2022/12/01 更新

写真a

ナカイ ヒロユキ
中井 博之
NAKAI Hiroyuki
所属
教育研究院 自然科学系 農学系列 准教授
農学部 農学科 准教授
職名
准教授
外部リンク

学位

  • 博士(農学) ( 2005年3月   北海道大学 )

研究キーワード

  • 物質生産

  • 酵素化学

  • 糖質科学

研究分野

  • ライフサイエンス / 応用生物化学

  • ライフサイエンス / 食品科学

経歴(researchmap)

  • 新潟大学   農学部 応用生物化学科   准教授

    2015年4月 - 現在

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  • 新潟大学   大学院自然科学研究科   助教

    2010年12月 - 2015年3月

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  • 独立行政法人 農業・食品産業技術総合研究機構 食品総合研究所   農研機構特別研究員

    2010年10月 - 2010年11月

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  • デンマーク工科大学   助教

    2008年9月 - 2010年9月

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  • デンマーク工科大学   博士研究員

    2007年9月 - 2008年8月

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  • 北海道大学   大学院農学研究科   博士研究員

    2005年4月 - 2007年9月

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▶ 全件表示

経歴

  • 新潟大学   農学部 農学科   准教授

    2017年4月 - 現在

  • 新潟大学   自然科学研究科 生命・食料科学専攻 応用生命・食品科学   准教授

    2015年4月 - 2017年3月

  • 新潟大学   応用生物化学科   准教授

    2015年4月 - 2017年3月

  • 新潟大学   経営戦略本部 若手研究者育成推進室   助教

    2010年12月 - 2015年3月

学歴

  • 北海道大学   大学院農学研究科   応用生命科学専攻

    - 2005年3月

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    国名: 日本国

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  • 北海道大学   農学部   応用生命科学科

    - 2000年3月

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    国名: 日本国

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所属学協会

 

論文

  • Discovery of solabiose phosphorylase and its application for enzymatic synthesis of solabiose from sucrose and lactose 査読

    Wataru Saburi, Takanori Nihira, Hiroyuki Nakai, Motomitsu Kitaoka, Haruhide Mori

    Scientific Reports   12 ( 1 )   259   2022年1月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Springer Science and Business Media LLC  

    <title>Abstract</title>Glycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized <italic>Paenibacillus borealis</italic> PBOR_28850 protein, belonging to glycoside hydrolase family 94. Screening of acceptor substrates for reverse phosphorolysis, in which α-<sc>d</sc>-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in <italic>Escherichia coli</italic> specifically utilized <sc>d</sc>-galactose as an acceptor and produced solabiose (β-<sc>d</sc>-Glc<italic>p</italic>-(1 → 3)-<sc>d</sc>-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to <sc>d</sc>-galactose and <sc>d</sc>-glucose by β-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.

    DOI: 10.1038/s41598-021-04421-2

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    その他リンク: https://www.nature.com/articles/s41598-021-04421-2

  • Enzymatic control and evaluation of degrees of polymerization of β-(1→2)-glucans 査読

    Masahiro Nakajima, Nobukiyo Tanaka, Kaito Kobayashi, Hiroyuki Nakai, Satoshi Kimura, Tadahisa Iwata, Hayao Taguchi

    Analytical Biochemistry   632   114366 - 114366   2021年11月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    DOI: 10.1016/j.ab.2021.114366

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  • Structure of a bacterial α-1,2-glucosidase defines mechanisms of hydrolysis and substrate specificity in GH65 family hydrolases. 国際誌

    Shuntaro Nakamura, Takanori Nihira, Rikuya Kurata, Hiroyuki Nakai, Kazumi Funane, Enoch Y Park, Takatsugu Miyazaki

    The Journal of biological chemistry   297 ( 6 )   101366 - 101366   2021年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Glycoside hydrolase family 65 (GH65) comprises glycoside hydrolases (GHs) and glycoside phosphorylases (GPs) that act on α-glucosidic linkages in oligosaccharides. All previously reported bacterial GH65 enzymes are GPs, whereas all eukaryotic GH65 enzymes known are GHs. In addition, to date, no crystal structure of a GH65 GH has yet been reported. In this study, we use biochemical experiments and X-ray crystallography to examine the function and structure of a GH65 enzyme from Flavobacterium johnsoniae (FjGH65A) that shows low amino acid sequence homology to reported GH65 enzymes. We found that FjGH65A does not exhibit phosphorolytic activity, but it does hydrolyze kojibiose (α-1,2-glucobiose) and oligosaccharides containing a kojibiosyl moiety without requiring inorganic phosphate. In addition, stereochemical analysis demonstrated that FjGH65A catalyzes this hydrolytic reaction via an anomer-inverting mechanism. The three-dimensional structures of FjGH65A in native form and in complex with glucose were determined at resolutions of 1.54 and 1.40 Å resolutions, respectively. The overall structure of FjGH65A resembled those of other GH65 GPs, and the general acid catalyst Glu472 was conserved. However, the amino acid sequence forming the phosphate-binding site typical of GH65 GPs was not conserved in FjGH65A. Moreover, FjGH65A had the general base catalyst Glu616 instead, which is required to activate a nucleophilic water molecule. These results indicate that FjGH65A is an α-1,2-glucosidase and is the first bacterial GH found in the GH65 family.

    DOI: 10.1016/j.jbc.2021.101366

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  • Next-generation prebiotic promotes selective growth of bifidobacteria, suppressing Clostridioides difficile 査読

    Rika Hirano, Mikiyasu Sakanaka, Kazuto Yoshimi, Naohisa Sugimoto, Syogo Eguchi, Yuko Yamauchi, Misaki Nara, Shingo Maeda, Yuta Ami, Aina Gotoh, Takane Katayama, Noriho Iida, Tamotsu Kato, Hiroshi Ohno, Satoru Fukiya, Atsushi Yokota, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki Nakai, Shin Kurihara

    Gut Microbes   13 ( 1 )   1973835   2021年1月

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    担当区分:責任著者   掲載種別:研究論文(学術雑誌)   出版者・発行元:Informa UK Limited  

    DOI: 10.1080/19490976.2021.1973835

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  • Alkoxycarbonyl elimination of 3-O-substituted glucose and fructose by heat treatment under neutral pH 査読

    Kazuhiro Chiku, Riku Tsukasaki, Yu Teshima, Mitsuru Yoshida, Hiroki Aramasa, Takanori Nihira, Hiroyuki Nakai, Hiroshi Ono, Motomitsu Kitaoka

    Carbohydrate Research   108129 - 108129   2020年8月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Elsevier BV  

    DOI: 10.1016/j.carres.2020.108129

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  • Large-scale preparation of β-1,2-glucan using quite a small amount of sophorose 査読

    Kobayashi K., Nakajima M., Aramasa H., Kimura S., Iwata T., Nakai H., Taguchi H.

    Bioscience, Biotechnology, and Biochemistry   83 ( 10 )   1867 - 1874   2019年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1080/09168451.2019.1630257

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  • Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family. 査読

    Tanaka N, Nakajima M, Narukawa-Nara M, Matsunaga H, Kamisuki S, Aramasa H, Takahashi Y, Sugimoto N, Abe K, Terada T, Miyanaga A, Yamashita T, Sugawara F, Kamakura T, Komba S, Nakai H, Taguchi H

    The Journal of biological chemistry   294 ( 19 )   7942 - 7965   2019年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.RA118.007087

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  • Colorimetric determination of β-1,2-glucooligosaccharides for an enzymatic assay using 3-methyl-2-benzothiazolinonehydrazone. 査読

    Kobayashi K, Aramasa H, Nakai H, Nakajima M, Taguchi H

    Analytical biochemistry   560   1 - 6   2018年11月

  • Synthesis of three deoxy-sophorose derivatives for evaluating the requirement of hydroxy groups at position 3 and/or 3' of sophorose by 1,2-β-oligoglucan phosphorylases. 査読

    Tanaka N, Nakajima M, Aramasa H, Nakai H, Taguchi H, Tsuzuki W, Komba S

    Carbohydrate research   468   13 - 22   2018年10月

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  • Characterization and Structural Analysis of a Novel exo-Type Enzyme Acting on β-1,2-Glucooligosaccharides from Parabacteroides distasonis. 査読

    Shimizu H, Nakajima M, Miyanaga A, Takahashi Y, Tanaka N, Kobayashi K, Sugimoto N, Nakai H, Taguchi H

    Biochemistry   57 ( 26 )   3849 - 3860   2018年7月

  • A heterozygous mutation in the SAM domain of p63 underlies a mild form of ectodermal dysplasia. 査読 国際誌

    Toru Kawai, Ryota Hayashi, Hiroyuki Nakai, Yutaka Shimomura, Mazen Kurban, Lamiaa Hamie, Hiroki Fujikawa, Atsushi Fujimoto, Riichiro Abe

    Journal of dermatological science   90 ( 3 )   360 - 363   2018年6月

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  • Structural and thermodynamic insights into β-1,2-glucooligosaccharide capture by a solute-binding protein in <i>Listeria innocua</i>. 査読

    Abe K, Sunagawa N, Terada T, Takahashi Y, Arakawa T, Igarashi K, Samejima M, Nakai H, Taguchi H, Nakajima M, Fushinobu S

    The Journal of biological chemistry   293 ( 23 )   8812 - 8828   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.RA117.001536

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  • Function and structure relationships of a β-1,2-glucooligosaccharide-degrading β-glucosidase. 査読

    Ishiguro R, Tanaka N, Abe K, Nakajima M, Maeda T, Miyanaga A, Takahashi Y, Sugimoto N, Nakai H, Taguchi H

    FEBS letters   591 ( 23 )   3926 - 3936   2017年12月

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  • Function and structure relationships of beta-1,2-glucooligosaccharide-degrading beta-glucosidase 査読

    Rikuto Ishiguro, Nobukiyo Tanaka, Koichi Abe, Masahiro Nakajima, Takuma Maeda, Akimasa Miyanaga, Yuta Takahashi, Naohisa Sugimoto, Hiroyuki Nakai, Hayao Taguchi

    FEBS LETTERS   591 ( 23 )   3926 - 3936   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    BT_3567 protein, a putative -glucosidase from Bacteroides thetaiotaomicron, exhibits higher activity toward Sop(3-5) (Sop(n), n: degree of polymerization of beta-1,2-glucooligosaccharides) than toward Sop(2), unlike a known beta-glucosidase from Listeria innocua which predominantly prefers Sop(2). In the complex structure determined by soaking of a D286N mutant crystal with Sop(4), a Sop(3) moiety was observed at subsites -1 to +2. The glucose moiety at subsite +2 forms a hydrogen bond with Asn81, which is replaced with Gly in the L. innocua beta-glucosidase. The K-m values of the N81G mutant for Sop(3-5) are much higher than those of the wild-type, suggesting that Asn81 contributes to the binding to substrates longer than Sop(3).

