Researcher basic information

• Ph. D. (S. Chiba) (Hokkaido University), Hokkaido University

• https://researchmap.jp/read0118392?lang=en

• https://jglobal.jst.go.jp/detail?JGLOBAL_ID=200901065754657289
• https://sites.google.com/elms.hokudai.ac.jp/molenz/home

• 200901065754657289

• 糖質分解酵素
• 分子進化
• 基質認識
• α-グルコシダーゼ
• 立体構造解析
• 触媒メカニズム
• α-グルコシダーゼII
• 糖転移反応
• 自殺基質
• タンパク質工学
• タンパク質の分子進化
• 小胞体グルコシダーゼ
• 触媒機構
• 基質特異性
• 糖質加水分解酵素
• デキストラナーゼ
• 部位特異的変異
• 糖転移
• 国際情報交換
• 蜂蜜生成
• 蛋白質工学
• アジア原産ミツバチ
• アミノ酸置換
• 多糖合成酵素
• タイ:韓国
• 構造因子
• 酵素反応
• グリコシダーゼ
• 合成酵素
• オリゴ糖合成
• 分子酵素学
• protein engineering
• molecular enzymology

• Life Science, Applied molecular and cellular biology
• Life Science, Applied biochemistry

• Bachelor's degree program, School of Agriculture
• Master's degree program, Graduate School of Agriculture
• Doctoral (PhD) degree program, Graduate School of Agriculture

Research activity information

• 2012, Committee of Plant and Seaweed Polysaccharides Workshop, Committee of Plant and Seaweed Polysaccharides Workshop, Poster prize
  OKUYAMA Masayuki
• 2010, 農芸化学奨励賞
  Japan
• 2007, CBM7 7th Carbohydrate Bioengineering Meeting Poster prize
• 2003, 日本農芸化学会, 日本農芸化学会BBB論文賞
  奥山 正幸

