2022/11/29 更新

写真a

キタオカ モトミツ
北岡 本光
KITAOKA Motomitsu
所属
教育研究院 自然科学系 農学系列 教授
農学部 農学科 教授
職名
教授
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外部リンク

学位

  • 博士(農学) ( 1993年12月   東京大学 )

研究キーワード

  • 糖関連酵素

  • ビフィズス菌

  • オリゴ糖

研究分野

  • ライフサイエンス / 応用生物化学  / 酵素学

経歴(researchmap)

  • 新潟大学   農学部   教授

    2019年4月 - 現在

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  • (国研)農業・食品産業技術総合研究機構 食品研究部門   食品分析研究領域   館長

    2018年4月 - 2019年3月

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  • (国研)農業・食品産業技術総合研究機構 食品研究部門   食品生物機能開発研究領域   酵素機能ユニット長

    2015年4月 - 2018年3月

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  • (独法)農業・食品産業技術総合研究機構 食品総合研究所   食品バイオテクノロジー研究領域   酵素研究ユニット長

    2006年4月 - 2015年3月

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  • (独法)食品総合研究所   生物機能開発部   酵素機能研究室長

    2004年4月 - 2006年3月

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  • (独法)食品総合研究所   生物機能開発部   主任研究員

    2001年4月 - 2004年3月

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  • 農林水産省食品総合研究所   生物機能開発部   主任研究員

    1999年10月 - 2001年3月

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  • 生物系特定産業技術研究推進機構   (農林水産省食品総合研究所)   派遣研究員

    1998年4月 - 1999年9月

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  • アイオワ州立大学   生化学・生物物理学科   博士研究員

    1995年5月 - 1998年2月

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▶ 全件表示

経歴

  • 新潟大学   農学部 農学科   教授

    2019年4月 - 現在

 

論文

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

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

    Scientific Reports   12 ( 1 )   2022年12月

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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

  • Priority effects shape the structure of infant-type Bifidobacterium communities on human milk oligosaccharides. 国際誌

    Miriam N Ojima, Lin Jiang, Aleksandr A Arzamasov, Keisuke Yoshida, Toshitaka Odamaki, Jinzhong Xiao, Aruto Nakajima, Motomitsu Kitaoka, Junko Hirose, Tadasu Urashima, Toshihiko Katoh, Aina Gotoh, Douwe van Sinderen, Dmitry A Rodionov, Andrei L Osterman, Mikiyasu Sakanaka, Takane Katayama

    The ISME journal   16 ( 9 )   2265 - 2279   2022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:Springer Science and Business Media {LLC}  

    Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation. Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, two avid HMO consumers, dominate through inhibitory priority effects. On the other hand, Bifidobacterium breve, a species with limited HMO-utilization ability, can benefit from facilitative priority effects and dominates by utilizing fucose, an HMO degradant not utilized by the other bifidobacterial species. Analysis of publicly available breastfed infant faecal metagenome data showed that the observed trends for B. breve were consistent with our in vitro data, suggesting that priority effects may have contributed to its dominance. Our study highlights the importance and history dependency of initial community assembly and its implications for the maturation trajectory of the infant gut microbiota.

    DOI: 10.1038/s41396-022-01270-3

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  • Isomerization of 6-O-substituted glucose and fructose under neutral pH conditions and subsequent β-elimination reactions

    Kazuhiro Chiku, Ayaka Ohfuji, Nozomi Ohtake, Mitsuru Yoshida, Hiroshi Ono, Motomitsu Kitaoka

    Carbohydrate Research   519   108626 - 108626   2022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:Elsevier BV  

    DOI: 10.1016/j.carres.2022.108626

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  • Production of lacto-&lt;i&gt;N&lt;/i&gt;-biose I using crude extracts of bifidobacterial cells

    Shuntaro Machida, Katsuichi Saito, Mamoru Nishimoto, Motomitsu Kitaoka

    Journal of Applied Glycoscience   69 ( 2 )   15 - 21   2022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:The Japanese Society of Applied Glycoscience  

    DOI: 10.5458/jag.jag.jag-2021_0012

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  • Diversification of a Fucosyllactose Transporter within the Genus Bifidobacterium. 国際誌

    Miriam N Ojima, Yuya Asao, Aruto Nakajima, Toshihiko Katoh, Motomitsu Kitaoka, Aina Gotoh, Junko Hirose, Tadasu Urashima, Satoru Fukiya, Atsushi Yokota, Maher Abou Hachem, Mikiyasu Sakanaka, Takane Katayama

    Applied and environmental microbiology   88 ( 2 )   e0143721   2022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)  

    Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. IMPORTANCE The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breast milk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated host-microbe symbiosis. While the coevolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allow bifidobacteria to consume different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

    DOI: 10.1128/AEM.01437-21

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  • Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