    DOI: 10.1002/1873-3468.12911

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  • Biochemical and structural analyses of a bacterial <i>endo</i>-β-1,2-glucanase reveal a new glycoside hydrolase family. 査読

    Abe K, Nakajima M, Yamashita T, Matsunaga H, Kamisuki S, Nihira T, Takahashi Y, Sugimoto N, Miyanaga A, Nakai H, Arakawa T, Fushinobu S, Taguchi H

    The Journal of biological chemistry   292 ( 18 )   7487 - 7506   2017年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.M116.762724

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  • Mechanistic insight into the substrate specificity of 1,2-β-oligoglucan phosphorylase from Lachnoclostridium phytofermentans. 査読

    Nakajima M, Tanaka N, Furukawa N, Nihira T, Kodutsumi Y, Takahashi Y, Sugimoto N, Miyanaga A, Fushinobu S, Taguchi H, Nakai H

    Scientific reports   7   42671   2017年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/srep42671

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  • Mutations in SDR9C7 gene encoding an enzyme for vitamin A metabolism underlie autosomal recessive congenital ichthyosis 査読

    Yohya Shigehara, Shujiro Okuda, Georges Nemer, Adele Chedraoui, Ryota Hayashi, Fadi Bitar, Hiroyuki Nakai, Ossama Abbas, Laetitia Daou, Riichiro Abe, Maria Bou Sleiman, Abdul Ghani Kibbi, Mazen Kurban, Yutaka Shimomura

    HUMAN MOLECULAR GENETICS   25 ( 20 )   4484 - 4493   2016年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of hereditary skin disorder characterized by an aberrant cornification of the epidermis. ARCI is classified into a total of 11 subtypes (ARCI1-ARCI11) based on their causative genes or loci. Of these, the causative gene for only ARCI7 has not been identified, while it was previously mapped on chromosome 12p11.2-q13.1. In this study, we performed genetic analyses for three Lebanese families with ARCI, and successfully determined the linkage interval to 9.47Mb region on chromosome 12q13.13-q14.1, which was unexpectedly outside of the ARCI7 locus. Whole-exome sequencing and the subsequent Sanger sequencing led to the identification of missense mutations in short chain dehydrogenase/ reductase family 9C, member 7 (SDR9C7) gene on chromosome 12q13.3, i. e. two families shared an identical homozygous mutation c.599T&gt; C (p.Ile200Thr) and one family had another homozygous mutation c.214C&gt; T (p.Arg72Trp). In cultured cells, expression of both the mutant SDR9C7 proteins was markedly reduced as compared to wild-type protein, suggesting that the mutations severely affected a stability of the protein. In normal human skin, the SDR9C7 was abundantly expressed in granular and cornified layers of the epidermis. By contrast, in a patient's skin, its expression in the cornified layer was significantly decreased. It has previously been reported that SDR9C7 is an enzyme to convert retinal into retinol. Therefore, our study not only adds a new gene responsible for ARCI, but also further suggests a potential role of vitamin A metabolismin terminal differentiation of the epidermis in humans.

    DOI: 10.1093/hmg/ddw277

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  • Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes 査読

    Darrell Cockburn, Casper Wilkens, Adiphol Dilokpimol, Hiroyuki Nakai, Anna Lewinska, Maher Abou Hachem, Birte Svensson

    PLOS ONE   11 ( 8 )   e0160112   2016年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

    DOI: 10.1371/journal.pone.0160112

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  • An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site. 査読

    Wilkens C, Andersen S, Petersen BO, Li A, Busse-Wicher M, Birch J, Cockburn D, Nakai H, Christensen HE, Kragelund BB, Dupree P, McCleary B, Hindsgaul O, Hachem MA, Svensson B

    Applied microbiology and biotechnology   100 ( 14 )   6265 - 6277   2016年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1007/s00253-016-7417-8

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  • A Solanum torvum GH3 β-glucosidase expressed in Pichia pastoris catalyzes the hydrolysis of furostanol glycoside. 査読

    Suthangkornkul R, Sriworanun P, Nakai H, Okuyama M, Svasti J, Kimura A, Senapin S, Arthan D

    Phytochemistry   127   4 - 11   2016年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.phytochem.2016.03.015

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  • Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis). 査読

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

    Bioscience, biotechnology, and biochemistry   80 ( 3 )   479 - 485   2016年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1080/09168451.2015.1116926

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  • Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua. 査読

    Nakajima M, Yoshida R, Miyanaga A, Abe K, Takahashi Y, Sugimoto N, Toyoizumi H, Nakai H, Kitaoka M, Taguchi H

    PloS one   11 ( 2 )   e0148870   2016年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1371/journal.pone.0148870

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  • A tale of two sisters: identical IL36RN mutations and discordant phenotypes 査読

    N. Rajan, N. Sinclair, H. Nakai, Y. Shimomura, S. . Natarajan

    BRITISH JOURNAL OF DERMATOLOGY   174 ( 2 )   417 - 420   2016年2月

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    記述言語:英語   出版者・発行元:WILEY-BLACKWELL  

    DOI: 10.1111/bjd.14003

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  • Novel splice site mutation in the fumarate hydratase (FH) gene is associated with multiple cutaneous leiomyomas in a Japanese patient 査読

    Yukina Yoshinaga, Hiroyuki Nakai, Ryota Hayashi, Akiko Ito, Naoyuki Kariya, Masaaki Ito, Yutaka Shimomura

    JOURNAL OF DERMATOLOGY   43 ( 1 )   85 - 91   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Cutaneous leiomyoma is a benign skin tumor that originates from the smooth muscle, such as the arrector pili muscle of the hair follicles. Familial cases with multiple cutaneous leiomyomas exist, which typically show an autosomal dominant inheritance trait. Most patients with the disease are known to carry heterozygous germ line mutations in the fumarate hydratase (FH) gene and can be complicated by tumors in internal organs, especially uterine leiomyoma and renal cell cancer in high frequency. In this study, we identified a Japanese male patient with multiple cutaneous leiomyomas and found a novel heterozygous splice site mutation, c. 738 + 2T&gt; A, in the FH gene of the patient, which was unexpectedly inherited from his unaffected father. Further analysis demonstrated loss of heterozygosity in the tumor tissue, which resulted in a hemizygote state of the mutant allele. Expression studies with the tumor tissue showed that the mutation led to skipping of exon 5 at mRNA levels, which was predicted to cause an in-frame deletion of FH protein (p.Ser186_ Gln246del). The protein structure analysis strongly suggested that the deletion would severely disrupt the conformation of the FH protein including the substratebinding domain, and thus would severely affect the expression and the function. Our findings further disclose the molecular basis of multiple cutaneous leiomyomas and also provide precious information to the mutation carriers in the family for an early diagnosis of renal cell cancer in the future.

    DOI: 10.1111/1346-8138.13019

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  • Characterization and crystal structure determination of β-1,2-mannobiose phosphorylase from Listeria innocua. 査読

    Tsuda T, Nihira T, Chiku K, Suzuki E, Arakawa T, Nishimoto M, Kitaoka M, Nakai H, Fushinobu S

    FEBS letters   589 ( 24 )   3816 - 3821   2015年12月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.febslet.2015.11.034

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  • An inverting β-1,2-mannosidase belonging to glycoside hydrolase family 130 from Dyadobacter fermentans. 査読

    Nihira T, Chiku K, Suzuki E, Nishimoto M, Fushinobu S, Kitaoka M, Ohtsubo K, Nakai H

    FEBS letters   589 ( 23 )   3604 - 3610   2015年11月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.febslet.2015.10.008

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  • Analysis of surface binding sites (SBS) within GH62, GH13 and GH77 査読

    Wilkens C, Cockburn D, Andersen S, Petersen BO, Ruzanski C, Field RA, Hindsgaul O, Nakai H, McCleary B, Smith AM, Abou Hachem M, Svensson B

    62 ( 3 )   87 - 93   2015年8月

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    記述言語:英語  

    DOI: 10.5458/jag.jag.JAG-2015_006

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  • Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes 査読

    Young-Woo Nam, Takanori Nihira, Takatoshi Arakawa, Yuka Saito, Motomitsu Kitaoka, Hiroyuki Nakai, Shinya Fushinobu

    JOURNAL OF BIOLOGICAL CHEMISTRY   290 ( 30 )   18281 - 18292   2015年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 angstrom. The active site is located near the dimer interface. At subsite + 1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite + 1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

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  • Large-scale preparation of 1,2-β-glucan using 1,2-β-oligoglucan phosphorylase 査読

    Abe K, Nakajima M, Kitaoka M, Toyoizumi H, Takahashi Y, Sugimoto N, Nakai H, Taguchi H

    Journal of Applied Glycoscience   62 ( 2 )   47 - 52   2015年5月

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    記述言語:英語   出版者・発行元:The Japanese Society of Applied Glycoscience  

    1,2-β-Glucan was produced enzymatically from 1.0 M sucrose and 0.5 M glucose by the combination of sucrose phosphorylase and 1,2-β-oligoglucan phosphorylase in the presence of 100 mM inorganic phosphate. Accumulation of 1,2-β-glucan in 2 L of the reaction mixture reached over 800 mM (glucose equivalent). Sucrose, glucose and fructose were removed after the reaction by yeast treatment. 1,2-β-Glucan was precipitated with ethanol to obtain 167 g of 1,2-β-glucan from 1 L of the reaction mixture.