• Porcine serum maltase-glucoamylase: structure, kinetics, and inhibition
  Ken Watanabe; Takayoshi Tagami; Chihiro Biwa; Masato Kawasaki; Naruhiko Adachi; Toshio Moriya; Toshiya Senda; Masayuki Okuyama
  Journal of Enzyme Inhibition and Medicinal Chemistry, 41, 1, Informa UK Limited, 14 Jan. 2026
  Scientific journal
• Structural basis of transglucosylation in dextran dextrinase, a homolog of anomer-inverting GH15 glucoside hydrolases.
  Takayoshi Tagami; Wataru Saburi; Juri Sadahiro; Yuya Kumagai; Weeranuch Lang; Naohiro Matsugaki; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura
  The Journal of biological chemistry, 301, 9, 110541, 110541, 30 Jul. 2025, [International Magazine]
  English, Scientific journal, Bacterial exopolysaccharide, dextran, primarily composed of α-(1→6)-linked d-glucosyl residues, is synthesized from α-(1→4)-glucan dextrin or sucrose through successive anomer-retaining transglucosylation reactions by dextran dextrinase (DDase) or dextransucrase, respectively. Although the structure-function relationship of dextransucrase has been extensively studied, that of DDase remains largely unknown. Herein, we revealed the Gluconobacter oxydans DDase structural basis through biochemical and structural analyses. The DDase comprises 1284 residues, with its N-terminal 902 residues being functionally essential. Crystal structure analysis of the minimal active DDase (Δ382C) complex with the pseudo-maltotetraose inhibitor, acarbose, revealed its homodimeric structure. A Δ382C protomer contains two β-sandwich domains, N1 and N2, and an (α/α)6-barrel domain A. Surprisingly, domains N2, A, and the helix-loop-helix connecting them structurally resemble those of bacterial anomer-inverting glucohydrolases in glycoside hydrolase family 15 (GH15). Domain N1 primarily forms intra- and inter-subunit domain interfaces. The DDase acarbose-binding residues in subsite -1 are conserved with GH15 glucohydrolases. The DDase Glu671 and Glu858 are positioned similarly to the GH15 glucohydrolase general acid and base catalysts, respectively. However, Glu858 is approximately 1.2 to 1.6 Å closer to the acarbose equivalent anomeric carbon, facilitating its role as a nucleophilic catalyst in the double displacement mechanism. The catalytic residue functions were biochemically confirmed using mutant enzymes. Spatial position of Glu858 is arranged by the local structure of the α11→α12 loop and subunit interactions involving domain N1. Enzymes classified in the same GH family catalyze reactions with different mechanisms, anomer-inverting or -retaining, due to differences in their catalytic residue spatial arrangement.
• Mutant β-fructofuranosidase synthesizing blastose [β-d-Fruf-(2→6)-d-Glcp].
  Atsuki Takagi; Takayoshi Tagami; Masayuki Okuyama
  Enzyme and microbial technology, 180, 110500, 110500, Oct. 2024, [International Magazine]
  English, Scientific journal, Fructooligosaccharides (FOS) are leading prebiotics that help keep the gut healthy and aid wellness by stimulating the growth and activity of beneficial intestinal bacteria. The best-studied FOS are inulin-type FOS, mainly oligosaccharides with β-Fruf-(2→1)-Fruf linkages, including 1-kestose [β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp] and nystose [β-Fruf-(2→1)-β-Fruf-(2→1)-β-Fruf-(2↔1)-α-Glcp]. However, the properties of other types of FOS-levan-type FOS with β-Fruf-(2→6)-Fruf linkages and neo-type FOS with β-Fruf-(2→6)-Glcp linkages-remain ambiguous because efficient methods have not been established for their synthesis. Here, using site-saturation mutation of residue His79 of β-fructofuranosidase from Zymomonas mobilis NBRC13756, we successfully obtained a mutant β-fructofuranosidase that specifically produces neo-type FOS. The H79G enzyme variant loses the native β-Fruf-(2→1)-Fru-transfer ability (which produces 1-kestose), and instead has β-Fruf-(2→6)-Glc-transfer ability and produces neokestose. Its hydrolytic activity specific to the β-Fruf-(2↔1)-α-Glcp bond of neokestose then yields blastose [β-Fruf-(2→6)-Glcp]. The enzyme produces 0.4 M blastose from 1.0 M sucrose (80 % of the theoretical yield). The production system for blastose established here will contribute to the elucidation of the physiological functions of this disaccharide.
• Substrate Specificity of GH29 α-L-Glucosidases from Cecembia lonarensis.
  Hye-Jin Kang; Takayoshi Tagami; Masayuki Okuyama
  Journal of applied glycoscience, 71, 3, 91, 94, 2024, [Domestic magazines]
  English, Scientific journal, We recently found two α-L-glucosidases, which can hydrolyze p-nitrophenyl α-L-glucopyranoside (PNP L-Glc) rather than p-nitrophenyl α-L-fucopyranoside, in glycoside hydrolase family 29. This study evaluated their substrate specificity for p-nitrophenyl α-L-rhamnopyranoside (PNP L-Rha), α-L-quinovopyranoside (PNP L-Qui), and α-L-xylopyranoside (PNP L-Xyl), of which structure is similar to PNP L-Glc. The two α-L-glucosidases had little activity toward PNP L-Rha. They exhibited higher k cat/K m values for PNP L-Qui but smaller for PNP L-Xyl than for PNP L-Glc. The molecular docking studies indicated that these specificities were correlated well with the active-site structure of the α-L-glucosidases. The finding that α-L-quinovoside, which has been suggested to occur in nature, is also a substrate for α-L-glucosidases indicates that this enzyme are not solely dedicated to α-L-glucoside hydrolysis.
• Structural and mutational analysis of glycoside hydrolase family 1 Br2 β-glucosidase derived from bovine rumen metagenome
  Wilaiwan Kaenying; Takayoshi Tagami; Eukote Suwan; Chariwat Pitsanuwong; Sinchai Chomngam; Masayuki Okuyama; Palangpon Kongsaeree; Atsuo Kimura; Prachumporn T. Kongsaeree
  Heliyon, 9, 11, e21923, e21923, Elsevier BV, Nov. 2023, [International Magazine]
  English, Scientific journal, Ruminant animals rely on the activities of β-glucosidases from residential microbes to convert feed fibers into glucose for further metabolic uses. In this report, we determined the structures of Br2, which is a glycoside hydrolase family 1 β-glucosidase from the bovine rumen metagenome. Br2 folds into a classical (β/α)8-TIM barrel domain but displays unique structural features at loop β5→α5 and α-helix 5, resulting in different positive subsites from those of other GH1 enzymes. Br2 exhibited the highest specificity toward laminaritriose, suggesting its involvement in β-glucan hydrolysis in digested feed. We then substituted the residues at subsites +1 and + 2 of Br2 with those of Halothermothrix orenii β-glucosidase. The C170E and C221T mutations provided favorable interactions with glucooligosaccharide substrates at subsite +2, while the A219N mutation probably improved the substrate preference for cellobiose and gentiobiose relative to laminaribiose at subsite +1. The N407Y mutation increased the affinity toward cellooligosaccharides. These results give further insights into the molecular determinants responsible for substrate specificity in GH1 β-glucosidases and may provide a basis for future enzyme engineering applications.
• 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, Elsevier BV, Nov. 2023, [Peer-reviewed], [International Magazine]
  English, Scientific journal, Isomaltomegalosaccharides with α-(1 → 4) and α-(1 → 6)-segments solubilize water-insoluble ligands since the former complexes with the ligand and the latter solubilizes the complex. Previously, we enzymatically synthesized isomaltomegalosaccharide with a single α-(1 → 4)-segment at the reducing end (S-IMS) by dextran dextrinase (DDase), but the chain length [average degree of polymerization (DP) ≤ 9] was insufficient for strong encapsulation. We hypothesized that the conjugation of longer α-(1 → 4)-segment afforded the promising function although DDase is incapable to do so. In this study, the cyclodextrin glucanotransferase-catalyzed coupling reaction of α-cyclodextrin to S-IMS synthesized a new α-(1 → 4)-segment at the nonreducing end (N-4S) of S-IMS to form D-IMS [IMS harboring double α-(1 → 4)-segments]. The length of N-4S was modulated by the ratio between α-cyclodextrin and S-IMS, generating N-4Ss with DPs of 7-50. Based on phase-solubility analysis, D-IMS-28.3/13/3 bearing amylose-like helical N-4S with DP of 28.3 displayed a water-soluble complex with aromatic drugs and curcumin. Small-angle X-ray scattering revealed the chain adapted to rigid in solution in which the radius of gyration was estimated to 2.4 nm. Furthermore, D-IMS with short N-4S solubilized flavonoids of less-soluble multifunctional substances. In our research, enzyme-generated functional biomaterials from DDase were developed to maximize the hydrophobic binding efficacy towards water-insoluble bioactive compounds.
• Partial depolymerization of tamarind seed xyloglucan and its functionality toward enhancing the solubility of curcumin
  Weeranuch Lang; Takayoshi Tagami; Hye-Jin Kang; Masayuki Okuyama; Nobuo Sakairi; Atsuo Kimura
  Carbohydrate Polymers, 307, 120629, 120629, Elsevier BV, May 2023, [International Magazine]
  English, Scientific journal, Polysaccharides of tamarind seed, a byproduct of the tamarind pulp industry, displayed a potential solubility improvement of lipophilic bioactive molecules but their textural characteristics hinder the dietary formulation. In contrast, the commonly available xyloglucan oligosaccharides (XOSs) with degrees of polymerization (DPs) of 7, 8, and 9 were too short to maintain their ability. The binding capacity of the between sizes is unknown due to a lack of appropriate preparation. We prepared xyloglucan megalosaccharides (XMSs) by partial depolymerization, where term megalosaccharide (MS) defines the middle chain-length saccharide between DPs 10 and 100. Digestion with fungal cellulase enabled reproducible active XMSs. Further identification of pure XMS segments indicated that XMS-B has an average DP of 17.2 (Gal3Glc8Xyl6) with a branched dimer of XOS 8 and 9 and was free of side-chain arabinose, the residue influencing high viscosity. Curcumin, a bioactive pigment, has poor bioavailability because of its water insolubility. XMSs with average DPs of 15.4-24.3 have similarly sufficient capacities to solubilize curcumin. The solubility of curcumin was improved 180-fold by the addition of 50 %, w/v, XMSs, which yielded a clear yellow liquid. Our findings indicated that XMSs were a promising added-value agent in foods and pharmaceuticals for the oral intake of curcumin.
• Nonreducing terminal chimeric isomaltomegalosaccharide and its integration with azoreductase for the remediation of soil-contaminated lipophilic azo dyes
  Weeranuch Lang; Sarote Sirisansaneeyakul; Takayoshi Tagami; Hye-Jin Kang; Masayuki Okuyama; Nobuo Sakairi; Atsuo Kimura
  Carbohydrate Polymers, 305, 120565, 120565, Elsevier BV, Apr. 2023, [Peer-reviewed], [International Magazine]
  English, Scientific journal, Lipophilic azo dyes are practically water-insoluble, and their dissolution by organic solvents and surfactants is harmful to biological treatment with living cells and enzymes. This study aimed to evaluate the feasibility of a newly synthesized nonreducing terminal chimeric isomaltomegalosaccharide (N-IMS) as a nontoxic solubilizer of four simulated lipophilic azo dye wastes for enzymatic degradation. N-IMS bearing a helical α-(1 → 4)-glucosidic segment derived from a donor substrate α-cyclodextrin was produced by a coupling reaction of cyclodextrin glucanotransferase. Inclusion complexing by N-IMS overcame the solubility issue with equilibrium constants of 1786-242 M-1 (methyl yellow > ethyl red > methyl red > azo violet). Circular dichroism spectra revealed the axial alignment of the aromatic rings in the N-IMS cavity, while UV-visible absorption quenching revealed that the azo bond of methyl yellow was particularly induced. Desorption of the dyes from acidic and neutral soils was specific to aqueous organic over alkali extraction. The dissolution kinetics of the incorporated dyes followed a sigmoid pattern facilitating the subsequent decolorization process with azoreductase. It was demonstrated that after soil extraction, the solid dyes dissolved with N-IMS assistance and spontaneously digested by coupled azoreductase/glucose dehydrogenase (for a cofactor regeneration system) with the liberation of the corresponding aromatic amine.
• Formulation and evaluation of a novel megalomeric microemulsion from tamarind seed xyloglucan-megalosaccharides for improved high-dose quercetin delivery
  Weeranuch Lang; Debashish Mondol; Aphichat Trakooncharoenvit; Takayoshi Tagami; Masayuki Okuyama; Tohru Hira; Nobuo Sakairi; Atsuo Kimura
  Food Hydrocolloids, 137, 108430, 108430, Elsevier BV, Apr. 2023, [Peer-reviewed]
  Scientific journal
• Crystal structure and identification of amino acid residues for catalysis and binding of GH3 AnBX β-xylosidase from Aspergillus niger
  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, Springer Science and Business Media LLC, 06 Mar. 2023, [Peer-reviewed], [International Magazine]
  English, Scientific journal, β-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 β-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction. The structure of the E88A mutant of AnBX, determined at 2.5-Å resolution, contains two molecules in the asymmetric unit, each of which is composed of three domains, namely an N-terminal (β/α)8 TIM-barrel-like domain, an (α/β)6 sandwich domain, and a C-terminal fibronectin type III domain. Asp288 and Glu500 of AnBX were experimentally confirmed to act as the catalytic nucleophile and acid/base catalyst, respectively. The crystal structure revealed that Trp86, Glu88 and Cys289, which formed a disulfide bond with Cys321, were located at subsite -1. Although the E88D and C289W mutations reduced catalytic efficiency toward all four substrates tested, the substitution of Trp86 with Ala, Asp and Ser increased the substrate preference for glucoside relative to xyloside substrates, indicating that Trp86 is responsible for the xyloside specificity of AnBX. The structural and biochemical information of AnBX obtained in this study provides invaluable insight into modulating the enzymatic properties for the hydrolysis of lignocellulosic biomass. KEY POINTS: • Asp288 and Glu500 of AnBX are the nucleophile and acid/base catalyst, respectively • Glu88 and the Cys289-Cys321 disulfide bond are crucial for the catalytic activity of AnBX • The W86A and W86S mutations in AnBX increased the preference for glucoside substrates.
• Discovery of α-l-Glucosidase Raises the Possibility of α-l-Glucosides in Nature
