, 森 春英, 木村淳夫
    日本農芸化学会2010年度大会  2010
  • Aspergillus niger a-glucosidaseのサブサイト+1の改変  [Not invited]
    田上貴祥, 奥山正幸, 森 春英, 木村淳夫
    日本応用糖質科学会平成22年度大会  2010
  • Specificity element recognizing long-chain substrates in GH family 31 a-glucosidase derived from sugar beet (Beta vulgaris L.)  [Not invited]
    T. Tagami, M. Okuyama, H. Nakai, H. Mori, K. Taguchi, A. Kimura
    8th Carbohydrate Bioengineering Meeting  2009/05
  • テンサイa-glucosidaseのサブサイト+2および+3の形成に重要なアミノ酸残基の決定  [Not invited]
    田上貴祥, 奥山正幸, 森 春英, 田口和憲, 木村淳夫
    日本応用糖質科学会平成21年度大会  2009
  • テンサイa-glucosidaseのcDNAクローニングとPichia pastorisによる組換え酵素の生産  [Not invited]
    田上貴祥, 奥山正幸, 森 春英, 田口和憲, 木村淳夫
    日本応用糖質科学会平成20年度大会  2008

Association Memberships

  • 日本蛋白質科学会   JAPAN SOCIETY FOR BIOSICENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY   THE JAPANESE SOCIETY OF APPLIED GLYCOSCIENCE   

Research Projects

  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2023/04 -2028/03 
    Author : 片桐 成二, 柳川 洋二郎, 杉浦 智親, 奥山 みなみ, 平山 博樹, 永野 昌志, 田上 貴祥
  • 牛の精漿蛋白質による子宮機能調節機序の解明と受胎性改善技術への応用
    日本学術振興会：科学研究費助成事業 基盤研究（A）
    Date (from‐to) : 2019/04 -2024/03 
    Author : 片桐 成二
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
    Date (from‐to) : 2019/10 -2023/03 
    Author : 木村 淳夫, 橋床 泰之, 崎浜 靖子, 奥山 正幸, 田上 貴祥
     
    我々は世界で初めてメガロ糖（MS）の生産に成功した。性質を調べると、BCS IIに属す化合物（難水溶性・高膜透過性の薬剤や食品素材など）を可溶化する画期的な機能が発見された。またMSは「BCS II化合物を溶質とする糖質水溶化剤」と捉えることもできた。一方、難溶性ベンジル系アゾ色素もBCS IIに属し、かつ「東南アジア諸国における名高い環境汚染物質」である点に注目し、MSとアゾ分解酵素を組合せることで、実験室レベルではあるが、色素の可溶化と酵素分解に成功した。以上は初めて生産したMS、すなわち従来型MSの知見である。極最近に従来型MSより高機能な新奇MS（新型MS）を発見した。本申請では、新型MSによるアゾ色素の酵素分解を目的とし、現地試験をタイで実施する。最終的な到達目標はアゾ色素の汚染解消（すなわち環境問題解決への貢献）である。 本年度は、新型コロナウイルス感染症の流行および相手国の政情不安から渡航が困難となり、現地調査に大きな支障が生じ、計画の遂行に予想外の大きな遅れが発生した。本状況下で得られた成果を述べる。多糖選抜および新型MS調製の項目に関し、昨年度に行った研究計画上の工夫（調査が不可な期間は、日タイ共通植物種を対象に研究を先行。渡タイ可能時に現地で結果検証）により研究を進めた。共通植物種から取得したMSが示すBCS II化合物の可溶化能を測定し、優れた能力を有する新奇MSを見出した。さらに調査対象の植物種を増やし、多糖調製・MS分離・構造と機能の解析を継続している。なお、現地で実施が必要なMS高機能化および色素汚染解消に関する計画に大幅な遅延が生じているが、渡航開始後に打開を目指したい。
  • 乳牛の低受胎対策技術の実行可能性検証事業
    公益財団法人全国競馬・畜産振興会：日本中央競馬会畜産振興事業
    Date (from‐to) : 2021/04 -2023/03
  • O-マンノース型糖鎖の酵素合成と分解酵素探索
    日本学術振興会：科学研究費助成事業 若手研究
    Date (from‐to) : 2019/04 -2022/03 
    Author : 田上 貴祥
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2017/04 -2021/03 
    Author : KIMURA Atsuo
     
    We succeeded in production of megalosaccharides and found their valuable function to solubilize water-insoluble compounds. However, the period of our research is very short, so that we have many problems that must be solved. This project challenges a resolution of three important problems, from which we will obtain the fundamental knowledge about megalosaccharides. Furthermore, it also contributes to the development of application research on megalosaccharides. The purposes of this program are 1) analysis of molecular mechanism of polysaccharide-forming enzyme to produce megalosaccharide, 2) synthesis of new megalosaccharide with high functionality, and 3) improvement of azo-dye pollution using megalosaccharide.
  • タンパク質工学による新奇オリゴ糖合成酵素の作出
    公益財団法人栗林育英学術財団：研究助成
    Date (from‐to) : 2018/10 -2020/03 
    Author : 田上 貴祥
  • 乳牛の低受胎対策新規技術開発事業
    日本中央競馬会：日本中央競馬会畜産振興事業
    Date (from‐to) : 2017/04 -2020/03 
    Author : 片桐 成二
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