    DOI: 10.5458/jag.jag.JAG-2014_011

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  • Discovery of two β-1,2-mannoside phosphorylases showing different chain-length specificities from Thermoanaerobacter sp. X-514. 査読

    Chiku K, Nihira T, Suzuki E, Nishimoto M, Kitaoka M, Ohtsubo K, Nakai H

    PloS one   9 ( 12 )   e114882   2014年12月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • One pot enzymatic production of nigerose from common sugar resources employing nigerose phosphorylase 査読

    Nihira T, Miyajima F, Nishimoto M, Kitaoka M, Ohtsubo K, Nakai H

    Journal of Applied Glycoscience   61 ( 3 )   75 - 80   2014年8月

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    担当区分:最終著者, 責任著者   記述言語:英語  

    DOI: 10.5458/jag.jag.JAG-2013_012

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  • Structural basis for reversible phosphorolysis and hydrolysis reactions of 2-O-α-glucosylglycerol phosphorylase. 査読

    Touhara KK, Nihira T, Kitaoka M, Nakai H, Fushinobu S

    The Journal of biological chemistry   289 ( 26 )   18067 - 18075   2014年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Characterization of two α-1,3-glucoside phosphorylases from Clostridium phytofermentans 査読

    Nihira T, Nishimoto M, Nakai H, Ohtsubo K, Kitaoka M

    Journal of Applied Glycoscience   61 ( 2 )   59 - 66   2014年5月

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    記述言語:英語  

    DOI: 10.5458/jag.jag.JAG-2013_013

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  • 1,2-β-Oligoglucan phosphorylase from Listeria innocua. 査読

    Nakajima M, Toyoizumi H, Abe K, Nakai H, Taguchi H, Kitaoka M

    PloS one   9 ( 3 )   e92353   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • 2-O-α-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on β-D-glucose 1-phosphate. 査読

    Nihira T, Saito Y, Ohtsubo K, Nakai H, Kitaoka M

    PloS one   9 ( 1 )   e86548   2014年1月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi 査読

    Takanori Nihira, Yuka Saito, Mamoru Nishimoto, Motomitsu Kitaoka, Kiyohiko Igarashi, Ken'Ichi Ohtsubo, Hiroyuki Nakai

    FEBS Letters   587 ( 21 )   3556 - 3561   2013年11月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY  

    A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-β-d-glucopyranosyl-d-gluconic acid: phosphate α-d-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α-d-glucose 1-phosphate and d-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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  • Potassium ion-dependent trehalose phosphorylase from halophilic Bacillus selenitireducens MLS10 査読

    Takanori Nihira, Yuka Saito, Kazuhiro Chiku, Motomitsu Kitaoka, Ken'ichi Ohtsubo, Hiroyuki Nakai

    FEBS LETTERS   587 ( 21 )   3382 - 3386   2013年11月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    We discovered a potassium ion-dependent trehalose phosphorylase (Bsel_1207) belonging to glycoside hydrolase family 65 from halophilic Bacillus selenitireducens MLS10. Under high potassium ion concentrations, the recombinant Bsel_1207 produced in Escherichia coli existed as an active dimeric form that catalyzed the reversible phosphorolysis of trehalose in a typical sequential bi bi mechanism releasing beta-D-glucose 1-phosphate and D-glucose. Decreasing potassium ion concentrations significantly reduced thermal and pH stabilities, leading to formation of inactive monomeric Bsel_1207.
    Structured summary of protein interactions:
    Bsel_1207 and Bsel_1207 bind by molecular sieving (View interaction)
    (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

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  • Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family 査読

    Muhammad Farooq, Hiroyuki Nakai, Atsushi Fujimoto, Hiroki Fujikawa, Klaus Wilbrandt Kjaer, Shahid Mahmood Baig, Yutaka Shimomura

    HUMAN GENETICS   132 ( 11 )   1253 - 1264   2013年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T &gt; C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

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  • Discovery of β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans. 査読

    Nihira T, Suzuki E, Kitaoka M, Nishimoto M, Ohtsubo K, Nakai H

    The Journal of biological chemistry   288 ( 38 )   27366 - 27374   2013年9月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • A snapshot into the metabolism of isomalto-oligosaccharides in probiotic bacteria 査読

    Abou Hachem M, Møller MS, Andersen JM, Fredslund F, Majumder A, Nakai H, Leggio LL, Goh YJ, Barrangou R, Klaenhammer TR, Svensson B

    Journal of Applied Glycoscience   60 ( 2 )   95 - 100   2013年7月

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    記述言語:英語   出版者・発行元:The Japanese Society of Applied Glycoscience  

    <i>In vitro</i> and <i>in vivo</i> studies have demonstrated the prebiotic potential of isomalto-oligosaccharides (IMO), comprising α-(1,6)-gluco-oligosaccharides and panose, which selectively stimulate the growth of probiotic bifidobacteria and lactobacilli. The protein machinery conferring the utilization of IMO by probiotics, however, remains vaguely described. We have used genomic, transcriptomic, enzymatic, and biophysical analyses to explore IMO utilization routes in probiotic lactobacilli and bifidobacteria as re­presented by <i>Lactobacillus acidophilus</i> NCFM and <i>Bifidobacterium animalis</i> subsp. <i>lactis</i> Bl-04, respectively. Utilization of IMO and malto-oligosaccharide (α-(1,4)-glucosides) appears to be linked both at the genetic and transcriptomic level in the acidophilus group lactobacilli as suggested by reverse transcriptase PCR (RT-PCR) and gene landscape analysis. Canonical intracellular GH13_31 glucan 1,6-α-glucosidases active on IMO longer than isomaltose occur widely in acidophilus group lactobacilli. Interestingly, however, isomaltose, isomaltulose and panose seem to be internalized through a phosphoenoyl pyruvate transferase system (PTS) and subsequently hydrolyzed by a GH4 6-phosphate-α-glucosidases based on whole genome microarray analysis. This sub-specificity within GH4 seems to be unique for lactobacilli mainly from the gut niche, as the sequences from this group segregate from characterized GH4 maltose-6-phosphate-α-glucosidases in other organisms. By comparison, IMO utilization in bifidobacteria is linked to soybean oligosaccharide utilization loci harboring GH36 α-galactosidases, GH13_31 oligo 1,6-α-glucosidases and a dual specificity ATP-binding cassette (ABC) transport system active in the uptake of both classes of α-(1,6)-glycosides. These data bring novel insight to advance our understanding of the basis of selectivity of IMO metabolism by important gut microbiome members.

    DOI: 10.5458/jag.jag.JAG-2012_022

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  • Colorimetric quantification of α-D-mannose 1-phosphate 査読

    Nihira T, Suzuki E, Kitaoka M, Nishimoto M, Ohtsubo K, Nakai H

    Journal of Applied Glycoscience   60 ( 2 )   137 - 139   2013年7月

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    担当区分:最終著者, 責任著者   記述言語:英語   出版者・発行元:The Japanese Society of Applied Glycoscience  

    We developed an enzymatic colorimetric method for the quantification of <i>α</i>-D-mannose 1-phosphate by adding phosphomannomutase, mannose 6-phosphate isomerase and glucose 6-phosphate isomerase to a conventional glucose 6-phosphate assay using glucose 6-phosphate dehydrogenase. In this method, <i>α</i>-D-mannose 1-phosphate is converted into D-glucose 6-phosphate <i>via </i>D-mannose 6-phosphate and D-fructose 6-phosphate and the resultant D-glucose 6-phosphate is ultimately converted into 6-phosphogluconolactone under concomitant reduction of thio-NAD<sup>+</sup> to thio-NADH, which can be quantified by its wavelength of 400 nm. This method is not altered by the presence of D-mannose, D-mannosamine, <i>N</i>-acetyl-D-mannosamine, L-mannose, <i>β</i>-1,4-mannobiose, <i>α</i>-1,2-mannobiose, methyl <i>α</i>-D-mannoside or dimethyl sulfoxide and it would be useful in studies involving enzymes such as phosphorylases belonging to glycoside hydrolase family 130, which release <i>α</i>-D-mannose 1-phosphate as the reaction product.

    DOI: 10.5458/jag.jag.JAG-2012_019

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  • In vitro growth of four individual human gut bacteria on oligosaccharides produced by chemoenzymatic synthesis 査読

    Louise K. Vigsnaes, Hiroyuki Nakai, Lene Hemmingsen, Joakim M. Andersen, Sampo J. Lahtinen, Louise E. Rasmussen, Maher Abou Hachem, Bent O. Petersen, Jens Ø. Duus, Anne S. Meyer, Tine R. Licht, Birte Svensson

    Food and Function   4 ( 5 )   784 - 793   2013年5月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    The present study aimed at examining oligosaccharides (OS) for potential stimulation of probiotic bacteria. Nineteen structurally well-defined candidate OS covering groups of β-glucosides, α-glucosides and α-galactosides with degree of polymerization 2-4 were prepared in &gt
    100 mg amounts by chemoenzymatic synthesis (i.e. reverse phosphorolysis or transglycosylation). Fourteen of the OS are not naturally occurring and five (β-d-glucosyl-fructose, β-d-glucosyl-xylitol, α-glucosyl-(1,4)- d-mannose, α-glucosyl-(1,4)-d-xylose
    α-glucosyl-(1,4)-l-fucose) have recently been synthesized for the first time. These OS have not been previously tested for effects of bacterial growth and here the ability of all 19 OS to support growth of four gastrointestinal bacteria: three probiotic bacteria Bifidobacterium lactis, Bifidobacterium longum, and Lactobacillus acidophilus, and one commensal bacterium, Bacteroides vulgatus has been evaluated in monocultures. The disaccharides β-d-glucosyl-xylitol and β-d-glucosyl-(1,4)-xylose noticeably stimulated growth yields of L. acidophilus NCFM, and additionally, β-d-glucosyl-(1,4)-xylose stimulated B. longum Bl-05. α-Glucosyl-(1,4)-glucosamine and α-glucosyl-(1,4)-N- acetyl-glucosamine enhanced the growth rate of B. animalis subsp. lactis and B. longum Bl-05, whereas L. acidophilus NCFM and Bac. vulgatus did not grow on these OS. α-Galactosyl-(1,6)-α-galactosyl-(1,6)-glucose advanced the growth rate of B. animalis subsp. lactis and L. acidophilus NCFM. Thus several of the structurally well-defined OS supported growth of beneficial gut bacteria. This reflects a broad specificity of their sugar transporters for OS, including specificity for non-naturally occurring OS, hence showing promise for design of novel prebiotics. © 2013 The Royal Society of Chemistry.

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  • Recent development of phosphorylases possessing large potential for oligosaccharide synthesis 査読

    Hiroyuki Nakai, Motomitsu Kitaoka, Birte Svensson, Ken'ichi Ohtsubo

    CURRENT OPINION IN CHEMICAL BIOLOGY   17 ( 2 )   301 - 309   2013年4月

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    担当区分:筆頭著者, 責任著者   記述言語:英語   出版者・発行元:ELSEVIER SCI LTD  

    Phosphorylases are one group of carbohydrate active enzymes involved in the cleavage and formation of glycosidic linkages together with glycoside hydrolases and sugar nucleotide-dependent glycosyltransferases. Noticeably, the catalyzed phosphorolysis is reversible, making phosphorylases suitable catalysts for efficient synthesis of particular oligosaccharides from a donor sugar 1-phosphate and suitable carbohydrate acceptors with strict regioselectivity. Although utilization of phosphorylases for oligosaccharide synthesis has been limited because only few different enzymes are known, recently the number of reported phosphorylases has gradually increased, providing the variation making these enzymes useful tools for efficient synthesis of diverse oligosaccharides.

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  • Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 alpha-glucosidase contribute to recognition of long-chain substrates 査読

    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   2013年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Glycoside hydrolase family 31 alpha-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger alpha-glucosidase (ANG) is specific for short-chain substrates with the highest k(cat)/K-m for maltotriose, while sugar beet alpha-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k(cat)/K-m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K-m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K-m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AG5. (C) 2012 Elsevier B.V. All rights reserved.