  Rikako Shishiuchi; Hyejin Kang; Takayoshi Tagami; Yoshitaka Ueda; Weeranuch Lang; Atsuo Kimura; Masayuki Okuyama
  ACS Omega, 7, 50, 47411, 47423, American Chemical Society (ACS), 09 Dec. 2022, [Peer-reviewed], [Corresponding author], [International Magazine]
  English, Scientific journal, Glucose, a common monosaccharide in nature, is dominated by the d-enantiomer. Meanwhile, the discovery of l-glucose-utilizing bacteria and the elucidation of their metabolic pathways 10 years ago suggests that l-glucose exists naturally. Most carbohydrates exist as glycosides rather than monosaccharides; therefore, we expected that nature also contains l-glucosides. Sequence analysis within glycoside hydrolase family 29 led us to identify two α-l-glucosidases, ClAgl29A and ClAgl29B, derived from Cecembia lonarensis LW9. ClAgl29A and ClAgl29B exhibited higher K m, k cat, and k cat/K m values for p-nitrophenyl α-l-glucoside than that for p-nitrophenyl α-l-fucoside. Structural analysis of ClAgl29B in complex with l-glucose showed that these enzymes have an active-site pocket that preferentially binds α-l-glucoside, but excludes α-l-fucoside. These results suggest that ClAgl29A and ClAgl29B evolved to hydrolyze α-l-glucoside, implying the existence of α-l-glucoside in nature. Furthermore, α-l-glucosidic linkages (α-l-Glc-(1 → 3)-l-Glc, α-l-Glc-(1 → 2)-l-Glc, and α-l-Glc-(1 → 6)-l-Glc) were synthesized by the transglucosylation activity of ClAgl29A and ClAgl29B. We believe that this study will lead to new research on α-l-glucosides, including determining the physiological effects on humans, and the discovery of novel α-l-glucoside-related enzymes.
• Physicochemical functionality of chimeric isomaltomegalosaccharides with α-(1 → 4)-glucosidic segments of various lengths
  Weeranuch Lang; Yuya Kumagai; Shinji Habu; Juri Sadahiro; Takayoshi Tagami; Masayuki Okuyama; Shinichi Kitamura; Nobuo Sakairi; Atsuo Kimura
  Carbohydrate Polymers, 291, 119562, 119562, Elsevier BV, Sep. 2022, [International Magazine]
  English, Scientific journal, Isomaltomegalosaccharide (IMS) is a long chimeric glucosaccharide composed of α-(1 → 6)- and α-(1 → 4)-linked segments at nonreducing and reducing ends, respectively; the hydrophilicity and hydrophobicity of these segments are expected to lead to bifunctionality. We enzymatically synthesized IMS with average degrees of polymerization (DPs) of 15.8, 19.3, and 23.5, where α-(1 → 4)-segments had DPs of 3, 6, and 9, respectively. IMS exhibited considerably higher water solubility than maltodextrin because of the α-(1 → 6)-segment and an identical resistance to thermal degradation as short dextran. Interaction of IMS with a fluorescent probe of 2-p-toluidinylnaphthalene-6-sulfonate demonstrated that IMS was more hydrophobic than maltodextrin, where the degree of hydrophobicity increased as DP of α-(1 → 4)-segment increased (9 > 6 > 3). Fluorescent pyrene-estimating polarity of IMS was found to be similar to that of methanol or 1-butanol. The bifunctional IMS enhanced the water solubility of quercetin-3-O-glucoside and quercetin: the solubilization of less-soluble bioactive substances is beneficial in carbohydrate industry.
• Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain
  Yuya Kumagai; Hideki Kishimura; Weeranuch Lang; Takayoshi Tagami; Masayuki Okuyama; Atsuo Kimura
  Marine Drugs, 20, 4, 250, 250, MDPI AG, 31 Mar. 2022, [International Magazine]
  English, Scientific journal, 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.
• 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, Springer Science and Business Media {LLC}, Jan. 2022, [International Magazine]
  English, Scientific journal, 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%.
• Structural insights reveal the second base catalyst of isomaltose glucohydrolase.
  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, 19 Oct. 2021, [International Magazine]
  English, Scientific journal, 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, respectively. 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.
• Molecular insight into regioselectivity of transfructosylation catalyzed by GH68 levansucrase and β-fructofuranosidase.
  Masayuki Okuyama; Ryo Serizawa; Masanari Tanuma; Asako Kikuchi; Juri Sadahiro; Takayoshi Tagami; Weeranuch Lang; Atsuo Kimura
  The Journal of biological chemistry, 296, 100398, 100398, Elsevier {BV}, 08 Feb. 2021, [Peer-reviewed], [Lead author], [International Magazine]
  English, Scientific journal, 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.
• Novel α-1,3/α-1,4-Glucosidase from Aspergillus niger Exhibits Unique Transglucosylation to Generate High Levels of Nigerose and Kojibiose.
  Min Ma; Masayuki Okuyama; Takayoshi Tagami; Asako Kikuchi; Patcharapa Klahan; Atsuo Kimura
  Journal of agricultural and food chemistry, 67, 12, 3380, 3388, 27 Mar. 2019, [Peer-reviewed], [International Magazine]
  English, Scientific journal, 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.
• Engineered dextranase from Streptococcus mutans enhances the production of longer isomaltooligosaccharides.
  Patcharapa Klahan; Masayuki Okuyama; Kohei Jinnai; Min Ma; Asako Kikuchi; Yuya Kumagai; Takayoshi Tagami; Atsuo Kimura
  Bioscience, biotechnology, and biochemistry, 82, 9, 1480, 1487, Informa {UK} Limited, Sep. 2018, [Peer-reviewed], [International Magazine]
  English, Scientific journal, 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.
• A novel glycoside hydrolase family 97 enzyme: Bifunctional β-l-arabinopyranosidase/α-galactosidase from Bacteroides thetaiotaomicron.
  Asako Kikuchi; Masayuki Okuyama; Koji Kato; Shohei Osaki; Min Ma; Yuya Kumagai; Kana Matsunaga; Patcharapa Klahan; Takayoshi Tagami; Min Yao; Atsuo Kimura
  Biochimie, 142, 41, 50, Nov. 2017, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Substrate recognition of the catalytic α-subunit of glucosidase II from Schizosaccharomyces pombe.
  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, Aug. 2017, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Effects of mutation of Asn694 in Aspergillus niger α-glucosidase on hydrolysis and transglucosylation.
  Min Ma; Masayuki Okuyama; Megumi Sato; Takayoshi Tagami; Patcharapa Klahan; Yuya Kumagai; Haruhide Mori; Atsuo Kimura
  Applied microbiology and biotechnology, 101, 16, 6399, 6408, Aug. 2017, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b).
  Masayuki Okuyama; Kana Matsunaga; Ken-Ichi Watanabe; Keitaro Yamashita; Takayoshi Tagami; Asako Kikuchi; Min Ma; Patcharapa Klahan; Haruhide Mori; Min Yao; Atsuo Kimura
  The FEBS journal, 284, 5, 766, 783, Mar. 2017, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Functional characterization of UDP-rhamnose-dependent rhamnosyltransferase involved in anthocyanin modification, a key enzyme determining blue coloration in Lobelia erinus.
  Yang-Hsin Hsu; Takayoshi Tagami; Kana Matsunaga; Masayuki Okuyama; Takashi Suzuki; Naonobu Noda; Masahiko Suzuki; Hanako Shimura
  The Plant journal : for cell and molecular biology, 89, 2, 325, 337, Jan. 2017, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Molecular insights into the mechanism of thermal stability of actinomycete mannanase.
  Yuya Kumagai; Misugi Uraji; Kun Wan; Masayuki Okuyama; Atsuo Kimura; Tadashi Hatanaka
  FEBS letters, 590, 17, 2862, 9, Sep. 2016, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Kinetic properties and substrate inhibition of α-galactosidase from Aspergillus niger.
  Julan Liao; Masayuki Okuyama; Keigo Ishihara; Yoshinori Yamori; Shigeo Iki; Takayoshi Tagami; Haruhide Mori; Seiya Chiba; Atsuo Kimura
  Bioscience, biotechnology, and biochemistry, 80, 9, 1747, 52, Sep. 2016, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl.
  Takayoshi Tagami; Eri Miyano; Juri Sadahiro; Masayuki Okuyama; Tomohito Iwasaki; Atsuo Kimura
  The Journal of biological chemistry, 291, 32, 16438, 47, 05 Aug. 2016, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Heat treatment of curdlan enhances the enzymatic production of biologically active β-(1,3)-glucan oligosaccharides.
  Yuya Kumagai; Masayuki Okuyama; Atsuo Kimura
  Carbohydrate polymers, 146, 396, 401, 01 Aug. 2016, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• A Solanum torvum GH3 β-glucosidase expressed in Pichia pastoris catalyzes the hydrolysis of furostanol glycoside.
  Rungarun Suthangkornkul; Pornpisut Sriworanun; Hiroyuki Nakai; Masayuki Okuyama; Jisnuson Svasti; Atsuo Kimura; Saengchan Senapin; Dumrongkiet Arthan
  Phytochemistry, 127, 4, 11, Jul. 2016, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• α-Glucosidases and α-1,4-glucan lyases: structures, functions, and physiological actions.
  Masayuki Okuyama; Wataru Saburi; Haruhide Mori; Atsuo Kimura
  Cellular and molecular life sciences : CMLS, 73, 14, 2727, 51, Jul. 2016, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Structural and biochemical studies of plant α-glucosidases with a series of long-chain inhibitors.
  Tagami T; Yamashita K; Okuyama M; Mori H; Yao M; Kimura A
  Bull Appl Glycosci, 6, 2, 103, 108, 2016, [Peer-reviewed]
  Japanese, Scientific journal
• Megalo-type α-1,6-glucosaccharides induce production of tumor necrosis factor α in primary macrophages via toll-like receptor 4 signaling.
  Ga-Hyun Joe; Midori Andoh; Aki Shinoki; Weeranuch Lang; Yuya Kumagai; Juri Sadahiro; Masayuki Okuyama; Atsuo Kimura; Hidehisa Shimizu; Hiroshi Hara; Satoshi Ishizuka
  Biomedical research (Tokyo, Japan), 37, 3, 179, 86, 2016, [Peer-reviewed], [Domestic magazines]
  English, Scientific journal
• Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis).
  Yasushi Masuda; Masayuki Okuyama; Takahisa Iizuka; Hiroyuki Nakai; Wataru Saburi; Taro Fukukawa; Janjira Maneesan; Takayoshi Tagami; Tetsushi Naraoka; Haruhide Mori; Atsuo Kimura
  Bioscience, biotechnology, and biochemistry, 80, 3, 479, 85, 2016, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• The loop structure of Actinomycete glycoside hydrolase family 5 mannanases governs substrate recognition.
  Yuya Kumagai; Keitaro Yamashita; Takayoshi Tagami; Misugi Uraji; Kun Wan; Masayuki Okuyama; Min Yao; Atsuo Kimura; Tadashi Hatanaka
  The FEBS journal, 282, 20, 4001, 14, Oct. 2015, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• 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, 9, 24 Mar. 2015, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• 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, 9, 13 Feb. 2015, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• Extracellular and cell-associated forms of Gluconobacter oxydans dextran dextrinase change their localization depending on the cell growth.
  Juri Sadahiro; Haruhide Mori; Wataru Saburi; Masayuki Okuyama; Atsuo Kimura
  Biochemical and biophysical research communications, 456, 1, 500, 5, 02 Jan. 2015, [Peer-reviewed], [International Magazine]
  English, Scientific journal
• 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, [Peer-reviewed]
  English, Scientific journal
• Biochemical properties and substrate recognition mechanism of GH31 alpha-glucosidase from Bacillus sp AHU 2001 with broad substrate specificity
  Wataru Saburi; Masayuki Okuyama; Yuya Kumagai; Atsuo Kimura; Haruhide Mori
  BIOCHIMIE, 108, 140, 148, Jan. 2015, [Peer-reviewed]
  English, Scientific journal
• Structural Advantage of Sugar Beet alpha-Glucosidase to Stabilize the Michaelis Complex with Long-chain Substrate
  Takayoshi Tagami; Keitaro Yamashita; Masayuki Okuyama; Haruhide Mori; Min Yao; Atsuo Kimura
  JOURNAL OF BIOLOGICAL CHEMISTRY, 290, 3, 1796, 1803, Jan. 2015, [Peer-reviewed]
  English, Scientific journal
• Different molecular complexity of linear-isomaltomegalosaccharides and beta-cyclodextrin on enhancing solubility of azo dye ethyl red: Towards dye biodegradation
  Weeranuch Lang; Yuya Kumagai; Juri Sadahiro; Janjira Maneesan; Masayuki Okuyama; Haruhide Mori; Nobuo Sakairi; Atsuo Kimura
  BIORESOURCE TECHNOLOGY, 169, 518, 524, Oct. 2014, [Peer-reviewed]
  English, Scientific journal
• Catalytic role of the calcium ion in GH97 inverting glycoside hydrolase
  Masayuki Okuyama; Takuya Yoshida; Hironori Hondoh; Haruhide Mori; Min Yao; Atsuo Kimura
  FEBS LETTERS, 588, 17, 3213, 3217, Aug. 2014, [Peer-reviewed]
  English, Scientific journal
• Biodecolorization of a food azo dye by the deep sea Dermacoccus abyssi MT1.1(T) strain from the Mariana Trench
  Weeranuch Lang; Sarote Sirisansaneeyakul; Ligia O. Martins; Lukana Ngiwsara; Nobuo Sakairi; Wasu Pathom-aree; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura
  JOURNAL OF ENVIRONMENTAL MANAGEMENT, 132, 155, 164, Jan. 2014, [Peer-reviewed]
  English, Scientific journal
• Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: Toward dye decolorization using a packed-bed metal affinity reactor
  Weeranuch Lang; Sarote Sirisansaneeyakul; Lukana Ngiwsara; Sonia Mendes; Ligia O. Martins; Masayuki Okuyama; Atsuo Kimura
  BIORESOURCE TECHNOLOGY, 150, 298, 306, Dec. 2013, [Peer-reviewed]
  English, Scientific journal
• 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, Nov. 2013, [Peer-reviewed]
  English, Scientific journal
• Aromatic Residue on beta ->alpha Loop 1 in the Catalytic Domain Is Important to the Transglycosylation Specificity of Glycoside Hydrolase Family 31 alpha-Glucosidase
  Kyung-Mo Song; Masayuki Okuyama; Mariko Nishimura; Takayoshi Tagami; Haruhide Mori; Atsuo Kimura
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77, 8, 1759, 1765, Aug. 2013, [Peer-reviewed]
  English, Scientific journal
• Molecular Basis for the Recognition of Long-chain Substrates by Plant alpha-Glucosidases
  Takayoshi Tagami; Keitaro Yamashita; Masayuki Okuyama; Haruhide Mori; Min Yao; Atsuo Kimura
  JOURNAL OF BIOLOGICAL CHEMISTRY, 288, 26, 19296, 19303, Jun. 2013, [Peer-reviewed]
  English, Scientific journal
• Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1
  Takayoshi Tagami; Yoshiyuki Tanaka; Haruhide Mori; Masayuki Okuyama; Atsuo Kimura
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 77, 2, 312, 319, Feb. 2013, [Peer-reviewed]
  English, Scientific journal
• Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 alpha-glucosidase contribute to recognition of long-chain substrates
  Takayoshi Tagami; Masayuki Okuyama; Hiroyuki Nakai; Young-Min Kim; Haruhide Mori; Kazunori Taguchi; Birte Svensson; Atsuo Kimura
  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1834, 1, 329, 335, Jan. 2013, [Peer-reviewed]
  English, Scientific journal
• A novel mechanism for the promotion of quercetin glycoside absorption by megalo alpha-1,6-glucosaccharide in the rat small intestine