    Toma Kashima, Kouki Okumura, Akihiro Ishiwata, Machika Kaieda, Tohru Terada, Takatoshi Arakawa, Chihaya Yamada, Kentaro Shimizu, Katsunori Tanaka, Motomitsu Kitaoka, Yukishige Ito, Kiyotaka Fujita, Shinya Fushinobu

    Journal of Biological Chemistry   297 ( 5 )   101324 - 101324   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.jbc.2021.101324

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  • Generation of 3-Deoxypentulose by the Isomerization and β-Elimination of 4-O-Substituted Glucose and Fructose

    Kazuhiro Chiku, Mitsuru Yoshida, Hiroshi Ono, Motomitsu Kitaoka

    Carbohydrate Research   108402 - 108402   2021年7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.2021.108402

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  • Three-dimensional alignment of cellulose II microcrystals under a strong magnetic field

    Masahisa Wada, Sayuri Wakiya, Kayoko Kobayashi, Satoshi Kimura, Motomitsu Kitaoka, Ryosuke Kusumi, Fumiko Kimura, Tsunehisa Kimura

    Cellulose   28 ( 11 )   6757 - 6765   2021年7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  

    DOI: 10.1007/s10570-021-03954-z

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    その他リンク: https://link.springer.com/article/10.1007/s10570-021-03954-z/fulltext.html

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

    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 )   2021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:Informa UK Limited  

    DOI: 10.1080/19490976.2021.1973835

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  • Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction. 国際誌

    Yuya Kuritani, Kohei Sato, Hideo Dohra, Seiichiro Umemura, Motomitsu Kitaoka, Shinya Fushinobu, Nobuyuki Yoshida

    Scientific reports   10 ( 1 )   20066 - 20066   2020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)  

    Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

    DOI: 10.1038/s41598-020-77133-8

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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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  • Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete <i>Phanerochaete chrysosporium</i>

    Kiyohiko Igarashi, Satoshi Kaneko, Motomitsu Kitaoka, Masahiro Samejima

    Journal of Applied Glycoscience   67 ( 2 )   51 - 57   2020年5月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:The Japanese Society of Applied Glycoscience  

    DOI: 10.5458/jag.jag.jag-2020_0003

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  • Surface structural analysis of selectively <sup>13</sup>C-labeled cellulose II by solid-state NMR spectroscopy 査読

    Yusuke Kita, Ryosuke Kusumi, Tsunehisa Kimura, Motomitsu Kitaoka, Yusuke Nishiyama, Masahisa Wada

    Cellulose   27 ( 4 )   1899 - 1907   2020年3月

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    掲載種別:研究論文(学術雑誌)  

    © 2019, Springer Nature B.V. The local structure on the surface of plate-like cellulose II oligomer crystals (CIIOCs) was investigated by NMR spectroscopy. CIIOCs with reducing ends that were selectively labeled with 13C were enzymatically synthesized using cellodextrin phosphorylase. The solid-state 13C cross polarization/magic angle spinning spectra of the 13C-labeled CIIOCs clearly exhibited two resonance peaks (labeled as C1Rα and C1Rβ) derived from the C1 atoms of the reducing ends. The 2D 13C double quantum/13C single quantum homonuclear correlation spectrum indicated that two magnetically nonequivalent glucopyranose rings (Rα and Rβ units) coexisted at the reducing end units. The 1H/13C heteronuclear correlation spectrum suggested that there was a large difference between the local environments around the anomeric carbons of Rα and Rβ units. The abundance ratio of C1Rβ to C1Rα in the solid state was 4:1, whereas that of C1α and C1β in solution was 1:1. When the oligomer chains are packed in the cellulose II crystal, the reducing end units located on the surface of the plate-like crystal may tend to have β-anomeric structure, which would be more sterically stable than the α-anomeric structure.

    DOI: 10.1007/s10570-019-02896-x

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  • Varied Pathways of Infant Gut-Associated <i>Bifidobacterium</i> to Assimilate Human Milk Oligosaccharides: Prevalence of the Gene Set and Its Correlation with Bifidobacteria-Rich Microbiota Formation. 査読

    Sakanaka M, Gotoh A, Yoshida K, Odamaki T, Koguchi H, Xiao JZ, Kitaoka M, Katayama T

    Nutrients   12 ( 1 )   71 - 71   2019年12月

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    掲載種別:研究論文(学術雑誌)  

    DOI: 10.3390/nu12010071

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  • Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis. 査読 国際誌

    Mikiyasu Sakanaka, Morten Ejby Hansen, Aina Gotoh, Toshihiko Katoh, Keisuke Yoshida, Toshitaka Odamaki, Hiroyuki Yachi, Yuta Sugiyama, Shin Kurihara, Junko Hirose, Tadasu Urashima, Jin-Zhong Xiao, Motomitsu Kitaoka, Satoru Fukiya, Atsushi Yokota, Leila Lo Leggio, Maher Abou Hachem, Takane Katayama

    Science advances   5 ( 8 )   eaaw7696   2019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)  

    The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2'-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.