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  • Mutation Analysis of the IL36RN Gene in 14 Japanese Patients with Generalized Pustular Psoriasis 査読

    Muhammad Farooq, Hiroyuki Nakai, Atsushi Fujimoto, Hiroki Fujikawa, Asako Matsuyama, Naoyuki Kariya, Atsuko Aizawa, Hiroshi Fujiwara, Masaaki Ito, Yutaka Shimomura

    HUMAN MUTATION   34 ( 1 )   176 - 183   2013年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    Generalized pustular psoriasis (GPP) is a rare, potentially life threatening, and aggressive form of psoriasis, which is characterized by sudden onset with repeated episodic skin inflammation leading to pustule formation. Familial GPP is known to be caused by recessively inherited mutations in the IL36RN gene, which encodes interleukin 36 receptor antagonist (IL-36Ra). In this article, we performed mutation analysis of the IL36RN gene in 14 Japanese patients with GPP, and identified mutations in two of these patients analyzed. One patient was compound heterozygous for mutations c.115+6T&gt;C and c.368C&gt;G (p.Thr123Arg), whereas the other carried compound heterozygous mutations c.28C&gt;T (p.Arg10*) and c.115+6T&gt;C in the IL36RN gene. Expression studies using total RNA from the patients' skin revealed that the mutation c.115+6T&gt;C resulted in skipping of exon 3, leading to a frameshift and a premature termination codon (p.Arg10Argfs*1). The protein structure analysis suggested that the missensemutation p.Thr123Arg caused misfolding and instability of IL-36Ra protein. In vitro studies in cultured cells showed impaired expression of the p.Thr123Arg mutant IL-36Ra protein, which failed to antagonize the IL-36 signaling pathway. Our data further underscore the critical role of IL36RN in pathogenesis of GPP. Hum Mutat 34:176-183, 2013. (C) 2012 Wiley Periodicals, Inc.

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  • Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates. 査読

    Tagami T, Okuyama M, Nakai H, Kim YM, Mori H, Taguchi K, Svensson B, Kimura A

    Biochimica et biophysica acta   1834 ( 1 )   329 - 335   2013年1月

  • Characterization of a laminaribiose phosphorylase from Acholeplasma laidlawii PG-8A and production of 1,3-β-D-glucosyl disaccharides. 査読

    Nihira T, Saito Y, Kitaoka M, Nishimoto M, Otsubo K, Nakai H

    Carbohydrate research   361   49 - 54   2012年11月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Identification of Bacillus selenitireducens MLS10 maltose phosphorylase possessing synthetic ability for branched α-D-glucosyl trisaccharides. 査読

    Nihira T, Saito Y, Kitaoka M, Otsubo K, Nakai H

    Carbohydrate research   360   25 - 30   2012年10月

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    担当区分:最終著者, 責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Amino acids in conserved region II are crucial to substrate specificity, reaction velocity, and regioselectivity in the transglucosylation of honeybee GH-13 α-glucosidases. 査読

    Ngiwsara L, Iwai G, Tagami T, Sato N, Nakai H, Okuyama M, Mori H, Kimura A

    Bioscience, biotechnology, and biochemistry   76 ( 10 )   1967 - 1974   2012年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Bacteroides thetaiotaomicron VPI-5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions 査読

    Young-Min Kim, Eiji Yamamoto, Min-Sun Kang, Hiroyuki Nakai, Wataru Saburi, Masayuki Okuyama, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Zui Fujimoto, Mikihiko Kobayashi, Doman Kim, Atsuo Kimura

    FEBS JOURNAL   279 ( 17 )   3185 - 3191   2012年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

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

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  • Enzymology and structure of the GH13_31 glucan 1,6-α-glucosidase that confers isomaltooligosaccharide utilization in the probiotic Lactobacillus acidophilus NCFM. 査読

    Møller MS, Fredslund F, Majumder A, Nakai H, Poulsen JC, Lo Leggio L, Svensson B, Abou Hachem M

    Journal of bacteriology   194 ( 16 )   4249 - 4259   2012年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1128/JB.00622-12

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  • Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach 査読

    Young-Min Kim, Yoshiaki Kiso, Tomoe Muraki, Min-Sun Kang, Hiroyuki Nakai, Wataru Saburi, Weeranuch Lang, Hee-Kwon Kang, Masayuki Okuyama, Haruhide Mori, Ryuichiro Suzuki, Kazumi Funane, Nobuhiro Suzuki, Mitsuru Momma, Zui Fujimoto, Tetsuya Oguma, Mikihiko Kobayashi, Doman Kim, Atsuo Kimura

    JOURNAL OF BIOLOGICAL CHEMISTRY   287 ( 24 )   19927 - 19935   2012年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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

    DOI: 10.1074/jbc.M111.339036

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  • A novel metabolic pathway for glucose production mediated by α-glucosidase-catalyzed conversion of 1,5-anhydrofructose. 査読

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

    The Journal of biological chemistry   287 ( 27 )   22441 - 22444   2012年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.C112.360909

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  • 3-O-α-D-glucopyranosyl-L-rhamnose phosphorylase from Clostridium phytofermentans. 査読

    Nihira T, Nakai H, Kitaoka M

    Carbohydrate research   350   94 - 97   2012年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.carres.2011.12.019

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  • Discovery of nigerose phosphorylase from Clostridium phytofermentans 査読

    Takanori Nihira, Hiroyuki Nakai, Kazuhiro Chiku, Motomitsu Kitaoka

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   93 ( 4 )   1513 - 1522   2012年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and beta-d-glucose 1-phosphate (beta-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k (cat) = 67 s(-1) and K (m) = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and beta-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-alpha-d-glucoside. All major products were alpha-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-alpha-d-glucoside produced significant amounts of alpha-1,2-glucosides as by-products. We propose 3-alpha-d-glucosyl-d-glucose:phosphate beta-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.

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  • Recombinant production and characterisation of two related GH5 endo-beta-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity 査読

    Adiphol Dilokpimol, Hiroyuki Nakai, Charlotte H. Gotfredsen, Martin J. Baumann, Natsuko Nakai, Maher Abou Hachem, Birte Svensson

    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS   1814 ( 12 )   1720 - 1729   2011年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    The glycoside hydrolase family 5 (GH5) endo-beta-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastor-is X33 and purified in high yields of 120 and 145 mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K-M) towards beta-1,4 mannooligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer beta-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA Site-directed mutagenesis to ManA S289W lowered K-M for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely. K-M for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-beta-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity. (C) 2011 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.bbapap.2011.08.003

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  • Recombinant production and characterisation of two related GH5 endo-β-1,4-mannanases from Aspergillus nidulans FGSC A4 showing distinctly different transglycosylation capacity. 査読

    Dilokpimol A, Nakai H, Gotfredsen CH, Baumann MJ, Nakai N, Abou Hachem M, Svensson B

    Biochimica et biophysica acta   1814 ( 12 )   1720 - 1729   2011年12月

  • An enzymatic colorimetric quantification of orthophosphate 査読

    Li B, Nihira T, Nakai H, Nishimoto M, Kitaoka M

    Journal of Applied Glycoscience   58 ( 3 )   125 - 127   2011年9月

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    記述言語:英語   出版者・発行元:The Japanese Society of Applied Glycoscience  

    Quantification of orthophosphate (Pi) in the presence of labile phosphate esters is required for biochemical assays. We developed a method for the enzymatic colorimetric quantification of Pi using pyruvate oxidase and peroxidase. The calibration curve was not affected by the presence of labile phosphate esters. Furthermore, this method allows continuous monitoring of the reaction of Pi-releasing enzymes.

    DOI: 10.5458/jag.jag.JAG-2011_002

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  • Truncation of N- and C-terminal regions of Streptococcus mutans dextranase enhances catalytic activity 査読

    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   2011年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER  

    Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCG Delta) or N-VR/C-VR (TM-Delta CG Delta) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-Delta CG Delta did not accept any further protease-degradation during long storage. TM-NCG Delta and TM-Delta CG Delta enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.

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  • Starch-binding domains in the CBM45 family--low-affinity domains from glucan, water dikinase and α-amylase involved in plastidial starch metabolism. 査読

    Glaring MA, Baumann MJ, Abou Hachem M, Nakai H, Nakai N, Santelia D, Sigurskjold BW, Zeeman SC, Blennow A, Svensson B

    The FEBS journal   278 ( 7 )   1175 - 1185   2011年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1111/j.1742-4658.2011.08043.x

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  • Enzymatic synthesis of β-xylosyl-oligosaccharides by transxylosylation using two β-xylosidases of glycoside hydrolase family 3 from Aspergillus nidulans FGSC A4. 査読

    Dilokpimol A, Nakai H, Gotfredsen CH, Appeldoorn M, Baumann MJ, Nakai N, Schols HA, Hachem MA, Svensson B

    Carbohydrate research   346 ( 3 )   421 - 429   2011年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.carres.2010.12.010

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  • Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum 査読

    Hiroyuki Nakai, Maher Abou Hachem, Bent O. Petersen, Yvonne Westphal, Karin Mannerstedt, Martin J. Baumann, Adiphol Dilokpimol, Henk A. Schols, Jens O. Duus, Birte Svensson

    BIOCHIMIE   92 ( 12 )   1818 - 1826   2010年12月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

    Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from alpha-D-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of alpha-D-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming beta-glucosyl disaccharides with beta-(1--&gt;4)- regioselectivity from five monosaccharides as well as branched beta-glucosyl trisaccharides with beta-(1--&gt;4)-regioselectivity from three (1--&gt;6)-linked disaccharides. CtCDP showed strict beta-(1--&gt;4)-regioselectivity and catalysed linear chain extension of the three beta-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two beta-glucosyl oligosaccharide product series to represent novel compounds, i.e. beta-D-glucopyranosyl-[(1--&gt;4)-beta-D-glucopyranosyl](n)-(1--&gt;2)-D-glucopyranose, and beta-D-glucopyranosyl-(1--&gt;4)-beta-D-glucopyranosyl](n)-(1--&gt;3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the Cl hydroxyl of beta-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of Cl substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using alpha-D-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides. (C) 2010 Elsevier Masson SAS. All rights reserved.

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  • Suicide substrate-based inactivation of endodextranase by ω-epoxyalkyl α-glucopyranosides 査読

    Kang HK, Kim YM, Nakai H, Kang MS, Hakamada W, Okuyama M, Mori H, Nishio T, Kimura A

    Journal of Applied Glycoscience   57 ( 4 )   269 - 272   2010年11月

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    記述言語:英語   出版者・発行元:日本応用糖質科学会  

    <i>Streptococcus mutans</i> ATCC 25175由来のエンド型デキストラナーゼ(SmDex)に対し,3種類のω-エポキシアルキルα-<small>D</small>-グルコピラノシド(3′,4′-エポキシブチルα-<small>D</small>-グルコピラノシド(E4G),4′,5′-エポキシペンチルα-<small>D</small>-グルコピラノシド(E5G)および5′,6′-エポキシヘキシルα-<small>D</small>-グルコピラノシド(E6G):アグリコンのアルキル鎖長が異なる)を作用させると,SmDexは擬一次的な活性低下を示した.アルキル鎖長に依存した失活が認められ,失活の度合いはE5G > E6G > E4Gであった.したがってω-エポキシアルキルα-<small>D</small>-グルコピラノシドのグルコース残基とエポキシ基の距離が,SmDexの失活に対し重要であることが判明した.E5Gは可逆的な中間体を形成する失活機構(自殺基質型の失活機構)を与え,不活性化の一次定数(<i>k</i>)と中間体の解離定数(<i>K</i><small>R</small>)はそれぞれ0.44 min<sup>-1</sup>および1.45 m<small>M</small>と算出された.SmDexの加水分解反応の生成物であるイソマルトースの存在によりE5Gの失活が防御されたため,E5GはSmDexの触媒部位に結合すると示唆された.本論文は,ω-エポキシアルキルα-<small>D</small>-グルコピラノシドがエンド型デキストラナーゼの自殺基質になることを示す初めての報告である.