  Aki Shinoki; Weeranuch Lang; Charin Thawornkuno; Hee-Kwon Kang; Yuya Kumagai; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura; Satoshi Ishizuka; Hiroshi Hara
  FOOD CHEMISTRY, 136, 2, 293, 296, Jan. 2013, [Peer-reviewed]
  English, Scientific journal
• Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates.
  Tagami T; Okuyama M; Nakai H; Kim YM; Mori H; Taguchi K; Svensson B; Kimura A
  Biochimica et biophysica acta, 1834, 1, 329, 335, Jan. 2013, [Peer-reviewed]
• Characterization of a glycoside hydrolase family 31 α-glucosidase involved in starch utilization in podospora anserina
  Kyung-Mo Song; Masayuki Okuyama; Kazuyuki Kobayashi; Haruhide Mori; Atsuo Kimura
  Bioscience, Biotechnology and Biochemistry, 77, 10, 2117, 2124, 2013, [Peer-reviewed]
  English, Scientific journal
• The Delay in the Development of Experimental Colitis from Isomaltosyloligosaccharides in Rats Is Dependent on the Degree of Polymerization
  Hitoshi Iwaya; Jae-Sung Lee; Shinya Yamagishi; Aki Shinoki; Weeranuch Lang; Charin Thawornkuno; Hee-Kwon Kang; Yuya Kumagai; Shiho Suzuki; Shinichi Kitamura; Hiroshi Hara; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura; Satoshi Ishizuka
  PLOS ONE, 7, 11, e50658, Nov. 2012, [Peer-reviewed]
  English, Scientific journal
• Amino Acids in Conserved Region II Are Crucial to Substrate Specificity, Reaction Velocity, and Regioselectivity in the Transglucosylation of Honeybee GH-13 alpha-Glucosidases
  Lukana Ngiwsara; Gaku Iwai; Takayoshi Tagami; Natsuko Sato; Hiroyuki Nakai; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 76, 10, 1967, 1974, Oct. 2012, [Peer-reviewed]
  English, Scientific journal
• 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, Sep. 2012, [Peer-reviewed]
  English, Scientific journal
• Structural Elucidation of Dextran Degradation Mechanism by Streptococcus mutans Dextranase Belonging to Glycoside Hydrolase Family 66
  Nobuhiro Suzuki; Young-Min Kim; Zui Fujimoto; Mitsuru Momma; Masayuki Okuyama; Haruhide Mori; Kazumi Funane; Atsuo Kimura
  JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 24, 19916, 19926, Jun. 2012, [Peer-reviewed]
  English, Scientific journal
• 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, Jun. 2012, [Peer-reviewed]
  English, Scientific journal
• A Novel Metabolic Pathway for Glucose Production Mediated by alpha-Glucosidase-catalyzed Conversion of 1,5-Anhydrofructose
  Young-Min Kim; Wataru Saburi; Shukun Yu; Hiroyuki Nakai; Janjira Maneesan; Min-Sun Kang; Seiya Chiba; Doman Kim; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura
  JOURNAL OF BIOLOGICAL CHEMISTRY, 287, 27, 22441, 22444, Jun. 2012, [Peer-reviewed]
  English, Scientific journal
• Chemical constituents and free radical scavenging activity of corn pollen collected from Apis mellifera hives compared to floral corn pollen at Nan, Thailand
  Atip Chantarudee; Preecha Phuwapraisirisan; Kiyoshi Kimura; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura; Chanpen Chanchao
  BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE, 12, 45, Apr. 2012, [Peer-reviewed]
  English, Scientific journal
• In vitro antiproliferative/cytotoxic activity on cancer cell lines of a cardanol and a cardol enriched from Thai Apis mellifera propolis
  Dungporn Teerasripreecha; Preecha Phuwapraisirisan; Songchan Puthong; Kiyoshi Kimura; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura; Chanpen Chanchao
  BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE, 12, 27, Mar. 2012, [Peer-reviewed]
  English, Scientific journal
• Degree of polymerization in dietary α-1,6-gluco¬sac¬cha¬rides modulates symptom of experimental colitis in rats.
  Iwaya H; Lee JS; Yamagishi S; Shinoki A; Lang W; Kang HK; Okuyama M; Mori H; Hara H; Kimura A; Ishizuka S
  Plos One, 7, 11, e50658, e50658, 2012, [Peer-reviewed]
  English, Scientific journal
• Characterization of some enzymatic properties of recombinant α-glucosidase III from the Thai honeybee, Apis cerana indica Fabricus.
  Kaewmuangmoon J; Yoshiyama M; Kimura K; Okuyama M; Mori H; Kimura A; Chanchao C
  Afr J Biotechnol, 11, 96, 16220, 16232, 2012, [Peer-reviewed]
  English, Scientific journal
• Function and Structure Studies of GH Family 31 and 97 alpha-Glycosidases
  Masayuki Okuyama
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 75, 12, 2269, 2277, Dec. 2011, [Peer-reviewed]
  English
• Crystallization and preliminary crystallographic analysis of dextranase from Streptococcus mutans
  Nobuhiro Suzuki; Young-Min Kim; Zui Fujimoto; Mitsuru Momma; Hee-Kwon Kang; Kazumi Funane; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura
  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, 67, Pt 12, 1542, 1544, Dec. 2011, [Peer-reviewed]
  English, Scientific journal
• 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, Aug. 2011, [Peer-reviewed]
  English, Scientific journal
• Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity
  Young-Min Kim; Ryoko Shimizu; Hiroyuki Nakai; Haruhide Mori; Masayuki Okuyama; Min-Sun Kang; Zui Fujimoto; Kazumi Funane; Doman Kim; Atsuo Kimura
  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 91, 2, 329, 339, Jul. 2011, [Peer-reviewed]
  English, Scientific journal
• The first alpha-1,3-glucosidase from bacterial origin belonging to glycoside hydrolase family 31
  Min-Sun Kang; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura
  BIOCHIMIE, 91, 11-12, 1434, 1442, Nov. 2009, [Peer-reviewed]
  English, Scientific journal
• Catalytic Reaction Mechanism Based on alpha-Secondary Deuterium Isotope Effects in Hydrolysis of Trehalose by European Honeybee Trehalase
  Haruhide Mori; Jin-Ha Lee; Masayuki Okuyama; Mamoru Nishimoto; Masao Ohguchi; Doman Kim; Atsuo Kimura; Seiya Chiba
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 73, 11, 2466, 2473, Nov. 2009, [Peer-reviewed]
  English, Scientific journal
• Catalytic Mechanism of Retaining alpha-Galactosidase Belonging to Glycoside Hydrolase Family 97
  Masayuki Okuyama; Momoyo Kitamura; Hironori Hondoh; Min-Sun Kang; Haruhide Mori; Atsuo Kimura; Isao Tanaka; Min Yao
  JOURNAL OF MOLECULAR BIOLOGY, 392, 5, 1232, 1241, Oct. 2009, [Peer-reviewed]
  English, Scientific journal
• 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, Dec. 2008
  English, Scientific journal
• Structural and Functional Analysis of a Glycoside Hydrolase Family 97 Enzyme from Bacteroides thetaiotaomicron
  Momoyo Kitamura; Masayuki Okuyama; Fumiko Tanzawa; Haruhide Mori; Yu Kitago; Nobuhisa Watanabe; Atsuo Kimura; Isao Tanaka; Min Yao
  JOURNAL OF BIOLOGICAL CHEMISTRY, 283, 52, 36328, 36337, Dec. 2008, [Peer-reviewed]
  English, Scientific journal
• 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, Sep. 2008, [Peer-reviewed]
  In book
• 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, May 2008, [Peer-reviewed]
  English, Scientific journal
• Glycoside hydrolase family 31 Escherichia coli alpha-xylosidase
  M-S. Kang; M. Okuyama; K. Yaoi; Y. Mitsuishi; Y-M. Kim; H. Mori; A. Kimura
  BIOCATALYSIS AND BIOTRANSFORMATION, 26, 1-2, 96, 103, 2008
  English, Scientific journal
• Rice α-glucosidase isozymes and isoforms showing different starch granules-binding and -degrading ability
  Hiroyuki Nakai; Shigeki Tanizawa; Tatsuya Ito; Koutaro Kamiya; Young-Min Kim; Takeshi Yamamoto; Kazuki Matsubara; Makoto Sakai; Hiroyuki Sato; Tokio Imbe; Masayuki Okuyama; Haruhide Mori; Seiya Chiba; Yoshio Sano; Atsuo Kimura
  Biocatalysis and Biotransformation, 26, 1-2, 104, 110, 2008, [Peer-reviewed]
  English, Scientific journal
• Aglycone specificity of Escherichia coli alpha-xylosidase investigated by transxylosylation
  Min-Sun Kang; Masayuki Okuyama; Katsuro Yaoi; Yasushi Mitsuishi; Young-Min Kim; Haruhide Mori; Doman Kim; Atsuo Kimura
  FEBS JOURNAL, 274, 23, 6074, 6084, Dec. 2007, [Peer-reviewed]
  English, Scientific journal
• Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are alpha-glucosidase and alpha-xylosidase
  Hiroyuki Nakai; Shigeki Tanizawa; Tatsuya Ito; Koutarou Kamiya; Young-Min Kim; Takeshi Yamamoto; Kazuki Matsubara; Makoto Sakai; Hiroyuki Sato; Tokio Imbe; Masayuki Okuyama; Haruhide Mori; Yoshio Sano; Seiya Chiba; Atsuo Kimura
  JOURNAL OF BIOCHEMISTRY, 142, 4, 491, 500, Oct. 2007, [Peer-reviewed]
  English, Scientific journal
• 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, Sep. 2007, [Peer-reviewed]
  English, Scientific journal
• Molecular cloning of cDNA for trehalase from the European honeybee, Apis mellifera L., and its heterologous expression in Pichia pastoris
  Jin-Ha Lee; Saori Saito; Haruhide Mori; Mamoru Nishimoto; Masayuki Okuyama; Doman Kim; Jintanart Wongchawalit; Atsuo Kimura; Seiya Chiba
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71, 9, 2256, 2265, Sep. 2007, [Peer-reviewed]
  English, Scientific journal
• Molecular cloning of cDNAs and genes for three alpha-glucosidases from European honeybees, Apis mellifera L., and heterologous production of recombinant enzymes in Pichia pastoris
  Mamoru Nishimoto; Haruhide Mori; Tsuneharu Moteki; Yukiko Takamura; Gaku Iwai; Yu Miyaguchi; Masayuki Okuyama; Jintanart Wongchawalit; Rudee Surarit; Jisnuson Svasti; Atsuo Kimura; Seiya Chiba
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 71, 7, 1703, 1716, Jul. 2007, [Peer-reviewed]
  English, Scientific journal
• Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare)
  Hiroyuki Nakai; Tatsuya Ito; Masatoshi Hayashi; Koutarou Kamiya; Takeshi Yamamoto; Kazuki Matsubara; Young-Min Kim; Wongchawalit Jintanart; Masayuki Okuyama; Haruhide Mori; Seiya Chiba; Yoshio Sano; Atsuo Kimura
  BIOCHIMIE, 89, 1, 49, 62, Jan. 2007, [Peer-reviewed]
  English, Scientific journal
• Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA
  Jintanart Wongchawalit; Takeshi Yamamoto; Hiroyuki Nakai; Young-Min Kim; Natsuko Sato; Mamoru Nishimoto; Masayuki Okuyama; Haruhide Mori; Osamu Saji; Chanpen Chanchao; Siriwat Wongsiri; Rudee Surarit; Jisnuson Svasti; Seiya Chiba; Atsuo Kimura
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 70, 12, 2889, 2898, Dec. 2006, [Peer-reviewed]
  English, Scientific journal
• Plant α-Glucosidase : Molecular Analysis of Rice α-Glucosidase and Degradation Mechanism of Starch Granules in Germination Stage
  NAKAI Hiroyuki; ITO Tatsuya; TANIZAWA Shigeki; MATSUBARA Kazuki; YAMAMOTO Takeshi; OKUYAMA Masayuki; MORI Haruhide; CHIBA Seiya; SANO Yoshio; KIMURA Atsuo
  Journal of Applied Glycoscience, 53, 2, 137, 142, The Japanese Society of Applied Glycoscience, Jul. 2006, [Peer-reviewed]
  English, In germination of plant seeds, storage starch is principally degraded by the combination of amylolytic enzymes. As starch is an insoluble granule, a conventional view of the degradation pathway is that the initial attack is performed by α-amylase having the starch granule-binding ability. Plant α-glucosidase was also capable of adsorbing and hydrolyzing starch granules directly, indicating a possible second pathway: the direct liberation of glucose from starch granules by plant α-glucosidase rather than the α-amylase-mediated system. We found that the starch-binding site of plant α-glucosidase was situated in its C-terminal region, of which function was independent of the catalytic domain. Site-directed mutagenesis analysis on the aromatic amino acid residues conserved in this region revealed that Trp803 and Phe895 of rice α-glucosidase were responsible for binding to starch granules. Mold α-glucosidases were devoid of the ability to attack starch granules. In plant seeds, multiple α-glucosidases have been observed. Two types of α-glucosidases, insoluble and soluble enzymes, were found in the germinating stage of rice. Expression patterns of their activities classified 14 rice varieties into two groups (Groups 1 and 2). In Group 1 varieties, insoluble enzyme decreased immediately after germination. The soluble enzyme increased by de novo synthesis. Group 2 maintained a constant activity level of insoluble and soluble α-glucosidases in germination. From Groups 1 and 2, we selected varieties of Akamai and Nipponbare, respectively, of which analysis elucidated interesting molecular mechanisms of insoluble and soluble enzymes: i) isoform and isozyme formations by post-translational proteolysis as well as by chromosomal gene expression; ii) characterization of purified enzymes exhibiting different activities to starch granules.
• Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase
  M Okuyama; A Kaneko; H Mori; S Chiba; A Kimura
  FEBS LETTERS, 580, 11, 2707, 2711, May 2006, [Peer-reviewed]
  English, Scientific journal
• Structural elements in dextran glucosidase responsible for high specificity to long chain substrate
  Wataru Saburi; Haruhide Mori; Saori Saito; Masayuki Okuyama; Atsuo Kimura
  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1764, 4, 688, 698, Apr. 2006, [Peer-reviewed]
  English, Scientific journal
• 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, Apr. 2006, [Peer-reviewed]
• Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase
  F Sato; M Okuyama; H Nakai; H Mori; A Kimura; S Chiba
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 69, 10, 1905, 1913, Oct. 2005, [Peer-reviewed]
  English, Scientific journal
• Crystallization and preliminary X-ray analysis of alpha-xylosidase from Escherichia coli
  M Kitamura; T Ose; M Okuyama; H Watanabe; M Yao; H Mori; A Kimura; Tanaka, I
  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS, 61, Pt 2, 178, 179, Feb. 2005, [Peer-reviewed]
  English, Scientific journal
• Enzymatic synthesis of alkyl alpha-2-deoxyglucosides by alkyl alcohol resistant alpha-glucosidase from Aspergillus niger
  YM Kim; M Okuyama; H Mori; H Nakai; W Saburi; S Chiba; A Kimura
  TETRAHEDRON-ASYMMETRY, 16, 2, 403, 409, Jan. 2005, [Peer-reviewed]
  English, Scientific journal
• 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, [Peer-reviewed]
  English
• Nakai H., Okuyama M., Kim YM., Saburi W., Wongchawalit J., Mori H., Chiba S. and Kimura A. "Molecular analysis of alpha-glucosidase belonging to GH-family 31" Biologia 60: 131-135(2005)*
  2005
• Okuyama M., Tanimoto Y., Ito T., Anzai A., Mori H., Kimura A., Matsui H. and Chiba S. "Purification and characterization of the hyper-glycosylated extracellular alpha-glucosidase from Schizosaccharomyces pombe" Enzyme and Microbial Technol., 37:472-48・・・
  2005
  Okuyama M., Tanimoto Y., Ito T., Anzai A., Mori H., Kimura A., Matsui H. and Chiba S. "Purification and characterization of the hyper-glycosylated extracellular alpha-glucosidase from Schizosaccharomyces pombe"
  