    DOI: 10.1126/sciadv.aaw7696

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  • Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum 査読

    Young Woo Nam, Mamoru Nishimoto, Takatoshi Arakawa, Motomitsu Kitaoka, Shinya Fushinobu

    Scientific reports   9 ( 1 )   11081   2019年7月

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    掲載種別:研究論文(学術雑誌)  

    Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD+/UDP, NAD+/UDP-GlcNAc, and NAD+/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.

    DOI: 10.1038/s41598-019-47591-w

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  • Epidermerization and Decomposition of Kojibiose and Sophorose by Heat Treatment under Neutral pH Conditions 査読

    Chiku Kazuhiro, Wada M, Atsuji Haruka, Hosonuma Arisa, Yoshida Mitsuru, Ono Hiroshi, Kitaoka Motomitsu

    JOURNAL OF APPLIED GLYCOSCIENCE   66 ( 1 )   1 - 9   2019年

  • Identification, functional characterization, and crystal structure determination of bacterial levoglucosan dehydrogenase. 査読

    Sugiura M, Nakahara M, Yamada C, Arakawa T, Kitaoka M, Fushinobu S

    The Journal of biological chemistry   293 ( 45 )   17375 - 17386   2018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)  

    DOI: 10.1074/jbc.RA118.004963

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  • Sharing of human milk oligosaccharides degradants within bifidobacterial communities in faecal cultures supplemented with Bifidobacterium bifidum. 査読 国際誌

    Aina Gotoh, Toshihiko Katoh, Mikiyasu Sakanaka, Yiwei Ling, Chihaya Yamada, Sadaki Asakuma, Tadasu Urashima, Yusuke Tomabechi, Ayako Katayama-Ikegami, Shin Kurihara, Kenji Yamamoto, Gaku Harata, Fang He, Junko Hirose, Motomitsu Kitaoka, Shujiro Okuda, Takane Katayama

    Scientific reports   8 ( 1 )   13958 - 13958   2018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)  

    Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.

    DOI: 10.1038/s41598-018-32080-3

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  • Expression and Characterization of Recombinant Sucrose Phosphorylase 査読

    Hui Zhang, Xiao Sun, Wenjie Li, Tuoping Li, Suhong Li, Motomitsu Kitaoka

    Protein Journal   37 ( 1 )   93 - 100   2018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 New York LLC  

    SPase is widely used in the food, cosmetics, and pharmaceutical industries. Previously, a SPase gene was cloned from Bifidobacterium longum JCM1217 and constructed into Escherichia coli BL21. In this paper, its expression conditions were optimized. The results showed that several induction factors determined the expression efficiency of SPase. The initial cell density, IPTG concentration, and induction time and temperature significantly (p OpenSPiltSPi 0.01) affected the total protein content and activity of expressed SPase. The highest expression efficiency was obtained at an initial cell density of OD600 = 0.5, with 0.05 mM IPTG, followed by shaking at 180 rpm and incubation at 30 °C for 15 h. The purified SPase had a specific activity of 122.1 U/mg, which was raised by 1.85 -fold more than that before optimization, and its recovery yield was 86%. Furthermore, SPase also showed higher thermostability. The results of this study provide essential information for the industrial production of SPase.

    DOI: 10.1007/s10930-017-9754-6

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  • Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42. 査読

    Viborg AH, Katayama T, Arakawa T, Abou Hachem M, Lo Leggio L, Kitaoka M, Svensson B, Fushinobu S

    The Journal of biological chemistry   292 ( 51 )   21092 - 21101   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)  

    DOI: 10.1074/jbc.M117.792598

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  • Molecular Insight into Evolution of Symbiosis between Breast-Fed Infants and a Member of the Human Gut Microbiome Bifidobacterium longum 査読

    Chihaya Yamada, Aina Gotoh, Mikiyasu Sakanaka, Mitchell Hattie, Keith A. Stubbs, Ayako Katayama-Ikegami, Junko Hirose, Shin Kurihara, Takatoshi Arakawa, Motomitsu Kitaoka, Shujiro Okuda, Takane Katayama, Shinya Fushinobu

    CELL CHEMICAL BIOLOGY   24 ( 4 )   515 - +   2017年4月

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    Breast-fed infants generally have a bifidobacteriarich microbiota with recent studies indicating that human milk oligosaccharides (HMOs) selectively promote bifidobacterial growth. Bifidobacterium bifidum possesses a glycoside hydrolase family 20 lacto-N-biosidase for liberating lacto-N-biose I from lacto-N-tetraose, an abundant HMO unique to human milk, while Bifidobacterium longum subsp. longum has a non-classified enzyme (LnbX). Here, we determined the crystal structure of the catalytic domain of LnbX and provide evidence for creation of a novel glycoside hydrolase family, GH136. The structure, in combination with inhibition and mutation studies, provides insight into the molecular mechanism and broader substrate specificity of this enzyme. Moreover, through genetic studies, we show that lnbX is indispensable for B. longum growth on lacto-N-tetraose and is a key genetic factor for persistence in the gut of breast-fed infants. Overall, this study reveals possible evolutionary routes for the emergence of symbiosis between humans and bifidobacterial species in the infant gut.