    DOI: 10.5458/jag.57.269

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  • Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase 査読

    Hiroyuki Nakai, Bent O. Petersen, Yvonne Westphal, Adiphol Dilokpimol, Maher Abou Hachem, Jens O. Duus, Henk A. Schols, Birte Svensson

    PROTEIN ENGINEERING DESIGN & SELECTION   23 ( 10 )   781 - 787   2010年10月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (alpha/alpha)(6)-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with beta-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (alpha/alpha)(6)-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413-Glu421, His413-Ile418 and His413-Glu415 from loop 3, that include His413 and Glu415 presumably recognising the alpha-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426-Ala431 in TP and Thr419-Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (alpha 1, alpha 1)- and alpha-(1,2)- regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.

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  • Aspergillus nidulans alpha-galactosidase of glycoside hydrolase family 36 catalyses the formation of alpha-galacto-oligosaccharides by transglycosylation 査読

    Hiroyuki Nakai, Martin J. Baumann, Bent O. Petersen, Yvonne Westphal, Maher Abou Hachem, Adiphol Dilokpimol, Jens O. Duus, Henk A. Schols, Birte Svensson

    FEBS JOURNAL   277 ( 17 )   3538 - 3551   2010年9月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g center dot L-1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with alpha-(1 -&gt; 6) regioselectivity from 40 mm 4-nitrophenol alpha-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol alpha-d-Galp-(1 -&gt; 6)-d-Galp, alpha-d-Galp-(1 -&gt; 6)-alpha-d-Galp-(1 -&gt; 6)-d-Glcp and alpha-d-Galp-(1 -&gt; 6)-alpha-d-Galp-(1 -&gt; 6)-d-Glcp-(alpha 1 -&gt;beta 2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol alpha-d-galactopyranoside (40 mm), alpha-(1 -&gt; 6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabinose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l-rhamnose. Structural modelling using Thermotoga maritima GH36 alpha-galactosidase as the template and superimposition of melibiose from the complex with human GH27 alpha-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred alpha-galactosyl to 6-OH of the terminal residue in the alpha-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the beta-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. alpha-d-Galp-(1 -&gt; 6)-d-Manp; alpha-d-Galp-(1 -&gt; 6)-beta-d-Glcp-(1 -&gt; 4)-d-Glcp; alpha-d-Galp-(1 -&gt; 6)-beta-d-Galp-(1 -&gt; 4)-d-Fruf; alpha-d-Galp-(1 -&gt; 6)-d-Glcp-(alpha 1 -&gt;alpha 1)-d-Glcp; and alpha-d-Galp-(1 -&gt; 6)-alpha-d-Glcp-(1 -&gt; 3)-d-Fruf.

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  • Efficient one-pot enzymatic synthesis of alpha-(1,4)-glucosidic disaccharides through a coupled reaction catalysed by Lactobacillus acidophilus NCFM maltose phosphorylase 査読

    Hiroyuki Nakai, Adiphol Dilokpimol, Maher Abou Hachem, Birte Svensson

    CARBOHYDRATE RESEARCH   345 ( 8 )   1061 - 1064   2010年5月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCI LTD  

    Lactobacillus acidophilus NCFM maltose phosphorylase (LaMalP) of glycoside hydrolase family 65 catalysed enzymatic synthesis of alpha-(1 -&gt; 4)-glucostdic disacchandes from maltose and five monosacchandes in a coupled phosphorolysis/reverse phosphorolysis one-pot reaction Thus phosphorolysis of maltose to 0-glucose I -phosphate circumvented addition of costly 0-glucose 1-phosphate for reverse phosphorolysis with different monosacchande acceptors, resulting in 91%, 89%, 88%, 86% and 84% yield of alpha-a-glucopyranosyl-(1 4)-N-acetyl-a-glucosain inopyranose IN-acetyl-maltosamine I, alpha-b-glucopyranosyl( 1 -&gt; 4)-o-glucosaminopyranose I maltosaminej, a-a-glucopyranosyl-(1 -&gt; 4)-b-mannopyranose, alpha-n-glucopyranosyl-(1 -&gt; 4)-t-fucopyranose and alpha-b-glucopyranosyl-(1 -&gt; 4)-D-xylopyranose, respectively, from 0 1 M maltose, 0.5 M N-acetyl glucosamine, 0.1 M glucosamine. 0.1 M mannose, 1 M t-fucose and 0.5 M xylose in 02 M phosphate-citrate p1-I 62 These current yields of 0.27-0.34 g of disaccharide products from 10 mL reaction mixtures are easy to scale up and moreover the strategy can be applied to large-scale production of other oligosacchandes from low-cost disacchandes as catalysed by phosphorylases with different substrate specificities 2010 Elsevier Ltd All rights reserved.

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  • The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase 査読

    Hiroyuki Nakai, Martin J. Baumann, Bent O. Petersen, Yvonne Westphal, Henk Schols, Adiphol Dilokpimol, Maher A. Hachem, Sampo J. Lahtinen, Jens O. Duus, Birte Svensson

    FEBS JOURNAL   276 ( 24 )   7353 - 7365   2009年12月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL PUBLISHING, INC  

    A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mm beta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1 -&gt; 4)-Glcp (maltose), alpha-Glcp-(1 -&gt; 4)-GlcNp (maltosamine), alpha-Glcp-(1 -&gt; 4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1 -&gt; 4)-Manp, alpha-Glcp-(1 -&gt; 4)-Xylp and alpha-Glcp-(1 -&gt; 4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

    DOI: 10.1111/j.1742-4658.2009.07445.x

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  • Molecular Mechanism of α-glucosidase 査読

    Masayuki Okuyama, Haruhide Mori, Hironori Hondoh, Hiroyuki Nakai, Wataru Saburi, Min Sung Kang, Young Min Kim, Mamoru Nishimoto, Jintanart Wongchawalit, Takeshi Yamamoto, Mee Son, Jin Ha Lee, San San Mar, Kenji Fukuda, Seiya Chiba, Atsuo Kimura

    Carbohydrate-Active Enzymes: Structure, Function and Applications   64 - 76   2008年9月

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    掲載種別:論文集(書籍)内論文  

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

    DOI: 10.1533/9781845695750.1.64

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  • Rice α-glucosidase isozymes and isoforms showing different starch granules-binding and -degrading ability 査読

    Nakai H, Tanizawa S, Ito T, Kamiya K, Kim YM, Yamamoto T, Matsubara K, Sakai M, Sato H, Imbe T, Okuyama M, Mori H, Chiba S, Sano Y, Kimura A

    Biocatalysis and Biotransformation   26 ( 1-2 )   104 - 110   2008年

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1080/10242420701788736

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  • Function-unknown glycoside hydrolase family 31 proteins, mRNAs of which were expressed in rice ripening and germinating stages, are alpha-glucosidase and alpha-xylosidase 査読

    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   2007年10月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three a-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. alpha-Xylosidase encoded in rice genome was found for the first time.

    DOI: 10.1093/jb/mvm174

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  • Multiple forms of alpha-glucosidase in rice seeds (Oryza sativa L., var Nipponbare) 査読

    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   2007年1月

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

    Two isoforms of alpha-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6 kDa-peptide and 88 kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88 kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88 kDa-peptide of ONG2-I. The peptides between 6 kDa and 88 kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three alpha-glucosidase cDNAs (ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of alpha-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice. (c) 2006 Elsevier Masson SAS. All rights reserved.

    DOI: 10.1016/j.biochi.2006.09.014

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  • Purification and characterization of alpha-glucosidase I from Japanese honeybee (Apis cerana japonica) and molecular cloning of its cDNA 査読

    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   2006年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    a-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

    DOI: 10.1271/bbb.60302

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  • Plant α-glucosidase: Molecular analysis of rice α-glucosidase and degradation mechanism of starch granules in germination stage 査読

    Nakai H, Ito T, Tanizawa S, Matsubara K, Yamamoto T, Okuyama M, Mori H, Chiba S, Sano Y, Kimura A

    Journal of Applied Glycoscience   53 ( 2 )   137 - 142   2006年7月

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    担当区分:筆頭著者   記述言語:英語   出版者・発行元:The Japanese Society of Applied Glycoscience  

    植物種子が発芽する際に貯蔵物質である澱粉は,各種加水分解酵素により酵素分解され,生長に必要な物質・エネルギーの供給源となる.澱粉は植物種子中に不溶性の澱粉粒として存在するため,現在までα-アミラーゼが唯一澱粉粒に直接作用できる酵素であると考えられてきた.しかし,我々は植物α-グルコシダーゼが澱粉粒への吸着・分解能をもつことを明らかにし,第2の澱粉分解経路 (澱粉粒にα-グルコシダーゼが作用し,直接グルコースを遊離する経路) の存在を示した.さらに,植物α-グルコシダーゼには,触媒ドメインとは独立して機能する澱粉粒吸着ドメインがC末端に存在することを解明した.本ドメインに保存された芳香族アミノ酸に対する部位特異的変異導入により,澱粉粒吸着に関与する残基を推定した.植物種子中には,複数のα-グルコシダーゼが存在する.我々は,イネ種子中に可溶性および不溶性 (界面活性剤により可溶化が可能) の性質を示す2種のアイソザイムを見出した.両酵素の発現様式から,14品種のイネ種子を二つのグループ (グループ1,2) に大別した.グループ1は,不溶性酵素のみが乾燥種子に検出されるイネ品種である.不溶性α-グルコシダーゼが登熟期で合成され,完熟に伴い種子中に保存されるが,発芽の進行につれ消失する.発芽後に可溶性酵素が新たに合成される.グループ2の品種では,可溶性および不溶性の酵素が乾燥種子に検出された.両酵素は登熟期で合成され,発芽期間中の活性は変化せず,一定のレベルで推移する.グループ1からは赤米を,グループ2では日本晴を実験対象に選び,α-グルコシダーゼの機能解析を行った.翻訳後修飾やゲノム遺伝子発現調節によるアイソフォームやアイソザイムの形成機構ならびに精製酵素を用いた性質の解明を行った.多様なα-グルコシダーゼが関与する澱粉代謝の一端が明らかにされた.N末領域に生じる翻訳後限定分解は,植物酵素に対し一般的に観察される現象であった.