  Enzyme and Microbial Technol., 37:472-480(2005)*
• KIM YM., Okuyama M., Mori H., Chiba S. and Kimura A.: "Enzymatic synthesis of alkyl alpha-2-deoxyglucosides by alkyl alcohol resistant alpha-glucosidase from Aspergillus niger", Tetrahedron-asymmetr. 16 403-409 (2005)*
  2005
• Localization of alpha-glucosidases I, II, and III in organs of European honeybees, Apis mellifera L., and the origin of alpha-glucosidase in honey
  M Kubota; M Tsuji; M Nishimoto; J Wongchawalit; M Okuyama; H Mori; H Matsui; R Surarit; J Svasti; A Kimura; S Chiba
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 68, 11, 2346, 2352, Nov. 2004, [Peer-reviewed]
  English, Scientific journal
• Overexpression and characterization of two unknown proteins, YicI and YihQ, originated from Escherichia coli
  M Okuyama; H Mori; S Chiba; A Kimura
  PROTEIN EXPRESSION AND PURIFICATION, 37, 1, 170, 179, Sep. 2004, [Peer-reviewed]
  English, Scientific journal
• Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage
  T Yamamoto; T Unno; Y Watanabe; M Yamamoto; M Okuyama; H Mori; S Chiba; A Kimura
  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 1700, 2, 189, 198, Aug. 2004, [Peer-reviewed]
  English, Scientific journal
• Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage.
  Yamamoto T; Unno T; Watanabe Y; Yamamoto M; Okuyama M; Mori H; Chiba S; Kimura A
  Biochimica et biophysica acta, 1700, 2, 189, 198, Aug. 2004, [Peer-reviewed]
• Purification, characterization, and sequence analysis of two alpha-amylase isoforms from azuki bean, Vigna angularis, showing different affinity towards beta-cyclodextrin sepharose
  SS Mar; H Mori; JH Lee; K Fukuda; W Saburi; A Fukuhara; M Okuyama; S Chiba; A Kimura
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 67, 5, 1080, 1093, May 2003, [Peer-reviewed]
  English, Scientific journal
• Evidence of Intramolecular Transglucosylation Catalyzed by an .ALPHA.-Glucosidase.
  Son Mee; Mori Haruhide; Okuyama Masayuki; Kimura Atsuo; Chiba Seiya
  Journal of Applied Glycoscience, 50, 1, 41, 44, The Japanese Society of Applied Glycoscience, 2003
  English, The hydrolytic reaction of carbohydrate-hydrolase is essentially accompanied by a reverse reaction (the condensation reaction), meaning that only the substrate capable of being hydrolyzed is produced by the reverse reaction. Honeybee α-glucosidase I can't hydrolyze isomaltose, but is capable of hydrolyzing maltose, kojibiose and slightly nigerose. Nevertheless, the enzyme catalyzes the formation and accumulation of isomaltose from glucose together with α-glucobioses such as maltose, kojibiose and nigerose. This finding is in conflict with the data that the enzyme has no hydrolytic activity toward isomaltose. However, the conflict for the peculiar phenomenon on the reaction was rationally explained by the evidence that isomaltose might be formed by the intramolecular transglucosylation via other α-glucobioses that are easily produced from glucose by the condensation reaction. It is suggested that the usual transglycosylation of carbohydrate-hydrolase may be accompanied by an intramolecular transfer reaction.
• Son, M., Mori, H., Okuyama, M., Kimura A. and Chiba, S.:" Evidence of Intramolecular Transglucosylation Catalyzed by an alpha-Glucosidase", Journal of Applied Glycoscience, 50: 41-44(2003)*
  2003
• Identification of essential ionizable groups and evaluation of subsite affinities in the active site of beta-D-glucosidase F-1 from a Streptomyces sp.
  K Fukuda; H Mori; M Okuyama; A Kimura; H Ozaki; M Yoneyama; S Chiba
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66, 10, 2060, 2067, Oct. 2002, [Peer-reviewed]
  English, Scientific journal
• Kinetic Studies on Substrate Specificity and Active Site of β-_D-Glucosidase F_1 from Streptomyces sp.
  FUKUDA Kenji; SHIRAKAWA Kou; MORI Haruhide; OKUYAMA Masayuki; KIMURA Atsuo; OZAKI Hachiro; YONEYAMA Michio; CHIBA Seiya
  Journal of applied glycoscience, 49, 3, 265, 272, The Japanese Society of Applied Glycoscience, 18 Jul. 2002
  English, The substrate specificity and the active site of β-D-glucosidase F^-1 (Mr, 50, 000; optimum pH, 5.5) purified from a Streptomyces sp. were kinetically investigated. The β-D-glucosidase showed a broad substrate specificity for synthetic glycosides and disaccharides having β-glycosidic linkage, but the former was more favorable substrate than the latter. The enzyme was characterized by the ability to hydrolyze rapidly not only ρ-nitrophenyl β-glucoside (K_m, 0.72 mM; k₀, 63 s^-1) but also ρ-nitrophenyl β-fucoside (K_m, 0.19 mM; k₀, 44 s^-1) and laminaribiose (K_m, 1.6 mM; k₀, 70 s^-1). The kinetic study was made as to whether the hydrolyses of these synthetic glycosides were catalyzed at a single active site or at dual active sites in the β-D-glucosidase. In the experiments with the mixed substrates of p-nitrophenyl β-glucoside (PNPG) and ρ-nitrophenyl β-fucoside (PNPF) or ρ-nitrophenyl β-galactoside (PNPGal), the kinetic features, the linearity of Lineweaver-Burk plots and the dependence of the apparent maximal veloities and K_m value on the mole fraction (f) of PNPG in the mixed substrate, f = [PNPG/([PNPG] + [PNPF or PNPGal]) agreed very closely with those theoretically predicted for a single catalytic site mechanism. The findings strongly support the notion that the β-D-glucosidase attacks the synthetic glycosides at a common active site.
• alpha-glucosidase mutant catalyzes "alpha-glycosynthase"-type reaction
  M Okuyama; H Mori; K Watanabe; A Kimura; S Chiba
  BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 66, 4, 928, 933, Apr. 2002, [Peer-reviewed]
  English, Scientific journal
• Kinetic Studies on Substrate Specificity and Active Site of β-D-Glucosidase F1 from Streptomyces sp.
  FUKUDA K; SHIRAKAWA K; MORI H; OKUYAMA M; KIMURA A; OZAKI H; YONEYAMA M; CHIBA S
  J. Appl. Glycosci., 49, 3, 265, 272, 2002
• Fukuda, K., Shirakawa, K., Mori, H., Okuyama, M., Kimura, A., Ozaki H., Yoneyama, M. and Chiba, S. "Kinetic Studies on Substrate Specificity and Active site of β-D-Glucosidase F1 from Streptomyces sp.", Journal of Applied Glycoscience, 49, 265-272(2002)
  2002
• Purification and identification of the essential ionizable groups of honeybee, Apis mellifera L., trehalase
  Jin-Ha Lee; Masahisa Tsuji; Mitsuru Nakamura; Mamoru Nishimoto; Masayuki Okuyama; Haruhide Mori; Atsuo Kimura; Hirokazu Matsui; Seiya Chiba
  Bioscience, Biotechnology and Biochemistry, 65, 12, 2657, 2665, Dec. 2001, [Peer-reviewed]
  English, Scientific journal
• Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe
  M Okuyama; A Okuno; N Shimizu; H Mori; A Kimura; S Chiba
  EUROPEAN JOURNAL OF BIOCHEMISTRY, 268, 8, 2270, 2280, Apr. 2001, [Peer-reviewed]
  English, Scientific journal
• Localization of α-Glucosidase in Yeast Cells
  SAEKI Takeshi; OKUYAMA Masayuki; MORI Haruhide; KIMURA Atsuo; CHIBA Seiya
  Journal of applied glycoscience, 45, 3, 281, 283, 日本応用糖質科学会, 31 Aug. 1998
  English