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  • Synthesis of 3-Keto-levoglucosan Using Pyranose Oxidase and Its Spontaneous Decomposition via beta-Elimination 査読

    Kitaoka Motomitsu

    JOURNAL OF APPLIED GLYCOSCIENCE   64 ( 4 )   99 - 107   2017年

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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  

    <p>3-Keto-levoglucosan (3ketoLG) has been postulated to be the product of a reaction catalyzed by levoglucosan dehydrogenase (LGDH), a bacterial enzyme involved in the metabolism of levoglucosan (LG). To investigate the LG metabolic pathway catalyzed by LGDH, 3ketoLG is needed. However, 3ketoLG has not been successfully isolated from the LGDH reaction. This study investigated the ability of pyranose oxidase to convert LG into 3ketoLG by oxidizing the C3 hydroxyl group. During the oxidation of LG, 3ketoLG was spontaneously crystallized in the reaction mixture. Starting with 500 mM LG, the isolation yield of 3ketoLG was 80 %. Nuclear magnetic resonance analyses revealed that a part of 3ketoLG dimerized in aqueous solution, explaining its poor solubility. Even under normal conditions, 3ketoLG was unstable in aqueous solution, with a half-life of 16 h at pH 7.0 and 30 °C. The decomposition proceeded through β-elimination of the C–O bonds at both C1 and C5, as evidenced by decomposition products. This instability explains the difficulty in obtaining 3ketoLG via the LGDH reaction.</p>

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  • Enzymatic Synthesis of 1,5-Anhydro-4-O-beta-D-glucopyranosyl-D-fructose Using Cellobiose Phosphorylase and Its Spontaneous Decomposition via beta-Elimination 査読

    Takahito Kajiki, Kazuhiro Yoshinaga, Shiro Komba, Motomitsu Kitaoka

    JOURNAL OF APPLIED GLYCOSCIENCE   64 ( 4 )   91 - 97   2017年

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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:JAPANESE SOC APPLIED GLYCOSCIENCE  

    Cellobiose phosphorylase from Cellvibrio gilvus was used to prepare 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-fructose [beta Glc(1 -> 4)AF] from 1,5-anhydro-D-fructose and alpha-D-glucose 1-phosphate. beta Glc(1 -> 4)AF decomposed into D-glucose and ascopyrone T via beta-elimination. Higher pH and temperature caused faster decomposition. However, decomposition proceeded significantly even under mild conditions. For instance, the half-life of beta Glc(1 -> 4)AF was 17 h at 30 degrees C and pH 7.0. Because beta Glc(1 -> 4)AF is a mimic of cellulose, in which the C2 hydroxyl group is oxidized, such decomposition may occur in oxidized cellulose in nature. Here we propose a possible oxidizing pathway by which this occurs.

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  • Galacto-N-biose is neuroprotective against glutamate-induced excitotoxicity in vitro 査読

    Yo Shinoda, Yui Nakajima, Hirotoshi Iguchi, Satoshi Tatsumi, Motomitsu Kitaoka, Masahiro Nakajima, Tsutomu Takahashi, Yasuyuki Fujiwara, Teiichi Furuichi

    EUROPEAN JOURNAL OF PHARMACOLOGY   791   711 - 717   2016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:ELSEVIER SCIENCE BV  

    Galacto-N-biose (GNB: Gal beta 1-3GalNAc) is an O-glycan disaccharide core moiety that is a core component of mucin in the gastrointestinal tract; however, the physiological properties of GNB are not well understood. Glutamate excitotoxicity causes neuronal death in acute neurological disorders including stroke, trauma, and neurodegenerative disease. Therefore the discovery of drugs to treat glutamate excitotoxicity is an important goal. Here, we report that GNB is neuroprotective against glutamate-induced excitotoxicity. We treated 14-15 days in vitro cultured rat cortical neurons with 0.1-1000 nM GNB together with 30 mu m glutamate for various durations. Short-term (3 h) GNB treatments showed a modest neuroprotective effect against glutamate neurotoxicity, however, long-term (24 h) GNB treatment conferred significant neuroprotective effects, as shown by both MTT and immunocytochemical assays. Prolonged GNB treatment did not alter glutamate-induced calcium influx, but did induce antioxidant-related gene expression. Furthermore, GNB treatment did not induce cell death or alter synaptic connections. These data suggest that GNB is a potential candidate drug that protects against glutamate excitotoxicity without affecting cell viability and synaptic connections.