    DOI: 10.5458/jag.53.137

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  • Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase 査読

    F Sato, M Okuyama, H Nakai, H Mori, A Kimura, S Chiba

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   69 ( 10 )   1905 - 1913   2005年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K-m) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.

    DOI: 10.1271/bbb.69.1905

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  • Enzymatic synthesis of alkyl α-2-deoxylglucosides by alkyl alcohol resistant α-glucosidase from Aspergillus niger 査読

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

    Tetrahedron Asymmetry   16 ( 2 )   403 - 409   2005年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.tetasy.2004.11.046

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  • Molecular analysis of α-glucosidase belonging to GH-family 31 査読

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

    Biologia, Bratislava   60   131 - 135   2005年

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    担当区分:筆頭著者   記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • 麹菌の細胞壁α-1,3-グルカン生合成に関与するα-アミラーゼAgtAの特性解析

    小泉亜未, 尾形慎, 矢野成和, 宮澤拳, 吉見啓, 吉見啓, 佐野元昭, 日高將文, 仁平高則, 中井博之, 木村聡, 岩田忠久, 阿部敬悦, 阿部敬悦

    日本農芸化学会大会講演要旨集(Web)   2021   2021年

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  • 次世代型プレバイオティクス・ガラクトシル-β-1,4-ラムノースのビフィズス菌特異的な増殖促進機構の解析

    平野里佳, 平野里佳, 阪中幹祥, 阪中幹祥, 杉本直久, 江口省吾, 奈良未沙希, 片山高嶺, 北岡本光, 北岡本光, 中井博之, 栗原新

    日本農芸化学会大会講演要旨集(Web)   2020   2020年

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  • ビフィズス菌を特異的に増殖させる次世代型プレバイオティクスの開発と応用

    平野里佳, 阪中幹祥, 杉本直久, 江口省吾, 奈良未沙希, 片山高嶺, 北岡本光, 中井博之, 栗原新

    日本乳酸菌学会誌   30 ( 2 )   2019年

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  • 酵素合成法による機能性オリゴ糖の創出

    中井博之

    応用糖質科学   8 ( 1 )   51‐55   2018年2月

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  • Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes

    Wade Abbott, Orly Alber, Ed Bayer, Jean-Guy Berrin, Alisdair Boraston, Harry Brumer, Ryszard Brzezinski, Anthony Clarke, Beatrice Cobucci-Ponzano, Darrell Cockburn, Pedro Coutinho, Mirjam Czjzek, Bareket Dassa, Gideon John Davies, Vincent Eijsink, Jens Eklof, Alfons Felice, Elizabeth Ficko-Blean, Geoff Pincher, Thierry Fontaine, Zui Fujimoto, Kiyotaka Fujita, Shinya Fushinobu, Harry Gilbert, Tracey Gloster, Ethan Goddard-Borger, Ian Greig, Jan-Hendrik Hehemann, Glyn Hemsworth, Bernard Henrissat, Masafumi Hidaka, Ramon Hurtado-Guerrero, Kiyohiko Igarashi, Takuya Ishida, Stefan Janecek, Seino Jongkees, Nathalie Juge, Satoshi Kaneko, Takane Katayama, Motomitsu Kitaoka, Naotake Konno, Daniel Kracher, Anna Kulminskaya, Alicia Lammerts van Bueren, Sine Larsen, Junho Lee, Markus Linder, Leila LoLeggio, Roland Ludwig, Ana Luis, Mirko Maksimainen, Brian Mark, Richard McLean, Gurvan Michel, Gurvan Michel, Cedric Montanier, Marco Moracci, Haruhide Mori, Hiroyuki Nakai, Wim Nerinckx, Takayuki Ohnuma, Richard Pickersgill, Kathleen Piens, Tirso Pons, Etienne Rebuffet, Peter Reilly, Magali Remaud-Simeon, Brian Rempel, Kyle Robinson, David Rose, Juha Rouvinen, Wataru Saburi, Yuichi Sakamoto, Mats Sandgren, Fathima Shaikh, Yuval Shoham, Franz St John, Jerry Stahlberg, Michael Suits, Gerlind Sulzenbacher, Tomomi Sumida, Ryuichiro Suzuki, Birte Svensson, Toki Taira, Ed Taylor, Takashi Tonozuka, Breeanna Urbanowicz, Gustav Vaaje-Kolstad, Wim Van den Ende, Annabelle Varrot, Maxime Versluys, Florence Vincent, David Vocadlo, Warren Wakarchuk, Tom Wennekes, Rohan Williams, Spencer Williams, David Wilson, Stephen Withers, Katsuro Yaoi, Vivian Yip, Ran Zhang

    GLYCOBIOLOGY   28 ( 1 )   3 - 8   2018年1月

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    記述言語:英語   出版者・発行元:OXFORD UNIV PRESS INC  

    CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

    DOI: 10.1093/glycob/cwx089

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  • Trichoderma reesei由来Cel1Aの糖転移生成物のセルラーゼ誘導活性

    戸谷一英, 佐野孝晃, 二階堂望, 中島将博, 中井博之, 中澤光

    日本農芸化学会大会講演要旨集(Web)   2017   ROMBUNNO.3J33a01 (WEB ONLY)   2017年3月

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  • Listeria innocua由来β‐グルコシダーゼの結晶構造解析

    中島将博, 吉田龍太, 宮永顕正, 中井博之, 田口速男

    KEK Progress Report (Web)   ( 2016-8 )   ROMBUNNO.283 (WEB ONLY)   2017年1月

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  • Lachnoclostridium phytofermentans由来1,2-β-オリゴグルカンホスホリラーゼの結晶構造解析

    中島将博, 古川那由太, 宮永顕正, 中井博之, 田口速男

    Photon Factory Activity Report   35   2017年

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  • β-1,2-グルカン関連酵素群の機能と構造

    中島将博, 阿部紘一, 阿部紘一, 田中信清, 宮永顕正, 中井博之, 伏信進矢, 五十嵐圭日子, 北岡本光, 鮫島正浩, 田口速男

    応用糖質科学   7 ( 3 )   2017年

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  • 2-O-α-D-グルコシル-D-グリセリン酸ホスホリラーゼ

    五十嵐充, 間島滉一郎, 仁平高則, 北岡本光, 荒川孝俊, 伏信進矢, 中井博之, 中井博之

    応用糖質科学   7 ( 3 )   2017年

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  • Trichoderma reesei由来β‐グルコシダーゼII(Cel1A)の糖転移生成物の解析

    戸谷一英, 二階堂望, 佐野孝晃, 小野寺一樹, 尾形慎, 中島将博, 中井博之

    応用糖質科学   6 ( 3 )   56 - 56   2016年8月

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    記述言語:日本語   出版者・発行元:(一社)日本応用糖質科学会  

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  • ABCトランスポーターソホロオリゴ糖結合タンパク質のリガンド認識の構造基盤

    阿部 紘一, 中島 将博, 砂川 直輝, 石田 卓也, 五十嵐 圭日子, 鮫島 正浩, 宮永 顕正, 中井 博之, 田口 速男, 荒川 孝俊, 伏信 進矢

    応用糖質科学   6 ( 3 )   62 - 62   2016年8月

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    記述言語:日本語   出版者・発行元:(一社)日本応用糖質科学会  

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  • Trichoderma reesei由来β‐グルコシダーゼの糖転移生成物の解析

    前川滉大, 田村真澄, 二階堂望, 中島将博, 中井博之, 戸谷一英

    日本農芸化学会大会講演要旨集(Web)   2016   2D026 (WEB ONLY)   2016年3月

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  • Listeria innocua由来ソホロオリゴ糖に特異的なABCトランスポーター基質結合タンパク質の機能と構造

    阿部紘一, 中島将博, 砂川直輝, 石田卓也, 五十嵐圭日子, 鮫島正浩, 宮永顕正, 中井博之, 田口速男, 荒川孝俊, 伏信進矢

    日本農芸化学会大会講演要旨集(Web)   2016   2016年

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  • ソホロオリゴ糖結合タンパク質のリガンド認識機構

    阿部紘一, 中島将博, 砂川直輝, 石田卓也, 五十嵐圭日子, 鮫島正浩, 宮永顕正, 中井博之, 田口速男, 荒川孝俊, 伏信進矢

    日本蛋白質科学会年会プログラム・要旨集   16th   2016年

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  • ソホロオリゴ糖に特異的なABCトランスポーター基質結合タンパク質の熱力学的解析と立体構造

    阿部紘一, 中島将博, 砂川直輝, 石田卓也, 五十嵐圭日子, 鮫島正浩, 宮永顕正, 中井博之, 田口速男, 荒川孝俊, 伏信進矢

    量子ビームサイエンスフェスタ(Web)   2015   2016年

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  • 酸化的セルロース分解に関与するセロビオン酸ホスホリラーゼ

    仁平高則, 杉本直久, 中井博之

    Cellul Commun   22 ( 4 )   175 - 179   2015年12月

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    記述言語:日本語   出版者・発行元:セルロース学会  

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  • Ap-8 マウス脾臓細胞のサイトカイン産生に及ぼすニゲロースの影響(その他有用糖質,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))

    杉本 直久, 伊藤 紗織, 佐藤 佳太, 仁平 高則, 大坪 研一, 北岡 本光, 原 崇, 中井 博之

    応用糖質科学 : 日本応用糖質科学会誌   5 ( 3 )   B39   2015年8月

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    記述言語:日本語   出版者・発行元:日本応用糖質科学会  

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  • Da-11 Bacteroides thetaiotaomicron由来β-グルコシダーゼの機能構造解析(糖質関連酵素,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))

    中島 将博, 宮永 顕正, 前田 拓磨, 中井 博之, 田口 速男

    応用糖質科学 : 日本応用糖質科学会誌   5 ( 3 )   B55   2015年8月

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  • 新規β‐マンノシドホスホリラーゼの発見

    知久和寛, 仁平高則, 鈴木絵里香, 西本完, 北岡本光, 中井博之, 大坪研一

    応用糖質科学   5 ( 2 )   120 - 127   2015年5月

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  • Trichoderma reesei由来β‐グルコシダーゼの基質特異性

    佐藤大輔, 平沢巽, 岡田宏文, 尾形慎, 中島将博, 中井博之, 戸谷一英

    日本農芸化学会大会講演要旨集(Web)   2015   3E32A14 (WEB ONLY)   2015年3月

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  • Trichoderma reesei由来のβ‐グルコシダーゼの基質特異性