• 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
  佐々木優希; 熊谷祐也; 貞廣樹里; LANG Weeranuch; 田上貴祥; 奥山正幸; 木村淳夫, 日本農芸化学会大会講演要旨集(Web), 2018, ROMBUNNO.2A26p03 (WEB ONLY), 05 Mar. 2018
  Japanese
• GH15 isomaltose glucohydrolaseの基質認識に関わる構造因子の同定
  田上貴祥; 陳明皓; 奥山正幸; 岩崎智仁; 田中良和; 姚閔; 木村淳夫, 応用糖質科学, 7, 3, 49, 20 Aug. 2017
  Japanese
• Bacteroides thetaiotaomicron β‐L‐arabinopyranosidase(BtAra97c)の立体構造解析
  菊池麻子; 加藤公児; 奥山正幸; 大嵜翔平; 姚閔; 木村淳夫, 応用糖質科学, 6, 3, 61, 20 Aug. 2016
  Japanese
• 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), 05 Mar. 2016
  Japanese
• S3-2 長鎖阻害剤の利用による植物α-グルコシダーゼの機能構造相関の解明(応用糖質科学シンポジウム,一般社団法人日本応用糖質科学会平成27年度大会(第64回))
  田上 貴祥; 山下 恵太郎; 奥山 正幸; 森 春英; 姚 閔; 木村 淳夫, 応用糖質科学 : 日本応用糖質科学会誌, 5, 3, B59, 20 Aug. 2015
  日本応用糖質科学会, Japanese
• Ba-3 Isomaltooligosaccharide 6-α-glucosyltransferaseの構造と触媒部位の機能性アミノ酸残基の同定(澱粉関連酵素,一般講演,一般社団法人日本応用糖質科学会平成27年度大会(第64回))
  川田 恭平; 藤本 瑞; 鐘ケ江 倫世; 西村 崇志; 奥山 正幸; 森 春英; 木村 淳夫, 応用糖質科学 : 日本応用糖質科学会誌, 5, 3, B41, 20 Aug. 2015
  日本応用糖質科学会, Japanese
• Ba-5 Podospora anserina α-glucosidaseの高い糖転移能に関わる構造因子(澱粉関連酵素,一般講演,一般社団法人日本応用糖質科学会平成27年度大会(第64回))
  福本 健太; Song Kyung-Mo; 奥山 正幸; 木村 淳夫, 応用糖質科学 : 日本応用糖質科学会誌, 5, 3, B41, 20 Aug. 2015
  日本応用糖質科学会, Japanese
• 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), 05 Mar. 2015
  Japanese
• 糖質関連酵素の最近の進歩―4 糖質加水分解酵素ファミリー内の機能の保存性と多様性
  OKUYAMA MASAYUKI, 化学と生物, 53, 2, 120, 126, 20 Jan. 2015
  Japan Society for Bioscience, Biotechnology, and Agrochemistry, Japanese
• テンサイα-グルコシダーゼの高重合度基質特異性はらせん構造の長鎖基質に適したサブサイト構造に起因する
  田上貴祥; 山下恵太郎; 奥山正幸; 森春英; YAO Min; YAO Min; 木村淳夫, 日本農芸化学会大会講演要旨集(Web), 2015, 2015
• Bp-6 Amphiphilic function of linear-isomaltomegalosaccharides (L-IMS) on ethyl red (ER) solubility
  Lang Weeranuch; Kumagai Yuya; Sadahiro Juri; Okuyama Masayuki; Mori Haruhide; Sakairi Nobuo; Kimura Atsuo, Bulletin of applied glycoscience, 4, 3, B42, 20 Aug. 2014
  The Japanese Society of Applied Glycoscience, English
• Ba-3 Dextran dextrinaseのPhe602の生成物特異性に関する影響(その他の糖質関連酵素,一般講演,日本応用糖質科学会平成26年度大会(第63回))
  坂谷 敦; 熊谷 祐也; 貞廣 樹里; Weeranuch Lang; 奥山 正幸; 森 春英; 木村 淳夫, 応用糖質科学：日本応用糖質科学会誌, 4, 3, B39, 2014
  一般社団法人 日本応用糖質科学会, Japanese
• Da-6 放線菌マンナナーゼの基質特異性に関するループ7の解析(ヘミセルラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))
  熊谷 祐也; 裏地 美杉; 奥山 正幸; 木村 淳夫; 畑中 唯史, 応用糖質科学：日本応用糖質科学会誌, 4, 3, B53, 2014
  一般社団法人 日本応用糖質科学会, Japanese
• 2P-052 Insight into the substrate specificity of galactosylmannooligosaccharide by actinomycete mannanase
  Kumagai Yuya; Uraji Misugi; Okuyama Masayuki; Kimura Atsuo; Hatanaka Tadashi, 日本生物工学会大会講演要旨集, 66, 119, 119, 2014
  日本生物工学会, Japanese
• 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, 27 Nov. 2013
  Japanese
• 糖質加水分解酵素ファミリー31に属するα‐グルコシダーゼの構造と基質・反応特異性
  OKUYAMA MASAYUKI, バイオサイエンスとインダストリー, 71, 5, 424, 428, 01 Sep. 2013
  バイオインダストリー協会, Japanese
• Ca-2 Schizosaccharomyces pombe由来グルコシダーゼIIα-サブユニットの大腸菌生産および機能解析(α-グルコシダーゼ・その他の糖質関連酵素1,一般講演,日本応用糖質科学会平成25年度大会(第62回))
  宮本 大司; 奥山 正幸; 松尾 一郎; 森 春英; 木村 淳夫, 応用糖質科学 : 日本応用糖質科学会誌, 3, 3, B37, 20 Aug. 2013
  日本応用糖質科学会, Japanese
• Ca-3 Streptococcus mutans由来dextran glucosidaseのβ→α loop 6への変異導入による鎖長特異性の改変(α-グルコシダーゼ・その他の糖質関連酵素1,一般講演,日本応用糖質科学会平成25年度大会(第62回))
  小林 桃子; 森 春英; 奥山 正幸; 木村 淳夫, 応用糖質科学 : 日本応用糖質科学会誌, 3, 3, B38, 20 Aug. 2013
  日本応用糖質科学会, Japanese
• 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), 05 Mar. 2013
  Japanese
• Basic study on production of useful oligosaccharides from starch using a non-recombinant yeast
  奥山 正幸, 年報, 29, 161, 167, 2013
  飯島藤十郎記念食品科学振興財団, Japanese
• Da-10 Gluconobacter oxydans由来dextran dextrinaseによる直鎖イソマルトメガロ糖の酵素合成(糖質の合成・生産,一般講演,日本応用糖質科学会平成25年度大会(第62回))
  熊谷 祐也; Lang Weeranuch; 貞廣 樹里; 奥山 正幸; 森 春英; 木村 淳夫, 応用糖質科学：日本応用糖質科学会誌, 3, 3, B46, 2013
  一般社団法人 日本応用糖質科学会, Japanese
• Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 .ALPHA.-glucosidase contribute to recognition of long-chain substrates
  TAGAMI Takayoshi; OKUYAMA Masayuki; NAKAI Hiroyuki; KIM Young-min; MORI Haruhide; TAGUCHI Kazunori; SVENSSON Birte; KIMURA Atsuo, Biochimica et Biophysica Acta, 1834, 1, 329, 335, Jan. 2013
  English
• Acarviosyl‐maltooligsaccharideの酵素合成とテンサイα‐glucosidaseに対する基質アナログとしての利用
  田上貴祥; 山下恵太郎; 田中良幸; 奥山正幸; 森春英; 姚閔; 木村淳夫, 応用糖質科学, 2, 3, (34), 20 Aug. 2012
  日本応用糖質科学会, Japanese
• Streptococcus mutans由来デキストラナーゼの結晶構造に基づく触媒機構の解明
  鈴木喜大; 金泳みん; 金泳みん; 藤本瑞; 門間充; 奥山正幸; 森春英; 舟根和美; 木村淳夫, PFシンポジウム要旨集, 29th, 28, 2012
  Japanese
• Isomaltooligosaccharide 6‐α‐glucosyltransferaseサブサイト+1変異酵素の機能解析
  青山泰; 西村崇志; 鐘ケ江倫世; 本同宏成; 奥山正幸; 森春英; 木村淳夫, 応用糖質科学, 1, 3, (36), 20 Jul. 2011
  Japanese
• Dextran glucosidase(DexB)ウォーターパス改変体の機能と構造
  小林桃子; 山下恵太郎; 田上貴祥; 本同宏成; 森春英; 奥山正幸; 姚閔; 木村淳夫, 応用糖質科学, 1, 3, (37), 20 Jul. 2011
  Japanese
• GH family 31に属するAspergillus niger由来α‐glucosidaseの鎖長特異性の改変
  田上貴祥; 奥山正幸; 森春英; 木村淳夫, 応用糖質科学, 1, 3, (36), 20 Jul. 2011
  Japanese
• アノマー反転型トレハラーゼ変異体により合成された新規非還元二糖の構造
  平内亨; 牧孝多朗; 森春英; 奥山正幸; 木村淳夫, 応用糖質科学, 1, 3, (36), 20 Jul. 2011
  Japanese
• 環状イソマルトオリゴ糖グルカノトランスフェラーゼのβ→αループ2はCI‐8の優先的生成に寄与する
  齊藤みどり; KANG Hee‐Kwon; 森春英; 奥山正幸; 藤本瑞; 舟根和美; 小林幹彦; 木村淳夫, 応用糖質科学, 1, 3, (39), 20 Jul. 2011
  Japanese
• ホタテガイ(Pastinopecton yessoensis)中腸腺由来α‐glucosidase—Cl⁻による反応速度の上昇と基質特異性の変化—
  桝田安志; 奥山正幸; 森春英; 木村淳夫, 日本農芸化学会大会講演要旨集, 2011, 43, 05 Mar. 2011
  Japanese
• Dextran glucosidase(DexB)のウォーターパス改変酵素の機能解析
  小林桃子; 本同宏成; 奥山正幸; 森春英; 木村淳夫, 日本農芸化学会大会講演要旨集, 2011, 43, 05 Mar. 2011
  Japanese
• アノマー反転型トレハラーゼ変異体によるオリゴ糖合成
  平内亨; 牧孝多朗; 森春英; 奥山正幸; 木村淳夫, 日本農芸化学会大会講演要旨集, 2011, 44, 05 Mar. 2011
  Japanese
• 環状イソマルトオリゴ糖グルカノトランスフェラーゼが示すCI‐8の優先的生成に寄与する構造因子
  齋藤みどり; KANG Hee‐Kwon; 森春英; 奥山正幸; 藤本瑞; 舟根和美; 小林幹彦; 木村淳夫, 日本農芸化学会大会講演要旨集, 2011, 42, 05 Mar. 2011
  Japanese
• Oligo‐1,6‐glucosidaseがCl⁻依存性を示すために必要なアミノ酸残基の決定
  寺田智明; 森春英; 奥山正幸; 木村淳夫, 日本農芸化学会東北支部大会プログラム・講演要旨集, 145th, 49, 27 Sep. 2010
  Japanese
• 加水分解反応の抑制により縮合反応の生成物を蓄積させるグルコアミラーゼ変異体
  砂守このみ; 森春英; 奥山正幸; 森本奈保喜; 松井博和; 木村淳夫, 日本農芸化学会東北支部大会プログラム・講演要旨集, 145th, 49, 27 Sep. 2010
  Japanese
• isomaltooligosaccharide6‐α‐glucosyltransferaseのサブサイト+1変異酵素の機能解析
  西村崇志; 鐘ケ江倫世; 本同宏成; 奥山正幸; 森春英; 木村淳夫, J Appl Glycosci, 57, Suppl., 43, 20 Jul. 2010
  Japanese
• Aspergillus niger α‐glucosidaseのサブサイト+1の改変
  田上貴祥; 奥山正幸; 森春英; 木村淳夫, J Appl Glycosci, 57, Suppl., 44, 20 Jul. 2010
  Japanese
• Oligo‐1,6‐glucosidaseの変異導入によるCl⁻イオン依存型への改変
  寺田智明; 森春英; 奥山正幸; 木村淳夫, 日本農芸化学会大会講演要旨集, 2010, 22, 05 Mar. 2010
  Japanese
• GH family 31 α‐グルコシダーゼにおける基質の鎖長認識機構の解明
  田上貴祥; 西村崇志; 奥山正幸; 森春英; 木村淳夫, 日本農芸化学会大会講演要旨集, 2010, 22, 05 Mar. 2010
  Japanese
• 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, 05 Mar. 2010
  Japanese
• Function analysis of subsite+1 mutants of isomaltooligosaccharide 6-α-glucosyltransferase
  nishimura takashi; kanegae michiyo; hondoh hironori; okuyama masayuki; mori haruhide; kimura atsuo, Journal of Applied Glycoscience Supplement, 2010, 71, 71, 2010
  The Japanese Society of Applied Glycoscience, Japanese
• Modification of subsite +1 in α-glucosidase derived from Aspergillus niger
  Tagami Takayoshi; Okuyama Masayuki; Mori Haruhide; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2010, 73, 73, 2010
  The Japanese Society of Applied Glycoscience, Japanese
• Suicide Substrate-based Inactivation of Endodextranase by .OMEGA.-Epoxyalkyl .ALPHA.-D-Glucopyranosides
  KAN HIGON; KIMU YONMIN; NAKAI HIROYUKI; KAN MINSON; HAKAMADA WATARU; OKUYAMA MASAYUKI; MORI HARUHIDE; NISHIO TOSHIYUKI; KIMURA ATSUO, J Appl Glycosci, 57, 4, 269-272 (J-STAGE), 272, 2010
  Three kinds of ω-epoxyalkyl α-glucopyranosides (3′,4′-epoxybutyl α-D-glucopyranoside (E4G), 4′,5′-epoxypentyl α-D-glucopyranoside (E5G) and 5′,6′-epoxyhexyl α-D-glucopyranoside (E6G)), having alkyl chains of different lengths at their aglycone moieties, inactivated the endodextranase from Streptococcus mutans ATCC 25175 (SmDex) irreversibly with the pseudo-first order kinetics. Alkyl chain length-dependent inactivation was observed and the degree of activity loss was E5G, E6G and E4G, in that order, implying that the distance between epoxide group and glucosyl residue of ω-epoxyalkyl α-glucopyranoside was important in the modification of endodextranase. Inactivation by E5G followed the model of reversible intermediate-complex formation mechanism (suicide inhibitor-based mechanism). The rate constant of irreversible inactivation (k) and the dissociation constant of intermediate-complex (KR) of SmDex and E5G were 0.44 min^-1 and 1.45 mM, respectively. Hydrolytic reaction product (isomaltose) protected SmDex from E5G-inactivation, suggesting that E5G bound to the catalytic site of SmDex. This is the first report that ω-epoxyalkyl α-glucopyranoside becomes a suicide substrate for endodextranase., The Japanese Society of Applied Glycoscience, English
• isomaltooligosaccharide6‐α‐glucosyltransferaseの糖転移反応に関わる構造因子
  西村崇志; 鐘ケ江倫世; KLM Young‐Min; 本同宏成; 奥山正幸; 森春英; 木村淳夫, J Appl Glycosci, 56, Suppl., 37, 20 Jul. 2009
  Japanese
• Bacteroides thetaiotaomicron由来SusBの触媒反応におけるCa²⁺の役割
  吉田拓弥; 奥山正幸; 本同宏成; 銚閔; 森春英; 木村淳夫, J Appl Glycosci, 56, Suppl., 38, 20 Jul. 2009
  Japanese
• 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, 20 Jul. 2009
  Japanese
• 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, 20 Jul. 2009
  The Japanese Society of Applied Glycoscience, Japanese
• テンサイα‐glucosidaseのサブサイト+2および+3の形成に重要なアミノ酸残基の決定
  田上貴祥; 奥山正幸; 森春英; 田口和憲; 木村淳夫, J Appl Glycosci, 56, Suppl., 38, 37, 20 Jul. 2009
  The Japanese Society of Applied Glycoscience, Japanese
• Structural Comparison of Streptococcus mutans Dextran Glucosidase with Glucoside Hydrolases in GH13
  HONDOH Hironori; OTSUKA RACHI Hiroaki; SABURI Wataru; MORI Haruhide; OKUYAMA Masayuki; KIMURA Atsuo, Journal of applied glycoscience, 56, 2, 111, 117, 20 Apr. 2009
  In glycoside hydrorase family (GH) 13, &alpha;-glucosidase, oligo-1,6-glucosidase and dextran glucosidase, which hydrolyze the non-reducing end glucosidic linkages of maltooligo- and/or isomaltoolligosaccharides, are categorized as &alpha;-glucoside hydrolase. Despite a high similarity in the sequence and overall structure of those family enzymes, GH 13 &alpha;-glucoside hydrolases show a wide range of substrate specificity. Until now, three crystal structures of &alpha;-glucoside hydrolase, dextran glucosidase from <i>Streptococcus mutans</i> (DGase), oligo-1,6-glucosidase from <i>Bacillus cereus</i> (O16G), and &alpha;-glucosidase from <i>Geobacillus</i> sp. HTA-462 (GSJ) have been determined. In this study, we have performed the structural comparison of these &alpha;-glucoside hydrolases. Their overall structures are generally similar, and consist of three major domains A, B and C as found in many &alpha;-amylase family enzymes. The significant structural differences in these enzymes are mainly found in loop regions. GSJ has a shorter &beta;&rarr;&alpha; loop 6 in a different orientation in addition to the disordered regions, whereas DGase and O16G show high similarity in their tertiary structures. Though these enzymes have the different substrate preference, they all possess the completely conserved configuration at subsite -1. Therefore, the substrate preference will be originated from the structure at subsite for the reducing end side of substrate. The substrate binding modes of these glucoside hydrolases were predicted by superimposing the substrate molecule of substrate-complex structures of DGase and &alpha;-amylase. DGase and O16G are thought to have a similar manner in substrate binding with conserved amino acid residues. The substrate recognition of GSJ at subsite -1 and +1 would be similar to that of &alpha;-amylases since the key residues in substrate binding are conserved in both primary and tertiary structures., 日本応用糖質科学会, English
• Isomal tooligosaccharide 6‐α‐glucosyltransferase(I6GT)のイソマルトオリゴ糖への作用と転移に関する構造因子
  西村崇志; 鐘ケ江倫世; KIM Young‐Min; 本同宏成; 奥山正幸; 森春英; 木村淳夫, 日本農芸化学会大会講演要旨集, 2009, 41, 05 Mar. 2009
  Japanese
• アズキα‐アミラーゼ(VAA)のMet398は過酸化水素により特異的に酸化される
  田中良幸; 森春英; 河合正悟; 奥山正幸; 木村淳夫, 日本農芸化学会大会講演要旨集, 2009, 44, 05 Mar. 2009
  Japanese
• 加水分解反応の抑制によりトレハロースを縮合蓄積させるトレハラーゼ変異酵素
  牧孝多朗; 森春英; 奥山正幸; 木村淳夫, 日本農芸化学会大会講演要旨集, 2009, 41, 05 Mar. 2009
  Japanese
• Role of Ca2+ in catalysis of SusB derived from Bacteroides thetaiotaomicron
  Yoshida Takuya; Okuyama Masayuki; Hondoh Hironori; Yao Min; Mori Haruhide; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2009, 38, 38, 2009
  The Japanese Society of Applied Glycoscience, Japanese
• Alteration of transglucosylation/hydrolysis ratio of Streptococcus mutans dextran glucosidase (2)
  Nakatsuka Daichi; Hondoh Hironori; Otsuka Hiroaki; Saburi Wataru; Mori Haruhide; Okuyama Masayuki; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2009, 34, 34, 2009
  The Japanese Society of Applied Glycoscience, Japanese
• Improving Activity of Honeybee α-Glucosidase III by Substitution of Q349 and L350 Position.
  Ngiwsara Lukana; Mori Haruhide; Okuyama Masayuki; Chiba Seiya; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2009, 36, 36, 2009
  The Japanese Society of Applied Glycoscience, Japanese
• Structural element for transglucosylation reaction of isomaltooligosaccharide 6-α-glucosyltransferase
  Nishimura Takashi; Kanegae Michiyo; KIM Young-Min; Hondoh Hironori; Okuyama Masayuki; Mori Haruhide; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2009, 35, 35, 2009
  The Japanese Society of Applied Glycoscience, Japanese
• Preliminary Structure Determination of Polysaccharide Extracted from Rhizobium sp.
  Wongchawalit Jintanart; Hashidoko Yasuyuki; Okuyama Masayuki; Mori Haruhide; Chiba Seiya; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2009, 0, 39, 39, 2009
  The Japanese Society of Applied Glycoscience, Japanese
• 大腸菌trehalaseの触媒アミノ酸残基の決定とglycosynthase反応
  森春英; 西塔沙織; 尾川陽; 牧孝多朗; 奥山正幸; 木村淳夫, J Appl Glycosci, 55, Suppl., 50, 103, 20 Jul. 2008
  The Japanese Society of Applied Glycoscience, Japanese
• Glycoside hydrolase family97アノマー保持型酵素の触媒機構
  奥山正幸; 姚閔; 本同宏成; 北村百世; 森春英; 田中勲; 木村淳夫, J Appl Glycosci, 55, Suppl., 59, 20 Jul. 2008
  Japanese
• Bacillus sp.由来isomaltooligosaccharide6‐α‐glucosyltrasferase(I6GT)の転移反応に関与する構造因子
  鐘ケ江倫世; KIM Young‐Min; 本同宏成; 奥山正幸; 森春英; 木村淳夫, J Appl Glycosci, 55, Suppl., 50, 20 Jul. 2008