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  • Introduction of H-antigens into oligosaccharides and sugar chains of glycoproteins using highly efficient 1,2-α-L-fucosynthase 査読

    Sugiyama Y, Gotoh A, Katoh T, Honda Y, Yoshida E, Kurihara S, Ashida H, Kumagai H, Yamamoto K, Kitaoka M, Katayama T

    Glycobiology   26 ( 11 )   1235 - 1247   2016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)  

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  • A glycosynthase derived from an inverting chitinase with an extended binding cleft 査読

    Takayuki Ohnuma, Satoshi Dozen, Yuji Honda, Motomitsu Kitaoka, Tamo Fukamizo

    JOURNAL OF BIOCHEMISTRY   160 ( 2 )   93 - 100   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:OXFORD UNIV PRESS  

    We created a glycosynthase from a GH19 chitinase from rye seeds (RSC-c), that has a long-extended binding cleft consisting of eight subsites; -4, -3, -2, -1, + 1, + 2, + 3 and + 4. When wild-type RSC-c was incubated with alpha-(G1cNAc)(3)-F [alpha-(GleNAc)(3) fluoride], (G1cNAc)(3) and hydrogen fluoride were produced through the Hehre resynthesis-hydrolysis mechanism. Glu89, which acts as a catalytic base, and Ser120, which fixes a nucleophilic water molecule, were mutated to produce two single mutants, E89G and S120A, and a double mutant, E89G/S120A. E89G only produced a small amount of (G1cNAc)(7) from alpha-(GlcNAc)(3)-F in the presence of (G1cNAc)(4). S120A, with the highest F- -releasing activity, produced a larger amount of (G1cNAc)(7), a fraction of which was decomposed by its own residual hydrolytic activity. However, the double mutant E89G/S120A, of which the hydrolytic activity was completely abolished while its F--releasing activity was only moderately affected, produced the largest amount of (G1eNAc)(7) from alpha-(GleNAc)(3)-F and (G1cNAc)(4) without decomposition. We concluded that E89G/S120A was an efficient glycosynthase, that enabled the addition of a three-sugar unit.

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  • Glycoside hydrolases 査読

    Motomitsu Kitaoka

    Handbook of Carbohydrate-Modifying Biocatalysts   2   215 - 235   2016年7月

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Pan Stanford Publishing Pte. Ltd.  

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  • Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies 査読

    Yuxin Ye, Wataru Saburi, Rei Odaka, Koji Kato, Naofumi Sakurai, Keisuke Komoda, Mamoru Nishimoto, Motomitsu Kitaoka, Haruhide Mori, Min Yao

    FEBS LETTERS   590 ( 6 )   828 - 837   2016年3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  

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

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  • Functional and structural analysis of a β-glucosidase involved in β-1,2-glucan metabolism in Listeria innocua 査読

    Masahiro Nakajima, Ryuta Yoshida, Akimasa Miyanaga, Koichi Abe, Yuta Takahashi, Naohisa Sugimoto, Hiroyuki Toyoizumi, Hiroyuki Nakai, Motomitsu Kitaoka, Hayao Taguchi

    PLoS ONE   11 ( 2 )   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)  

    © 2016 Nakajima et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.

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  • The crystal structure of an inverting glycoside hydrolase family 9 exo-β-D-glucosaminidase and the design of glycosynthase 査読

    Yuji Honda, Sachiko Arai, Kentaro Suzuki, Motomitsu Kitaoka, Shinya Fushinobu

    Biochemical Journal   473 ( 4 )   463 - 472   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)  

    © 2016 Authors; published by Portland Press Limited.Exo-β-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9.We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. Asp139, Asp143 and Glu555 in the active site were located near the β-O1 hydroxy group of GlcN (D-glucosamine), with Asp139 and Asp143 holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp139, we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (&lt;0.1%), and the F--release activity also decreased (&lt;3%). Therefore the glycosynthase activity of D139Ewas lower than that of glycosynthases created previously from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs.However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp139 might inevitably reduce F.-release activity.

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

    Tomohiro Tsuda, Takanori Nihira, Kazuhiro Chiku, Kazuhiro Chiku, Erika Suzuki, Takatoshi Arakawa, Mamoru Nishimoto, Motomitsu Kitaoka, Hiroyuki Nakai, Shinya Fushinobu

    FEBS Letters   589 ( 24 )   3816 - 3821   2015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)  

    © 2015 Federation of European Biochemical Societies.Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for β-mannosides. Here, we characterized β-1,2-mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with β-1,2-linked mannooligosaccharides. β-1,2-Mannotriose was bound in a U-shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open-close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided.