    戸谷一英, 平沢巽, 鈴木尊久, 尾形慎, 中島将博, 中井博之, 岡田宏文

    応用糖質科学   4 ( 3 )   (53)   2014年8月

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  • Cp-7 1,2-β-グルカナーゼの同定(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    阿部 紘一, 中島 将博, 豊泉 大幸, 山下 哲郎, 中井 博之, 北岡 本光, 田口 速男

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B49   2014年8月

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  • Cp-11 Listeria innocua由来1,2-β-オリゴマンナンホスホリラーゼのX線結晶構造解析(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    津田 智弘, 仁平 高則, 北岡 本光, 中井 博之, 荒川 孝俊, 伏信 進矢

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B50   2014年8月

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  • Cp-9 Saccharophagus degradans 2-40由来4-O-β-D-マンノシル-D-グルコースホスホリラーゼによる非還元性二糖の生産(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    鈴木 絵里香, 知久 和寛, 仁平 高則, 西本 完, 北岡 本光, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B50   2014年8月

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    記述言語:日本語   出版者・発行元:日本応用糖質科学会  

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  • Cp-10 新規1,2-β-オリゴマンナンホスホリラーゼ(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    仁平 高則, 知久 和寛, 鈴木 絵里香, 西本 完, 北岡 本光, 伏信 進矢, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B50   2014年8月

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  • Cp-5 ホスホリラーゼを用いた1,2-β-グルコオリゴ糖調製と1,2-β-グルカン大量合成(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    豊泉 大幸, 阿部 紘一, 中島 将博, 中井 博之, 北岡 本光, 田口 速男

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B49   2014年8月

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  • S3-7 複合糖鎖の代謝に関与する新規βマンノシドホスホリラーゼの発見(応用糖質科学シンポジウム,日本応用糖質科学会平成26年度大会(第63回))

    知久 和寛, 仁平 高則, 鈴木 絵里香, 西本 完, 北岡 本光, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B63   2014年8月

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    記述言語:日本語   出版者・発行元:日本応用糖質科学会  

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  • Cp-8 Talaromyces funiculosus由来1,2-β-グルカン分解酵素の遺伝子同定及び機能解析(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    田中 信清, 阿部 紘一, 中島 将博, 成川 恵, 北岡 本光, 中井 博之, 山下 哲郎, 田口 速男

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B50   2014年8月

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  • Cp-6 Listeria innocua由来β-グルコシダーゼの機能構造相関(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    吉田 龍太, 中島 将博, 宮永 顕正, 中井 博之, 北岡 本光, 田口 速男

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B49   2014年8月

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  • Cp-14 GH94セロビオン酸ホスホリラーゼの基質特異性の構造基盤(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    Nam Young-Woo, 仁平 高則, 北岡 本光, 中井 博之, 荒川 孝俊, 伏信 進矢

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B51   2014年8月

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  • Cp-12 GH130に属する糖質加水分解酵素β-1,2-マンノシダーゼの発見(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    知久 和寛, 仁平 高則, 鈴木 絵里香, 西本 完, 北岡 本光, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B51   2014年8月

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  • Cp-13 新規ホスホリラーゼを用いたセロビオン酸の合成(ホスホリラーゼ,一般講演,日本応用糖質科学会平成26年度大会(第63回))

    斉藤 由華, 仁平 高則, 西本 完, 北岡 本光, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B51   2014年8月

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  • S2-5 Analysis of Surface Binding Sites (SBS) within GH62, GH13 and GH77(Recent Progress of Carbohydrate Bioengineering)

    Wilkens C., Cockbum D., Andersen S., Petersen B.O., Ruzanski C., Field R., Hindsgaul O., Nakai H., McCleary B., Smith A., Abou Hachem M., Svensson B.

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B29   2014年8月

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    記述言語:英語   出版者・発行元:日本応用糖質科学会  

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  • ニゲラン代謝に関わるClostridium phytofermentans由来の新規ホスホリラーゼの発見

    仁平高則, 宮嶋双葉, 知久和寛, 北岡本光, 中井博之, 大坪研一

    応用糖質科学   4 ( 2 )   147 - 153   2014年5月

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    DOI: 10.5458/bag.4.2_147

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  • Heterozygote IL36RN mutations in a European case of early-onset generalized pustular psoriasis challenge the concept of private mutation

    N. Rajan, N. Sinclair, H. Nakai, Y. Shimomura, S. Natarajan

    BRITISH JOURNAL OF DERMATOLOGY   170 ( 4 )   E11 - E11   2014年4月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:WILEY-BLACKWELL  

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  • セロビオン酸ホスホリラーゼの発見

    仁平高則, 斉藤由華, 西本完, 北岡本光, 五十嵐圭日子, 伏信進矢, 中井博之, 大坪研一

    日本農芸化学会大会講演要旨集(Web)   2014   2014年

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  • S3-4 ニゲラン代謝に関わるClostridium phytofermentans由来の新規ホスホリラーゼの発見(応用糖質科学シンポジウム,日本応用糖質科学会平成25年度大会(第62回))

    仁平 高則, 宮嶋 双葉, 知久 和寛, 北岡 本光, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   3 ( 3 )   B52   2013年8月

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  • Cp-9 2-O-α-グルコシルグリセロールホスホリラーゼによるβGlc1P加水分解の分子機構および構造基盤(ホスホリラーゼ,一般講演,日本応用糖質科学会平成25年度大会(第62回))

    北岡 本光, 仁平 高則, 斉藤 由華, 東原 幸起, 伏信 進矢, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   3 ( 3 )   B42   2013年8月

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  • Cp-7 新規酵素β-1,4-マンノシルN-アセチルグルコサミンホスホリラーゼ(ホスホリラーゼ,一般講演,日本応用糖質科学会平成25年度大会(第62回))

    鈴木 絵里香, 仁平 高則, 北岡 本光, 西本 完, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   3 ( 3 )   B41   2013年8月

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  • Cp-8 Chloroflexus aurantiacus J-10-fl由来の耐熱性コージビオースホスホリラーゼの機能解析(ホスホリラーゼ,一般講演,日本応用糖質科学会平成25年度大会(第62回))

    斉藤 由華, 仁平 高則, 知久 和寛, 北岡 本光, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   3 ( 3 )   B41   2013年8月

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  • ホスホリラーゼを利用した安価原料からの機能性オリゴ糖合成

    仁平高則, 中井博之, 大坪研一

    Bio Ind   30 ( 5 )   47 - 54   2013年5月

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  • 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   2013年1月

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  • 糖質関連酵素が触媒する糖質合成反応を活用した新規オリゴ糖の生産開発

    中井博之, 仁平高則, 北岡本光, 大坪研一

    応用糖質科学   2 ( 4 )   223 - 224   2012年11月

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    記述言語:日本語  

    DOI: 10.5458/bag.2.4_223

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  • マルトースホスホリラーゼを用いた分岐グルコ3糖の生産

    仁平 高則, 斉藤 由華, 中井 博之, Kitaoka Motomitsu, Otsubo Ken'ichi, 仁平 高則, 斉藤 由華, 中井 博之, 北岡 本光, 大坪 研一

    新潟大学農学部研究報告   65 ( 1 )   67 - 75   2012年9月

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    記述言語:英語   出版者・発行元:新潟大学農学部  

    We discovered an inverting maltose phosphorylase (Bsel2056) belonging to glycoside hydrolase family 65 from Bacillus selenitireducens MLS10, which possesses synthetic ability for α-D-glucosyl disaccharides and trisaccharides through the reverse phosphorolysis with β-D-glucose 1-phosphate as the donor. Bsel2056 showed the preference for monosaccharide acceptors with alternative C2 substituent (2-amino-2-deoxy-D-glucose, 2-deoxy-D-arabino-hexose, 2-acetamido-2-deoxy-D-glucose, D-mannose), resulting in production of 1,4-α-D-glucosyl disaccharides with strict regioselectivity. In addition, Bsel2056 synthesized two maltose derivatives possessing additional D-glucosyl residue bound to C2 position of the D-glucose residue at the reducing end, 1,4-α-D-glucopyranosyl-[1,2-α-D-glucopyranosyl]-D-glucose and 1,4-α-D-glucopyranosyl-[1,2-β-D-glucopyranosyl]-D-glucose, from 1,2-α-D-glucopyranosyl-D-glucose (kojibiose) and 1,2-β-D-glucopyranosyl-D-glucose (sophorose), respectively, as the acceptors. Modeling structure analysis using Lactobacillus brevis GH65 maltose phosphorylase as the template and superimposition of an inhibitory pseudomaltotetrasacchaide acarbose from a complex with Aspergillus awamori GH15 glucoamylase suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of D-glucose.糖質加リン酸分解酵素(ホスホリラーゼ)は、その反応の可逆性と厳密な反応位置特異性からオリゴ糖合成に利用可能である。しかし既知のホスホリラーゼは16種類のみ報告されており、新たな特異性を有するホスホリラーゼの発見が望まれている。そこで今回Bacillus selenitireducens MLS10が有するGlycoside Hydrolase Family 65に属するマルトースホスホリラーゼ遺伝子(bsel2056)をクローニングし、組換え酵素の糖受容体特異性を調査した。結果、グルコースの2位にアミノ基を有するグルコサミンなど8種の単糖類を糖受容体とし、α-1,4- グルコ2糖を生産することが明らかになった。さらに既知のマルトースホスホリラーゼとは異なり、本酵素は1、2結合を有するグルコ2糖すなわちコウジビオースおよびソフォロースを糖受容体として認識し、分岐グルコ3糖を生産した。また3D モデリング解析の結果から、糖受容体として結合するグルコースの2位の水酸基周辺に本酵素特有の嵩高い置換基(アミノ基やグルコース残基)を収納するための結合スペースが認められた。

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    その他リンク: http://hdl.handle.net/10191/22583

  • Bp1-7 Acholeplasma laidlawii PG-8A由来ラミナリビオースホスホリラーゼ(ホスホリラーゼおよび関連酵素,一般講演,日本応用糖質科学会平成24年度大会(第61回))

    仁平 高則, 斉藤 由華, 北岡 本光, 西本 完, 中井 博之, 大坪 研一

    応用糖質科学 : 日本応用糖質科学会誌   2 ( 3 )   B37   2012年8月

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  • S3-5 The utilization routes of iso maltooligosaccharides by probiotics : an enzymatic and genetic overview

    Abou Hachem M., Moller M., Fredslund F., Andersen J.M., Majumder A., Nakai H., Lahtinen S.J., Yong J.-G., Klaenhammer T.R., Barrangou R., Lo Leggio L., Svensson B.