  Japanese
• 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, 20 Jul. 2008
  日本応用糖質科学会, Japanese
• 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, 20 Jul. 2008
  日本応用糖質科学会, Japanese
• 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, 20 Jul. 2008
  The Japanese Society of Applied Glycoscience, Japanese
• テンサイα‐glucosidaseのcDNAクローニングとpichia pastorisによる組換え酵素の生産
  田上貴祥; 奥山正幸; 森春英; 田口和憲; 木村淳夫, J Appl Glycosci, 55, Suppl., 48, 94, 20 Jul. 2008
  The Japanese Society of Applied Glycoscience, Japanese
• 新しく見出されたイソマルトオリゴ糖不均化酵素の遺伝子クローニングと異種宿主発現
  鐘ケ江倫世; KIM Young‐Min; 中井博之; 本同宏成; 奥山正幸; 森春英; 木村淳夫, 日本農芸化学会大会講演要旨集, 2008, 191, 05 Mar. 2008
  Japanese
• Schwanniomyces occidentalis由来GH31 α‐glucosidaseへの変異導入によるグルコシド結合選択性の変換
  西村茉利子; 奥山正幸; 森春英; 木村淳夫, 日本農芸化学会大会講演要旨集, 2008, 33, 05 Mar. 2008
  Japanese
• 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, 05 Mar. 2008
  Japanese
• The structural factor for transglucosylation of isomaltooligosaccharide 6-α-glucosyltrasferase(I6GT) from Bacillus sp.
  Kanegae Michiyo; KIM Young-Min; Hondoh Hironori; Okuyama Masayuki; Mori Haruhide; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2008, 104, 104, 2008
  The Japanese Society of Applied Glycoscience, Japanese
• Catalytic mechanism of retaining glycosidase in glycoside hydrolase family 97
  OKUYAMA MASAYUKI; YAO MIN; Hondoh Hironori; KITAMURA MOMOYO; MORI HARUHIDE; TANAKA ISAO; KIMURA ATSUO, Journal of Applied Glycoscience Supplement, 2008, 139, 139, 2008
  The Japanese Society of Applied Glycoscience, Japanese
• Coversion of Lactobacillus johnsonii novel α-glucosidase into glycosynthase
  Kang Min-Sun; Okuyama Masayuki; Mori Haruhide; Kimura Atsuo, Journal of Applied Glycoscience Supplement, 2008, 96, 96, 2008
  The Japanese Society of Applied Glycoscience, Japanese
• 新規酵素isomal tool igosaccharide6‐α‐glucosyl transferase(I6GT)の機能解析
  鐘ケ江倫世; KIM Young‐Min; 本同宏成; 奥山正幸; 森春英; 木村淳夫, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2008, 25, 2008
  Japanese
• 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
  Japanese
• Substrate Recognition of Escherichia coli YicI (.ALPHA.-Xylosidase)
  OKUYAMA MASAYUKI; KAN MINSON; YAOI KATSURO; MITSUISHI YASUSHI; MORI HARUHIDE; KIMURA ATSUO, J Appl Glycosci, 55, 2, 111-118 (J-STAGE), 118, 2008
  Glycoside hydrolase family 31 (GH 31) is one of the most intriguing glycoside hydrolase families. This family contains α-glucosidase, α-xylosidase, α-glucan lyase and isomaltosyltransferase. Escherichia coli YicI (α-xylosidase) is a representative enzyme of GH 31 because its biochemical and structural studies have been thoroughly carried out. YicI is a strict α-xylosidase, which rigidly recognizes α-xyloside at the non-reducing terminal end, even though its amino acid sequence apparently displays similarity with α-glucosidases. Phe277, Cys307, Trp345 and Lys414 at the subsite-1 are important for α-xylosidase activity. The mutant YicI enzymes, which possesses Ile307/Asp308 instead of Cys307/Phe308 and which has a shorter β→α loop 1 of (β/α)8 barrel in place of the original longer loop, respectively, possess α-glucosidase activity. In the transxylosylation of YicI, glucose, mannose and allose are able to act as acceptors, but galactose, talose and gulose never do, implying that equatorial OH-4 of the aldopyranose is crucial for acting as an acceptor. YicI transfers α-xylosyl moieties to a specific hydroxy group in the acceptor sugar (except fructopyranose) showing 1,6 regioselectivity, which is in agreement with the structural feature of the aglycone-biding site. Among the transxylosylation products of YicI, α-D-xylopyranosyl-(1→6)-D-mannopyranose, α-D-xylopyranosyl-(1→6)-D-fructofuranose, and α-D-xylopyranosyl-(1→3)-D-fructopyranose are novel sugars. α-D-Xylopyranosyl-(1→6)-D-mannopyranose and α-D-xylopyranosyl-(1→6)-D-fructofuranose have the ability to inhibit rat intestinal α-glucosidases., The Japanese Society of Applied Glycoscience, English
• Substrate recognition of Escherishia coli YicI (alpha-xylosidase)
  Journal of Applied Glycoscience, 55, 2, 111, 118, 2008
• 新規なイソマルトオリゴ糖生成酵素の単離と性質
  鐘ケ江倫世; KIM Young‐Min; 中井博之; 奥山正幸; 森春英; 木村淳夫, J Appl Glycosci, 54, Suppl., 43, 20 Jul. 2007
  Japanese
• 大腸菌Yicl(α‐xylosidase)の基質認識の解析
  奥山正幸; KANG Min‐Sun; 矢追克郎; 矢追克郎; 三石安; 森春英; 木村淳夫, J Appl Glycosci, 54, Suppl., 55, 20 Jul. 2007
  Japanese
• ホタテガイ由来塩素イオン依存性α‐amylaseの構造と機能
  飯塚貴久; 小林和之; 中井博之; 奥山正幸; 森春英; 奈良岡哲志; 千葉誠哉; 木村淳夫, J Appl Glycosci, 54, Suppl., 32, 20 Jul. 2007
  Japanese
• 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, 20 Jul. 2007
  Japanese
• デキストラン関連酵素が有するデキストラン結合ドメインの機能解析
  中井博之; 金泳民; 原口慶子; 奥山正幸; 森春英; 舟根和美; 小林幹彦; 木村淳夫, 日本農芸化学会大会講演要旨集, 2007, 65, 05 Mar. 2007
  Japanese
• B.thetaiotaomicron BT1871の求核触媒残基変異酵素の性質
  奥山正幸; KANG Minsun; 森春英; 木村淳夫, 日本農芸化学会大会講演要旨集, 2007, 206, 05 Mar. 2007
  Japanese
• ホタテガイ閉殻筋由来α‐glucosidaseのマルチプルフォームの構造解析
  飯塚貴久; 中井博之; 奥山正幸; 森春英; 奈良岡哲志; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2007, 206, 05 Mar. 2007
  Japanese
• 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, 05 Mar. 2007
  Japanese
• クロマルハナバチ(Bombus terrestris)α‐グルコシダーゼ:アロステリック酵素の性質解明と遺伝子発現
  佐藤なつ子; 鳥羽瀬輝; 中井博之; 西本完; 森春英; 奥山正幸; 木村淳夫, 生化学, 3P-0212, 2007
  Japanese
• 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
  Japanese
• ホタテガイ閉殻筋に存在するイオン要求性α‐glucosidaseの精製とその諸性質
  飯塚貴久; 中井博之; 奥山正幸; 森春英; 奈良岡哲志; 千葉誠哉; 木村淳夫, J Appl Glycosci, 53, Suppl., 30, 30 Aug. 2006
  Japanese
• Bacteroides thetaiotaomicron由来SusBの触媒残基解析
  丹澤史子; 奥山正幸; 北村百世; 森春英; 田中勲; 木村淳夫, J Appl Glycosci, 53, Suppl., 33, 30 Aug. 2006
  Japanese
• クロマルハナバチ,Bombus ignitus,α‐GlucosidaseアイソザイムIの長鎖基質認識に関わるアミノ酸残基の決定
  佐藤なつ子; 中井博之; 森春英; 奥山正幸; 千葉誠哉; 木村淳夫, J Appl Glycosci, 53, Suppl., 31, 30 Aug. 2006
  Japanese
• GST融合タンパク質を用いたイネα‐glucosidaseの澱粉粒吸着および分解機構の解析
  谷沢茂紀; 中井博之; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, J Appl Glycosci, 53, Suppl., 43, 30 Aug. 2006
  Japanese
• 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, 30 Aug. 2006
  Japanese
• 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, 30 Aug. 2006
  Japanese
• Glycoside hydrolase family 97酵素BT1871の反応機構
  奥山正幸; 北村百世; 森春英; 田中勲; 木村淳夫, 日本農芸化学会大会講演要旨集, 2006, 307, 05 Mar. 2006
  Japanese
• セイヨウミツバチα‐glucosidaseアイソザイムIIのβ→αループ8変異酵素の機能解析
  佐藤なつ子; 中井博之; 森春英; 奥山正幸; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2006, 154, 05 Mar. 2006
  Japanese
• 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, 05 Mar. 2006
  Japanese
• 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, 05 Mar. 2006
  Japanese
• Purification and characterization of the hyper-glycosylated extracellular α-glucosidase from Schizosaccharomyces pombe
  Masayuki Okuyama; Yoshihiro Tanimoto; Tatsuya Ito; Akiko Anzai; Haruhide Mori; Atsuo Kimura; Hirokazu Matsui; Seiya Chiba, Enzyme and Microbial Technology, 37, 5, 472, 480, 03 Oct. 2005
  English
• セイヨウミツバチα‐Glucosidase IIのV167N変異酵素は高いK_mおよびk₀を与え,マルハナバチα‐Glucosidase IIの性質を示した
  佐藤なつ子; 中井博之; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, J Appl Glycosci, 52, Suppl., 24, 20 Jul. 2005
  Japanese
• —ホタテガイ中腸腺由来イオン依存性α‐glucosidase—
  飯塚貴久; 福川太郎; 西岡謙吾; 中井博之; 奥山正幸; 森春英; 吉田孝; 千葉誠哉; 木村淳夫, J Appl Glycosci, 52, Suppl., 26, 20 Jul. 2005
  Japanese
• 植物α‐GlucosidaseのC末端領域は単独で澱粉粒に吸着する‐GST融合タンパク質による解析‐
  谷沢茂紀; 中井博之; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, J Appl Glycosci, 52, Suppl., 25, 20 Jul. 2005
  Japanese
• 植物α‐Glucosidase,特にイネ酵素の分子解析と発芽時の澱粉粒分解に関する研究
  中井博之; 谷沢茂紀; 松原一樹; 奥山正幸; 森春英; 千葉誠哉; 佐野芳雄; 木村淳夫, J Appl Glycosci, 52, Suppl., 52, 20 Jul. 2005
  Japanese
• Bacteroides thetaiotaomicron SusBの機能解析
  奥山正幸; 北村百世; 丹沢史子; 北郷悠; 森春英; 田中勲; 木村淳夫, J Appl Glycosci, 52, Suppl., 25, 20 Jul. 2005
  Japanese
• 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, 20 Jul. 2005
  Japanese
• Molecular analysis of alpha-glucosidase isozymes from Japanese honeybees (Apis cerana)
  J Wongchawalit; T Yamamoto; M Okuyama; H Mori; R Surarit; J Svasti; S Chiba; AK Kimura, FEBS JOURNAL, 272, 101, 101, Jul. 2005
  English, Summary international conference
• Bacteroides thetaiomicron VPI‐5482由来Glycoside hydrolase family 66様タンパク質の発現と精製および機能解析
  山本英治; 金泳民; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2005, 196, 05 Mar. 2005
  Japanese
• Glycoside hydrolase family 31酵素の構造と機能
  奥山正幸; 森春英; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2005, 30, 05 Mar. 2005
  Japanese
• イネGlycoside Hydrolase Family 31様遺伝子の発現解析
  中井博之; 伊藤真吾; 奥山正幸; 森春英; 千葉誠哉; 佐藤芳雄; 木村淳夫, 日本農芸化学会大会講演要旨集, 2005, 30, 05 Mar. 2005
  Japanese
• クロマルハナバチ,Bombus ignitus,α‐Glucosidaseアイソザイムの精製・諸性質および一次構造の解析
  佐藤なつ子; 高橋有志; 中井博之; 光畑雅宏; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2005, 30, 05 Mar. 2005
  Japanese
• イネGlycoside hydrolase family 31様タンパク質の機能解析
  谷沢茂紀; 中井博之; 奥山正幸; 森春英; 千葉誠哉; 佐野芳雄; 木村淳夫, 日本農芸化学会大会講演要旨集, 2005, 31, 05 Mar. 2005
  Japanese
• ミツバチ由来結合型トレハラーゼのcDNAクローニングと組換え酵素の生産
  西塔沙織; LEE Jin‐Ha; 西本完; 森春英; 奥山正幸; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2005, 196, 05 Mar. 2005
  Japanese
• 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, 05 Mar. 2005
  Japanese
• 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, 05 Mar. 2005
  Japanese
• 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, 05 Mar. 2005
  Japanese
• 大腸菌Trehalase(TreA)の触媒残基の部位特異的変異導入による解析
  西塔沙織; 森春英; 奥山正幸; 千葉誠哉; 木村淳夫, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2005, 16, 2005
  Japanese
• Glycoside hydrolase family31酵素の活性中心に保存されているArg残基の役割
  汐川由希子; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 2005, 16, 2005
  Japanese
• 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
  Japanese
• 植物α‐Glucosidaseの生澱粉吸着に関与するアミノ酸残基の解析
  中井博之; 谷沢茂紀; 奥山正幸; 森春英; 山本健; 佐野芳雄; 千葉誠哉; 木村淳夫, J Appl Glycosci, 51, Suppl., 41, 20 Jul. 2004
  Japanese
• 大腸菌yicl産物の機能およびX線構造解析
  奥山正幸; 尾瀬農之; 北村百世; 森春英; 千葉誠哉; 木村淳夫; 田中勲, J Appl Glycosci, 51, Suppl., 40, 20 Jul. 2004
  Japanese
• クロマルハナバチ,Bombus ignitus,α‐Glucosidase Iの精製と諸性質
  佐藤なつ子; 高橋有志; 中井博之; 光畑雅宏; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, J Appl Glycosci, 51, Suppl., 40, 20 Jul. 2004
  Japanese
• Streptomyces sp. No.35由来β‐glucosidase F₃の活性化現象
  峰島希; 福田健二; 森春英; 奥山正幸; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2004, 255, 05 Mar. 2004
  Japanese
• 植物α‐Glucosidaseに見出された新規な生澱粉結合部位の解析
  中井博之; 奥山正幸; 森春英; 山本健; 千葉誠哉; 佐野芳雄; 木村淳夫, 日本農芸化学会大会講演要旨集, 2004, 254, 05 Mar. 2004
  Japanese
• Glycosidase様タンパク質のglycosyl fluorideに対する反応の解析
  奥山正幸; 森春英; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2004, 257, 05 Mar. 2004
  Japanese
• ミツバチα‐glucosidase III触媒残基変異酵素D206Gはアノマー反転型反応を行う。
  岩井岳; 坪野真子; 森春英; 奥山正幸; 木村淳夫, 日本農芸化学会大会講演要旨集, 2004, 255, 05 Mar. 2004
  Japanese
• Bacillus subtilis 168S^r由来α‐glucosidase Phe144の機能解析
  八巻勉; 森春英; 奥山正幸; 木村淳夫, 日本農芸化学会大会講演要旨集, 2004, 255, 05 Mar. 2004
  Japanese
• オオムギα‐アミラーゼ1 Asn209変異酵素の機能解析
  福原有信; 森春英; 奥山正幸; SVENSSON B; 木村淳夫, 日本農芸化学会大会講演要旨集, 2004, 108, 05 Mar. 2004
  Japanese
• 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, 05 Mar. 2004
  Japanese
• α‐Glucosidase変異酵素がα‐Glycosynthase反応を触媒する
  奥山正幸; 森春英; 渡辺琴美; 木村淳夫; 千葉誠哉, 日本農芸化学会誌, 77, 11, 1140, 1141, 01 Nov. 2003
  Japan Society for Bioscience, Biotechnology, and Agrochemistry, Japanese
• 加水分解活性を失ったグリコシダーゼがオリゴ糖合成を触媒する : グリコシンターゼによる効率的なオリゴ糖合成反応
  奥山 正幸, 化学と生物, 41, 7, 422, 425, 25 Jul. 2003
  日本農芸化学会, Japanese
• オオムギα‐アミラーゼ1(AMY1)の部位特異的変異による基質特異性の改変
  福原有信; 森春英; 奥山正幸; SVENSSON B; 木村淳夫, 日本農芸化学会大会講演要旨集, 2003, 98, 05 Mar. 2003
  Japanese
• 赤米および日本晴で発芽時に作用するα‐Glucosidaseの一次構造の解析
  中井博之; 伊藤達也; 松原一樹; 奥山正幸; 森春英; 千葉誠哉; 佐野芳雄; 木村淳夫, 日本農芸化学会大会講演要旨集, 2003, 100, 05 Mar. 2003
  Japanese
• 糸状菌Acremonium implicatum IF030538由来α‐Glucosidase遺伝子のクローニング
  山本健; 大畑祐一郎; 海野剛裕; 小川浩一; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2003, 100, 05 Mar. 2003
  Japanese
• 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, 05 Mar. 2003
  Japanese
• Catalytic Amino Acid Residue Providing Proton Donor in .ALPHA.-Glucosidase Family II.
  OKUYAMA MASAYUKI; MORI HARUHIDE; KIMURA ATSUO; CHIBA SEIYA, J Appl Glycosci, 49, 2, 211, 219, 01 Apr. 2002
  cDNA encoding Schizosaccharomyces pombe a-glucosidase was cloned, and expressed in Saccharomyces cerevisiae. The deduced amino acid sequence categorized under the α-glucosidase family II showed a high homology to those of a-glucosidase from molds, plants and mammals. By site direct mutagenesis, Asp481, G1u484, and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COON) of the Asp647 residue was for the first time pointed out to be the candidate of proton donor in the a-glucosidase of family II. The carboxylate group (-COO-) of the Asp481 residue was assumed to be the secondary carboxylate group, which stabilize the oxocarbenium ion through electrostatic interaction, and the Asp481 was considered to be modified by the chemical modification with conduritol B epoxide. The role of the G1u484 residue, which was the third residue, was presumed to be to fix the reaction intermediate of substrates., The Japanese Society of Applied Glycoscience, Japanese
• α‐Glucosidase変異酵素はGlycosynthase反応を触媒する
  奥山正幸; 森春英; 木村淳夫; 千葉誠哉, 日本農芸化学会大会講演要旨集, 2002, 127, 05 Mar. 2002
  Japanese
• Aspergillus niger α‐Galactosidase遺伝子のPichia pastorisでの発現
  矢守美典; 石原啓吾; 奥山正幸; 森春英; 千葉誠哉; 木村淳夫, 日本農芸化学会大会講演要旨集, 2002, 265, 05 Mar. 2002
  Japanese
• 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, 05 Mar. 2002
  Japanese
• Analysis of amino acid residue concerning catalytic activity of .ALPHA.-glucosidase in Schizosaccharomyces pombe.
  OKUYAMA MASAYUKI; MORI HARUHIDE; KIMURA ATSUO; CHIBA SEIYA, 日本農芸化学会北海道支部・日本土壌肥料学会北海道支部・日本生物工学会北日本支部・日本応用糖質科学会北海道支部・北海道農芸化学協会合同学術講演会講演要旨, 1998, 15, 1998
  Japanese
• Schizosaccharomyces pombe. Inference of the primary structure and active free radical of .ALPHA.-glucosidase.
  OKUYAMA MASAYUKI; MORI HARUHIDE; KIMURA ATSUO; CHIBA MASAYA, 日本農芸化学会東北支部講演要旨, 1996, Godo Gakujutsu Koenkai, 17, Sep. 1996
  Japanese
• Schizosaccharomyces pombe. Estimation of primary structure and active dissociated group of .ALPHA.-glucosidase.
  OKUYAMA MASAYUKI; MORI HARUHIDE; KIMURA ATSUO; CHIBA SEIYA, 日本農芸化学会北海道支部・北海道農芸化学協会シンポジウム及び合同学術講演会講演要旨, 1996, 17, 1996
  Japanese