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

    Takanori Nihira, Kazuhiro Chiku, Erika Suzuki, Mamoru Nishimoto, Shinya Fushinobu, Motomitsu Kitaoka, Ken'ichi Ohtsubo, Hiroyuki Nakai

    FEBS Letters   589 ( 23 )   3604 - 3610   2015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)  

    © 2015 Federation of European Biochemical Societies.The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of β-d-mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer-3176 from Dyadobacter fermentans showed no synthetic activity using α-d-mannose 1-phosphate but it released α-d-mannose from β-1,2-mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer-3176 is a β-1,2-mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of β-1,2-mannotriose. The two residues are not conserved among GH130 phosphorylases and are predicted to assist the nucleophilic attack of a water molecule in the hydrolysis of the β-d-mannosidic bond.

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  • Diversity of phosphorylases in glycoside hydrolase families

    Motomitsu Kitaoka

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   99 ( 20 )   8377 - 8390   2015年10月

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    記述言語:英語   出版者・発行元:SPRINGER  

    Phosphorylases are useful catalysts for the practical preparation of various sugars. The number of known specificities was 13 in 2002 and is now 30. The drastic increase in available genome sequences has facilitated the discovery of novel activities. Most of these novel phosphorylase activities have been identified through the investigations of glycoside hydrolase families containing known phosphorylases. Here, the diversity of phosphorylases in each family is described in detail.

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

    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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  • Functional reassignment of Cellvibrio vulgaris EpiA to cellobiose 2-epimerase and an evaluation of the biochemical functions of the 4-O-β-D-mannosyl-D-glucose phosphorylase-like protein, UnkA 査読

    Wataru Saburi, Yuka Tanaka, Hirohiko Muto, Sota Inoue, Rei Odaka, Mamoru Nishimoto, Motomitsu Kitaoka, Haruhide Mori

    Bioscience, Biotechnology and Biochemistry   79 ( 6 )   969 - 977   2015年6月

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    © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry.The aerobic soil bacterium Cellvibrio vulgaris has a β-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimer-ase for converting D-mannose to D-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-β-D-mannosyl-D-glu-cose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and β-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for β-(1→4)-mannobiose was 5.5 × 10&lt;sup&gt;4&lt;/sup&gt;-fold higher than it was for D-mannose. Recombinant UnkA phosphorolyzed β-D-mannosyl-(1→4)-D-glucose and specifically utilized D-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-β-D-mannosyl-D-glucose phosphorylase.

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  • Novel substrate specificities of two lacto-N-biosidases towards β-linked galacto-N-biose-containing oligosaccharides of globo H, Gb5, and GA1 査読

    Aina Gotoh, Toshihiko Katoh, Yuta Sugiyama, Shin Kurihara, Yuji Honda, Haruko Sakurama, Taiho Kambe, Hisashi Ashida, Motomitsu Kitaoka, Kenji Yamamoto, Takane Katayama, Takane Katayama

    Carbohydrate Research   408   18 - 24   2015年5月

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    © 2015 Elsevier Ltd.We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galβ1-3GalNAc) and 2′-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of β-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids.

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  • Open-close structural change upon ligand binding and two magnesium ions required for the catalysis of N-acetylhexosamine 1-kinase

    Mayo Sato, Takatoshi Arakawa, Young-Woo Nam, Mamoru Nishimoto, Motomitsu Kitaoka, Shinya Fushinobu

    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS   1854 ( 5 )   333 - 340   2015年5月

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    Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-beta 1,3-G1cNAc) and galacto-N-biose (Gal-beta 1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric Cl position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16 degrees. In the nucleotide binding site, two magnesium ions bridging the alpha-gamma and beta-gamma phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism. (C) 2015 Elsevier B.V. All rights reserved.

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

    Koichi Abe, Masahiro Nakajima, Motomitsu Kitaoka, Hiroyuki Toyoizumi, Yuta Takahashi, Naohisa Sugimoto, Hiroyuki Nakai, Hayao Taguchi

    Journal of Applied Glycoscience   62 ( 2 )   47 - 52   2015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)   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.

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  • Facile enzymatic synthesis of sugar 1-phosphates as substrates for phosphorylases using anomeric kinases

    Yuan Liu, Mamoru Nishimoto, Motomitsu Kitaoka

    CARBOHYDRATE RESEARCH   401   1 - 4   2015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 SCI LTD  

    Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. alpha-D-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. alpha-D-Mannose 1-phosphate and 2-acetamido-2-deoxy-alpha-D-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts. (C) 2014 Elsevier Ltd. All rights reserved.