    応用糖質科学 : 日本応用糖質科学会誌   2 ( 3 )   B59   2012年8月

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  • プレバイオティクスとして有用な新規機能性オリゴ糖の生産開発

    中井博之, SVENSSON Birte, 大坪研一

    応用糖質科学   2 ( 2 )   117 - 121   2012年5月

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    記述言語:日本語  

    DOI: 10.5458/bag.2.2_117

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  • 新規ニゲロースホスホリラーゼを用いたα-1,3-グルコ2糖の生産

    仁平 高則, 中井 博之, 北岡 本光

    新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, Niigata University   64 ( 2 )   107 - 113   2012年3月

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    記述言語:英語   出版者・発行元:新潟大学農学部  

    糖質リン酸分解酵素(ホスホリラーゼ)は、その厳密な基質特異性と反応の可逆性からオリゴ糖合成に利用可能である。しかし既知のホスホリラーゼは15種類のみで、新たな基質特異性を有する新規ホスホリラーゼの発見が強く望まれている。そこで、これまで数種のホスホリラーゼが単離されているClostridium phytofermentansが有するGlycoside Hydrolase Family65に属する遺伝子(cphy1874)をクローニングし、組換え酵素の酵素化学的性質を調査した。結果、Cphy1874はリン酸存在下でニゲロースに対し高い加リン酸分解活性を示すことが明らかになった。β-グルコース1リン酸と各種糖を作用させたところ,グルコースをアクセプターとした際に高い合成活性を示した。キシロース、1,5-アンヒドログルシトール、ガラクトース、メチル-α-グルコシドにおいてもアクセプターとなり、主生成物はいずれもα-1,3結合であった。このことから本酵素は,これまで報告の無かった新規加リン酸分解酵素ニゲロースホスホリラーゼであることが判明した。

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  • 乳酸菌由来糖質加水分解酵素ファミリー32タンパク質の基質認識機構の解明

    中井 博之, 大坪 研一

    新潟大学農学部研究報告 = Bulletin of the Faculty of Agriculture, Niigata University   64 ( 2 )   115 - 126   2012年3月

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    乳酸菌Lactobacillus acidophilus NCFMでのフルクトオリゴ糖代謝に関与する2つの糖質加水分解酵素(BfrA. β-フルクトシダーゼ;ScrB,スクロース6-リン酸ハイドロラーゼ)基質認識機構を明らかにすることを目的とし、各種組み換え酵素を用いてフルクトオリゴ糖に対する加水分解反応の速度論的解析を行った。ScrBはフルクトオリゴ糖よりスクロースに対して高い加水分解活性を示し、BfrAは短鎖なフルクトオリゴ糖に対して特異性を示すことを明らかにした。加えて、両酵素の基質特異性に関与する領域(BfrAループ1)およびアミノ酸残基(ScrB His71)を、ホモロジーモデリング法により推定した。

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  • 高機能性食品素材として有用なオリゴ糖の生産技術開発とその高度利用 加リン酸分解酵素(ホスホリラーゼ)を利用して高収率生産

    中井博之

    化学と生物   49 ( 10 )   669 - 671   2011年10月

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    記述言語:日本語   出版者・発行元:Japan Society for Bioscience, Biotechnology, and Agrochemistry  

    DOI: 10.1271/kagakutoseibutsu.49.669

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  • α-D-ガラクトース1-リン酸の比色定量法

    仁平 高則, 中島 将博, 井上 公輔, 西本 完, 中井 博之, 北岡 本光, 仁平 高則, 中島 将博, 井上 公輔, 西本 完, 中井 博之, 北岡 本光

    新潟大学農学部研究報告   64 ( 1 )   81 - 85   2011年9月

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    記述言語:英語   出版者・発行元:新潟大学農学部  

    α-D-ガラクトース1-リン酸(Gal 1-P)の比色定量として、ガラクトース定量法にホスファターゼを組み合わせる方法が知られるが、ガラクトースおよびガラクトース含有オリゴ糖が混在する試料中でのGal 1-Pの定量法としては不都合があった。そこで我々はガラクトース代謝系酵素群に着目し、Gal 1-Pのみを検出およびGal 1-Pとガラクトースを同時に検出する2つの比色定量法を開発した。各種濃度のGal 1-Pに発色試薬(UDP-グルコース-ヘキソース-1-リン酸ウリジリルトランスフェラーゼ、ホスホグルコムターゼ、グルコース-6-リン酸デヒドロゲナーゼ、NAD+またはThio-NAD+、UDP-グルコース、グルコース1、6-ビスリン酸、MgCl2含有トリス緩衝液)を混合し、経時的に340nm(NAD+)または400nm(Thio-NAD+)の吸収を測定した結果、20分程度で平衡に達し吸光度はGal 1-P濃度に比例した。また上記試薬にムタロターゼ、ガラクトキナーゼおよびATPを追加することにより、各種濃度のガラクトースに対し同様の結果が得られた。また加リン酸分解酵素ラクト-N-ビオースホスホリラーゼの反応によって生じるGal 1-Pを、発色試薬中にて反応停止操作を伴なわず連続的にモニタリングした結果、算出された活性値は各酵素濃度に比例しており、本定量法を用いた活性測定法の妥当性・有用性が示された。

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    その他リンク: http://hdl.handle.net/10191/18620

  • 乳酸菌由来マルトースホスホリラーゼを用いたα-(1→4)-グルコ二糖の高収率生産

    中井 博之, 仁平 高則, SVENSSON Birte, 大坪 研一, 中井 博之, 仁平 高則, Svensson Birte, 大坪 研一

    新潟大学農学部研究報告   64 ( 1 )   71 - 79   2011年9月

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    記述言語:英語   出版者・発行元:新潟大学農学部  

    A gene cluster involved in maltose/maltodextrin metabolism was identified in the genome of Lactobacillus acidophilusNCFM. Enzymatic properties was described for MalP belonging to glycoside hydrolase family 65 (GH65), which is encodedby malP (GenBank: AAV43670.1) located in the gene cluster. MalP catalyses phosphorolysis of maltose with inversion ofthe anomeric configuration releasing β-glucose 1-phosphate and glucose. Broad acceptor specificity was demonstrated byreverse phosphorolysis using various carbohydrate acceptors and β-glucose 1-phosphate as donor. MalP showed strongpreference for monosaccharide acceptors with equatorial 3-OH and 4-OH, and reacted also with the 2-deoxy amino sugars.By contrast none of the tested di- and trisaccharides served as acceptor. Disaccharide yields obtained from 50 mM β-glucose 1-phosphate and 50 mM glucose, glucosamine, N-acetyl glucosamine, mannose, xylose, or L-fucose were 99, 80, 53,93, 81, and 13%, respectively. Product structures were determined by NMR and ESI-MS to be α-D-glucopyranosyl-(1→4)-Dglucopyranose,α -D-glucopyranosyl-(1→4)-D-glucosaminopyranose,α -D-glucopyranosyl-(1→4)-N-acetyl-D-glucosaminopyranose,α -D-glucopyranosyl-(1→4)-D-mannopyranose, α-D-glucopyranosyl-(1→4)-D-xylopyranose, and α-D-glucopyranosyl-(1→4)-L-fucopyranose. Additionally MalP catalysed synthesis of the α-(1 → 4)-glucosidic disaccharides from maltose in a coupledphosphorolysis/reverse phosphorolysis one-pot reaction. Thus phosphorolysis of maltose to β-glucose 1-phosphatecircumvented addition of costly β-glucose 1-phosphate for reverse phosphorolysis with different monosacchaide acceptors.This strategy can be applied to large-scale production of valuable oligosaccharides from low-cost carbohydrates as catalysedby phosphorylases with different substrate specificity.Lactobacillus acidophilus NCFM のゲノム内に、マルトース/マルトデキストリン代謝に関与する遺伝子クラスターを見出した。本クラスター中には、グルコシドハイドロレースファミリー65に属する機能未知な糖質関連酵素(MalP)をコードする遺伝子が含まれている。そこで大腸菌を宿主として組み換え酵素を生産し、その酵素化学的諸性質を調査した結果、MalP はマルトースに特異的に作用する糖質加リン酸分解酵素であることが明らかになった。加えてMalP が触媒する糖質合成反応を用いて、6種のα(- 1→4)- グルコ二糖を高収率生産に成功した。

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    その他リンク: http://agriknowledge.affrc.go.jp/RN/2010814991

  • 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   2008年3月

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  • 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   2007年7月

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  • 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   2007年7月

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  • 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   2007年3月

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  • 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   2007年3月

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  • 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年

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  • 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   2006年8月

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  • 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   2006年8月

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  • 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   2006年8月

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  • 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   2006年3月

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  • セイヨウミツバチα‐Glucosidase IIのV167N変異酵素は高いK<sub>m</sub>およびk<sub>0</sub>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   2005年7月

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  • —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   2005年7月

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  • 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   2005年7月

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  • 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   2005年7月

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  • 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   2005年3月

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  • 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   2005年3月

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  • 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   2005年3月

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  • 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   2004年7月

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  • 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   2004年7月

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  • 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   2004年3月

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  • 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   2003年3月

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  • 赤米α‐Glucosidaseの限定分解とそれによる諸性質への影響

    中井博之, 伊藤達也, 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   127   2002年3月

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  • 赤米α‐Glucosidaseの精製と諸性質

    中井博之, 林正敏, 森春英, 木村淳夫, 千葉誠哉

    日本農芸化学会誌   75   65   2001年3月

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    2021年9月   日本応用糖質科学会  

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    2020年3月   日本農芸化学会  

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    2017年9月   日本応用糖質科学会  

    中井 博之

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    2017年3月   日本農芸化学会  

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    2016年4月   公益財団法人 長瀬科学技術振興財団  

    中井 博之

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  • 安藤百福賞発明発見奨励賞

    2016年3月   公益財団法人 安藤スポーツ・食文化振興財団  

    中井 博之

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    2014年10月   公益財団法人 食の新潟国際賞財団  

    中井 博之

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    2013年5月   公益財団法人 インテリジェント・コスモス学術振興財団  

    中井 博之

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    2012年6月   一般財団法人 天野エンザイム科学技術振興財団  

    中井 博之

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  • Poster Award

    2010年7月   Plant Polysaccharide and Applied Glycoscience Workshop 2010  

    中井 博之

     詳細を見る

  • 1st Poster Award

    2007年4月   7th Carbohydrate Bioengineering Meeting  

    中井 博之

     詳細を見る

  • Best Poster Award

    2004年7月   Plant Polysaccharide Workshop  

    中井 博之

     詳細を見る

  • 平成16年度大会記者会見報道用トピックス賞

    2004年3月   日本農芸化学会  

    中井 博之

     詳細を見る

▶ 全件表示

 

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  • 応用生命・食品科学特論

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    機関名:新潟大学

  • 食と健康の科学

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  • 農学入門Ⅰ

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    機関名:新潟大学

  • 生物化学実験

    2016年
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    機関名:新潟大学

  • 食品安全学

    2016年
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    機関名:新潟大学

  • 食品製造学

    2016年
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  • Topics in Food Sciences

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  • 自然科学総論Ⅳ

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  • 実地見学

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  • 研究発表演習(中間発表)

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  • 米科学論

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  • 生命・食料科学セミナーBⅠ

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  • 文献詳読Ⅰ

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▶ 全件表示