• Discovery of α-L-glucosidase in nature
  Masayuki Okuyama; Hye-jin Kang; Rikako Shishiuchi; Takayoshi Tagami
  3rd Japan-Switzerland-Germany Workshop on Biocatalysis and Bioprocess Development, 11 Oct. 2023, English, Oral presentation
  10 Oct. 2023 - 12 Oct. 2023
• 糖質加水分解酵素ファミリー97における触媒機構と多様性
  奥山 正幸
  日本農芸化学会2015年度岡山大会, 29 Mar. 2015, Japanese, Nominated symposium
  [Invited], [Domestic Conference]

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• 応用生命科学概論, 2024年, 学士課程, 農学部
• 基礎分子生物学, 2024年, 学士課程, 農学部
• 応用生命科学演習Ⅰ, 2024年, 学士課程, 農学部
• 生物学実験, 2024年, 学士課程, 農学部

• 日本農芸化学会
• 日本応用糖質科学会

• 「Glycoside hydrolase family 31酵素の構造と機能」、日本農芸化学会平成17年度大会シンポジウム－糖質酵素の最先端研究を担う若きサイエンティストの視点－
  2005
• 「glycosidase変異酵素が触媒するglycosynthase反応」, 『第29回糖質科学懇話会』

• Problem-based researches utilized by novel megalosaccharides dissolving poorly-soluble BCS II compounds
  Grants-in-Aid for Scientific Research Fund for the Promotion of Joint International Research (Fostering Joint International Research (B))
  Oct. 2019 - Mar. 2023
  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。
  Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Hokkaido University, Coinvestigator, 19KK0147
• Further development of megalosaccharide research: synthesis and application of novel megalosaccharides displaying excellent functions
  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
  Apr. 2017 - Mar. 2021
  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.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Coinvestigator, 17H03801
• Search for L-glucoside degrading enzymes in nature and synthesis of unknown functional L-oligosaccharides
  Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
  Jun. 2018 - Mar. 2020
  OKUYAMA MASAYUKI
  This study was conducted on the basis that most of the natural carbohydrates are composed of the D-series, but I believe that even if L-series monosaccharides and their oligosaccharides are naturally occuring, they are not theoretically strange. We discovered a new specificity for L-glycoside in an existing enzyme, and succeeded in obtaining an enzyme with a novel L-glycoside specificity from a sequence database. In addition, these L-glycosidases were used to synthesize oligosaccharides consisting of L-series carbohydrates and a mixture of D- and L-series oligosaccharides.
  Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Research (Exploratory), Hokkaido University, Principal investigator, 18K19159
• Molecular mechanism and application of structural elements that govern carbohydrase-catalyzed transfer reaction
  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
  Apr. 2014 - Mar. 2017
  KIMURA, Atsuo
  This project aims at identification and application of structural elements that regulate and improve the carbohydrase-catalyzed transfer activities by elucidating four promising phenomena, which we newly found. Results obtained are as follows: 1) we identify the structural elements to control the α-1,3-transfer reaction displayed by α-1,3-glucoside-transfer enzyme; 2) we reveal the reaction mechanism of the catalytic residue-mutated glycosidase that shows the high transfer yield; 3) a pore canal at the active site of glycosidase plays a role that supplies the catalytic water by study at the molecular level. 4) we analyze the polysaccharide-producing enzyme and identify its structural elements to contribute to the synthesis of oligosaccharides.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, Coinvestigator, 26292049
• Development of glycosides synthesis system using a novel enzymatic activation of carbohydrates with enzymes
  Grants-in-Aid for Scientific Research
  2014 - 2016
  Okuyama Masayuki
  The purpose of this study is to make donor substrates for transglycosylation, which glycosidases catalyze. Using β-fructofranosidase and levansucrase, I succeeded in transferring a fructosyl group of sucrose to each saccharide, such as xylose, galactose, mannose, isomaltooligosaccharides, maltooligosaccharides, and glucuronic acid. The reaction product can be a donor substrate of the transglycosylation by glycosidases. Furthermore, I investigated the transglycosylation properties of several glycosidases and modified their specificity through molecular analyses. In addition, nucleotide sugar was successfully synthesized by the reaction of sucrose synthase with fructosyl saccharide as substrate.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, Principal investigator, Competitive research funding, 26450114
• Substrate recognition of glucosidase II in ER and its related enzymes
  Grants-in-Aid for Scientific Research(若手研究(B))
  2010 - 2011
  Masayuki OKUYAMA
  The purpose of this project was to clarify molecular evolution ofα-glucosidases distributed largely in organisms. An.-glucosidase, which hydrolyzesα-1, 3-glucosidic linkage in endoplasmic reticulum, also showed specificity forα-1, 4-glucosidic linkage. Structural studies of an.-glucosidase indicated that the loop structure, which covers the active pocket, was involved in substrate recognition. Trp residue in the active pocket of an yeast.-glucosidase had important role for substrate specificity. Novel.-glucosidase, which preferredα-1, 3-glucosidic linkage, was discovered from bacteria. It seems that an ancestor enzyme originally hadα-1, 3 specificity and its derivatives have acquired various specificity during the molecular evolution. Moreover, it appears that protein with a new function remained in the related family
  Ministry of Education, Culture, Sports, Science and Technology, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 22780082
• Analysis of structural elements controlling transglycosylation and its application
  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
  2008 - 2010
  KIMURA Atsuo; OKUYAMA Masayuki
  Glycosylases are enzymes, which are currently utilized for the production of oligosaccharides. Interestingly, production-ability is dependent on the kinds of glycosylases, meaning that their structural elements contribute to the reaction specificity. Our research aims at elucidating structural elements of glycosylase-catalyzed transglycosylation. Our research achieved i) elucidation of structural elements contributing to the oligosaccharide production ; ii) establishment of the theoretical background of oligosaccharide production ; iii) finding the ways to regulate the oligosaccharide production.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 20380056
• 1残基のアミノ酸置換で「糖質分解酵素を多糖合成酵素に変換」する現象の分子機構
  科学研究費助成事業 挑戦的萌芽研究
  2008 - 2009
  木村 淳夫; 森 春英; 奥山 正幸
  1残基のアミノ酸置換で「多糖の加水分解酵素(デキストラナーゼ)を合成酵素に転換できる現象」を見出した。この反応機構を分子解析することが、本申請の目的である。このような合成反応は例がなく、世界で初めての現象である。また、産業利用への発展にも期待したい。この残基は触媒アミノ酸と考えられる。本現象は試験管内の観察であるが、このような点突然変異した酵素が実際に生物で機能している可能性を得た。進化の過程においてアミノ酸置換は容易に生じ、1つのアミノ酸を変異させることで酵素分子を「加水分解→合成」にする戦略は、進化的に効率が良い。この戦略の検証も本申請の目的である。本年度は次の結果を得た。多糖合成の分子解析(デキストラナーゼ):1)酵素の結晶化と立体構造解析:昨年度に大量精製した親酵素とGly置換体を用いて結晶化条件を検討した。良好な結晶化条件を確定でき、X線構造解析を進行中である。2)他のアミノ酸による変異酵素:Asp→Gly置換体が合成反応を示したが、より効率の良いアミノ酸置換も想定されたため、他の残基への点変異を試みた。その結果、Gly置換体が最も高い反応効率を与えた。Glyは最もサイズの小さな残基であり、Asp→Gly置換で生じた大きな空間が重要と考えられた。すなわち、このサイズの大きい空間に陰イオンが侵入し合成反応が進行したと考えられた。3)陰イオンの解析:アザイドイオンが最も反応効率の良い陰イオンであった。従って本イオンのサイズ・強度が合成反応に最適であることが分かった。反応の至適pHを確定でき、pK_a値に大きな変化がないと予想できた。4)生成物の構造解析:生成多糖はデキストラン様の構造であった。触媒残基の変異酵素の解析(ウニ酵素):5)遺伝子の発現:酵素遺伝子の異種宿主発現を行った。酵素蛋白質は封入体を形成せず発現しているが、塩存在下であっても酵素活性が極めて低かった。
  日本学術振興会, 挑戦的萌芽研究, 北海道大学, 20658025
• Conversion of catalytic mechanism of glycoside hydrolases
  Grants-in-Aid for Scientific Research(若手研究(B))
  2008 - 2009
  Masayuki OKUYAMA
  I discovered an unique glycoside hydrolases family which contains inverting- and retaining-catalytic enzymes. This divergence was due to the simple difference of the position of catalytic residues. We attempted to convert the catalytic mechanism of these enzyme each other. However, the attempt was failed. These results indicated that two similar enzymes diverged from a common ancestor in early stage of evolution and each enzyme evolved in different ways.
  Ministry of Education, Culture, Sports, Science and Technology, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 20780068
• Molecular Analysis of Carbohydrases Showing Different Reaction by Novel Structure and Its Application
  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
  2005 - 2007
  KIMURA Atsuo; MORI Haruhide; OKUYAMA Masayuki
  This project is about the enzymes having the structure similar to α-glucosidase; i.e. α-xylosidase, glucan lyase, cyclic-tetrasaccharide-forming enzyme, and α-glucosidase, each of which catalyzes the different reaction. Three-dimensional structure available recently allows us to analyze the molecular mechanism of reactions exhibited by four enzymes. The purposes of research are 1) to elucidate the relationship between substrate and amino acid residue(s) in the catalytic site; 2) to analyze the function of catalytic residues; 3) to elucidate the structural element(s) to display the above-described different reactions; 4) to synthesize the useful enzyme. Results are as follows.
  (1) We analyzed the amino acid residues in the catalytic site of α-xylosidase to recognize α-xyloside-structure, and succeeded in conversion of α-xylosidase into α-glucosidase by the mutagenesis of its structural elements. (2) The catalytic residues were identified and their functions were investigated. (3) Amino acid replacement of α-glucosidase (a hydrolyzing enzyme) lost its hydrolytic activity and enhanced the transglucosidation ability, meaning the conversion of hydrolyzing enzyme into transferring enzyme. (4) We have succeed in change of transferring products.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 17380060
• 基質認識に基づいた特異性の高い自殺基質の分子設計
  科学研究費補助金(若手研究(B))
  2005 - 2006
  奥山 正幸
  構造プロテオミクス、プロテオーム解析など網羅的なタンパク質の機能解析の一方で、各論的な分子機能解析も今後の重要な課題となる。本研究の目的は分子機能解析の一環としての自殺基質を用いた糖質関連酵素の活性中心の一般的な探索法の開発である。自殺基質(EAG)は、酵素が基質を認識する機構を考慮した糖分子と反応基にはカルボキシ基と反応性の高いエポキシ環を用い、これら二つの分子群の距離をアルキル基鎖長を増減することで調節し、より特異性の高い自殺基質を設計した。酵素失活の分子機構解析に関しては失活が自殺基質的であることを反応動力学的に実証した。応用例も開発した。1.自殺基質の失活反応の解析;自殺基質の効果を解析するモデル酵素としてisomalto-dextranaseを用いた。アルキル基の長さ(炭素数3-6)が異なる自殺基質(E3G〜E6G)を混合し阻害効果を調べた。各自殺基質(EAG)濃度で失活の擬一次速度定数を求め、EAG濃度に対して擬一次速度定数をプロットすると両者の関係は直線ではなく飽和曲線となりMichaelis-Menten型の失活反応を示すことがわかった。すなわち失活はメカニカルベースであることがわかった。またE5Gの二次速度定数は他のEAGの6倍から10倍高くなっており、アルキル基の鎖長が自殺基質としての能力に重要なファクターであることがわかった。2.EAGを用いたα-amylase活性の特異的測定法;EAG利用の応用として生物試料破砕液からα-amylase活性を特異的に測定する方法の開発を試みた。β-amylase阻害剤E4Gとα-glucosidase阻害剤CBEをα-amylase:β-amylase:α-glucosidase混液と混合し後者二酵素を特異的に失活させ、α-amylaseのみの活性測定に成功した。このことは酵素を精製することなく粗酵素液中などで特定の酵素を失活させることができること示している。またこの結果は標識した自殺基質を用いて修飾後、ペプチドマスフィンガープリントなどにより、粗酵素液でも特定の酵素の活性中心のアミノ酸配列を知ることができるツールに成り得る可能性を示している。3.自殺基質との複合体立体構造の解析については期間内に解析を完了することはできなかった。現在も引き続き進行中である。
  文部科学省, 若手研究(B), 北海道大学, Principal investigator, Competitive research funding, 17780072
• Investigation on Molecular Mechanism of Sucrose-degrading Enzyme in Asian Honeybee.
  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
  2004 - 2005
  KIMURA Atsuo; MORI Haruhide; OKUYAMA Masayuki
  We have analyzed three sucrose-degrading isozymes (α-glucosidase I,II, and III) in European honeybees, since these enzymes exhibited the interesting activity to sucrose of high concentration. α-Glucosidase I was a unique allosteric enzyme to display negative cooperativity in extremely high sucrose concentration. α-Glucosidase II also exhibited the allosteric property of positive cooperativity. α-Glucosidase III was a Michaelis-Menten-type enzyme to be secreted to nectar (bees gathered from flower) and to contribute to the formation of honey. Purpose of this project is i)existence of allosteric α-glucosidase in Asian honeybees, ii)investigation of honey-forming mechanism, and iii)regulation in expression of three α-glucosidase isozymes.
  Four kinds of honeybee species were investigated in this research. From typical Asian honeybees (living in Thailand, Korea and Japan), three isozymes were isolated by chromatographic method. One of them was an allosteric enzyme having similar properties to European honeybee α-glucosidase I, allowing to molecular analysis of negative cooperativity. The second enzyme was α-glucosidase II-type isozyme. Enzyme in honey was also investigated, and showed similar to one of enzymes (α-glucosidase III-type isozyme). cDNAs of these three enzyme were cloned. It was found that the expression of three isozymes was regulated. In small honeybees living in Thailand, three α-glucosidases were also found, in which two α-glucosidases were isolated. Their properties investigated were almost identical to those of α-glucosidases I and II. Enzyme in honey was compared with adult bee enzymes. All honeybee species studied in this project contained allosteric enzyme. Asian honeybees were divided into two types : i)having three α-glucosidase isozymes and ii)having two α-glucosidase isozymes. Recently, we found that bumblebees also had two α-glucosidase isozymes. Further research will elucidate the interesting molecular mechanism of sucrose-degrading enzymes in flower bees.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 16405025
• 触媒アミノ酸を置換した酵素が、発揮する新たな機能とその応用
  科学研究費助成事業 萌芽研究
  2004 - 2005
  木村 淳夫; 森 春英; 奥山 正幸
  糖質の加水分解酵素は、2つの酸性アミノ酸(AspやGlu)を触媒残基とし、それぞれが-COO^-と-COOHの荷電状態を形成し、協奏的に加水分解を触媒する。応用性の高い糖転移作用も示すが、分解と転移は2つの酸性アミノ酸でなされ分割できない。最近、α-グルコシダーゼにある-COO^-型の触媒基であるAspをCysに置換した。本酵素(Asp→Cys)には活性はないが、温和な酸化で活性を発揮した。Cysの-SHが酸化され-SOOHとなり、活性中心内で-SOO^-に解離し-COO^-の代わりを行うと考えている。この酵素は分解能を失い、糖転移能が上昇し95%の収率を与えた。本研究の目的は、-SOO^-酵素に見出された「非分解・高転移」の現象を解析することである。具体的には、1)酸化したCys残基の構造決定、2)糖転移反応の解析、3)他の酵素を合成酵素にする先駆けとして、触媒基を-SOO^-にした糖質酵素の構築と機帯解析、である。計画は順調に進行し、1)と2)が完了した。本年度は、この現象の応用を図るために3)の課題を中心に研究を進行させた。レバン合成酵素とキチン分解酵素を取り上げ、触媒残基をCysに置換し、酸化処理を行った。両酵素のCys変異体には活性がなかったが、穏やかな酸化により活性が回復した。導入したSH基が-SOOHに変化したことを確認した。Cys酸化酵素は、親酵素と異なる性質を示した(レバン合成酵素:至適pHや転移作用の変化、キチン分解酵素:至適pHや協同性の変化)。
  日本学術振興会, 萌芽研究, 北海道大学, 16658136
• Research on Glycosylases which Obtained New Functions by Mutation on Catalytic Residue.
  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
  2002 - 2004
  KIMURA Atsuo; MORI Haruhide; OKUYAMA Masayuki
  Glycosylases are enzymes that hydrolyze the glycosidic linkage. These enzymes also catalyze the transglycosidation, in which the glycosyl residue is transferred to the acceptor substrate. The transglycosidation is an important reaction i)to produce oligosaccharides valuable for foods and ii)to synthesize bio-active sugar-chains. Transglycosidation and hydrolysis proceed in the same time, meaning that the substrate for transglycosidation as well as its product(s) is cleaved by hydrolysis even under conditions of transglycosidation. We have studied the reactions of glycosylase, and have found the phenomena that catalyzed the transglycosidation only. In this study, we analyze the mechanism of valuable phenomena and perform their application. The results are summarized as follows. 1)We have determined the catalytic residue of negatively charged by the method using suicide substrate. Mutant enzyme (synthase), of which catalytic residue was replaced, was produce and purified. The enzyme showed no hydrolytic reaction, only catalyzed the synthesis of oligosaccharide(s) from fluoride-substrate and acceptor. Acceptor of aryl glycoside is a good substrate, meaning that the hydrophobic interaction between aryl-group and subsite +2 is important. 2)Mutant enzyme, which recognized the plane-shaped substrate, a mimic compound of reaction intermediate, was constructed, and its ability of oligosaccharide-synthesis was studied. The low production was observed. We changed the substrate concentration, and succeeded in the improvement of yield. Addition of alcohol to reaction mixture was also effective, but the high concentration of alcohol decreased the production of oligosaccharide. We have found a glycosidase resistant for alcohol. Currently, the conversion of alcohol-stable enzyme to mutant enzyme of same type is trying.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), HOKKAIDO UNIVERSITY, 14360043

• 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
  Patent right
  特許公開２００７－１８１４５２

• 北海道旭川西高等学校SSH
  2010 - Present
  Lecturer, Organizing member
• 北海道札幌藻岩高校環境講座
  Sep. 2016
  Lecturer