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  • A β1-6/β1-3 galactosidase from Bifidobacterium animalis subsp. lactisBl-04 gives insight into sub-specificities of β-galactoside catabolism within Bifidobacterium

    Alexander Holm Viborg, Folmer Fredslund, Takane Katayama, Stinne Kirketerp Nielsen, Birte Svensson, Motomitsu Kitaoka, Leila Lo Leggio, Maher Abou Hachem

    Molecular Microbiology   94 ( 5 )   1024 - 1040   2014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)  

    © 2014 John Wiley &amp; Sons Ltd.The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host-derived β-galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) β-galactosidase from the probiotic Bifidobacterium animalis subsp. lactisBl-04 (BlGal42A). BlGal42A displays a preference for undecorated β1-6 and β1-3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N-acetyl substituted β1-3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite +1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite -1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting β1-6 and β1-3 galactosides likely reflects structural differences between these substrates and β1-4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub-specificities that support metabolic specialization in the gut niche.

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

    Kazuhiro Chiku, Takanori Nihira, Erika Suzuki, Mamoru Nishimoto, Motomitsu Kitaoka, Ken'ichi Ohtsubo, Hiroyuki Nakai

    PloS one   9 ( 12 )   2014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)  

    We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

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

    Takanori Nihira, Futaba Miyajima, Kazuhiro Chiku, Mamoru Nishimoto, Motomitsu Kitaoka, Ken’ichi Ohtsubo, Hiroyuki Nakai

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

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

    Kouki K. Touhara, Takanori Nihira, Motomitsu Kitaoka, Hiroyuki Nakai, Shinya Fushinobu

    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)  

    2-O-α-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-α-glucosylglycerol (GG) and the hydrolysis of β-D-glucose 1-phosphate (βGlc1P). GGP belongs to the glycoside hydrolase (GH) family 65 and can efficiently and specifically produce GG. However, its structural basis has remained unclear. In this study, the crystal structures of GGP complexed with glucose and the glucose analog isofagomine and glycerol were determined. Subsite - 1 of GGP is similar to those of other GH65 enzymes, maltose phosphorylase and kojibiose phosphorylase, whereas subsite +1 is largely different and is well designed for GG recognition. An automated docking analysis was performed to complement these crystal structures, βGlc1P being docked at an appropriate position. To investigate the importance of residues at subsite +1 in the bifunctionality of GGP, we constructed mutants at these residues. Y327F and K587A did not show detectable activities for either reverse phosphorolysis or βGlc1P hydrolysis. Y572F also showed significantly reduced activities for both of these reactions. In contrast, W381F showed significantly reduced reverse phosphorolytic activity but retained/3Glc1P hydrolysis. The mode of substrate recognition and the reaction mechanisms of GGP were proposed based on these analyses. Specifically, an extensive hydrogen bond network formed by Tyr-327, Tyr-572, Lys-587, and water molecules contributes to fixing the acceptor molecule in both reverse phosphorolysis (glycerol) and βGlc1P hydrolysis (water) for a glycosyl transfer reaction. This study will contribute to the development of a large scale production system of GG by facilitating the rational engineering of GGP. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Takanori Nihira, Mamoru Nishimoto, Hiroyuki Nakai, Ken’ichi Ohtsubo, Motomitsu Kitaoka

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

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  • Facile preparation of highly crystalline lamellae of (1→3)-β-d-glucan using an extract of euglena gracilis

    Yu Ogawa, Yu Ogawa, Kazuhiro Noda, Satoshi Kimura, Satoshi Kimura, Motomitsu Kitaoka, Masahisa Wada, Masahisa Wada

    International Journal of Biological Macromolecules   64   415 - 419   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)  

    In vitro synthesis of (1. →. 3)-β-. d-glucan was performed using laminaribiose phosphorylase obtained by an extraction of Euglena gracilis with sucrose phosphorylase. The synthetic product was a linear (1. →. 3)-β-. d-glucan with a narrow distribution of degree of polymerization (DP) centered on DP. = 30. X-ray diffraction and electron microscopy revealed that the glucan molecules obtained were self-organized as highly crystalline hexagonal lamellae. This synthetic product has quite high structural homogeneity at every level from primary to higher-order structure, which is a great advantage for the detailed analyses of physiological functions of (1. →. 3)-β-. d-glucan. © 2013 Elsevier B.V.

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  • 1,2-β-oligoglucan phosphorylase from Listeria innocua

    Masahiro Nakajima, Masahiro Nakajima, Hiroyuki Toyoizumi, Koichi Abe, Hiroyuki Nakai, Hayao Taguchi, Motomitsu Kitaoka

    PLoS ONE   9 ( 3 )   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)  

    We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-D-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein. © 2014 Nakajima et al.

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  • Distinct substrate specificities of three glycoside hydrolase family 42 β-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697

    Alexander H. Viborg, Alexander H. Viborg, Takane Katayama, Maher Abou Hachem, Mathias C F Andersen, Mamoru Nishimoto, Mads H. Clausen, Tadasu Urashima, Birte Svensson, Motomitsu Kitaoka

    Glycobiology   24 ( 2 )   208 - 216   2014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)  

    Glycoside hydrolase family 42 (GH42) includes β-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different β-d-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of β-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite -1 to be key specificity determinants. Bga42A thus prefers the β1-3-galactosidic linkage from human milk and other β1-3- and β1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Galβ1-4Galβ1-4Glc) as well as 4-galactobiose (Galβ1-4Gal) and 4-galactotriose (Galβ1-4Galβ1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan- derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the β-galactoside-rich gut niche and emphasize the importance and diversity of β-galactoside metabolism in probiotic bifidobacteria. © The Author 2013.

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  • Characterization of a thermophilic 4-O-β-D-mannosyl-D-glucose phosphorylase from Rhodothermus marinus

    Nongluck Jaito, Wataru Saburi, Rei Odaka, Yusuke Kido, Ken Hamura, Mamoru Nishimoto, Motomitsu Kitaoka, Hirokazu Matsui, Haruhide Mori

    Bioscience, Biotechnology and Biochemistry   78 ( 2 )   263 - 270   2014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)  

    © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry.4-O-β-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-β-D-mannosyl-D-glucose (Man-Glc) to α-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts β-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-β-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75°C, and it was stable between pH 5.5-8.3 and below 80°C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-D-glucose and D-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl β-D-glucoside and 1,5-anhydro-D-glucitol as acceptor substrates.

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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

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

    PLoS ONE   9 ( 1 )   2014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)  

    The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel-2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s-1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-D-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel-2816 possessed hydrolytic activity on βGlc1P with a kcat of 2.8 s-1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H218O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-D-glucosylglycerol (GG) at the rate of 180 s-1. Bsel-2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s-1. We propose 2-O-α-D- glucopyranosylglycerol: phosphate β-D-glucosyltransferase as the systematic name and 2-O-α-D-glucosylglycerol phosphorylase as the short name for Bsel-2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates. © 2014 Nihira et al.

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  • Error-prone rolling circle amplification greatly simplifies random mutagenesis

    Ryota Fujii, Motomitsu Kitaoka, Kiyoshi Hayashi

    Methods in Molecular Biology   1179   23 - 29   2014年

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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:Humana Press Inc.  

    We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl&lt
    inf&gt
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  • Random insertional-deletional strand exchange mutagenesis (RAISE): A simple method for generating random insertion and deletion mutations

    Ryota Fujii, Motomitsu Kitaoka, Kiyoshi Hayashi

    Methods in Molecular Biology   1179   151 - 158   2014年

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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:Humana Press Inc.  

    Although proteins can be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult. Here we describe a simple method called random insertional-deletional strand exchange mutagenesis (RAISE). This method is based on gene shuffling and can be used to introduce a wide variety of insertions, deletions, and substitutions. RAISE involves three steps: DNA fragmentation, attachment of a random short sequence, and reconstruction. This yields unique mutants and can be a powerful technique for protein engineering. © 2014 Springer Science+Business Media New York.

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  • Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi

    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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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    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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  • Directed evolution to enhance thermostability of galacto-N-biose/lacto-N-biose I phosphorylase

    Yoshiyuki Koyama, Masafumi Hidaka, Mamoru Nishimoto, Motomitsu Kitaoka

    PROTEIN ENGINEERING DESIGN & SELECTION   26 ( 11 )   755 - 761   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:OXFORD UNIV PRESS  

    Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-N-biose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 20C higher thermostability than the wild type.

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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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    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-beta-D-glucopyranosyl-D-gluconic acid: phosphate alpha-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 alpha-D-glucose 1-phosphate and D-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively. (C) 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

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

    Takanori Nihira, Erika Suzuki, Motomitsu Kitaoka, Mamoru Nishimoto, Ken'ichi Ohtsubo, Hiroyuki Nakai

    Journal of Biological Chemistry   288 ( 38 )   27366 - 27374   2013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)  

    A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-D-mannosyl-N-acetyl-Dglucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-D-mannosyl-N-acetyl-D-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-D-mannose 1-phosphate and N-acetyl-D-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the D-mannose residue of β-1,4-D-mannosyl-Nacetyl-D-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-D-mannopyranosyl-N-acetyl-Dglucosamine: phosphate α-D-mannosyltransferase as the systematic name and β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase as the short name for BT1033. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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  • Structure of a bacterial glycoside hydrolase family 63 enzyme in complex with its glycosynthase product, and insights into the substrate specificity

    Takatsugu Miyazaki, Megumi Ichikawa, Gaku Yokoi, Motomitsu Kitaoka, Haruhide Mori, Yoshikazu Kitano, Atsushi Nishikawa, Takashi Tonozuka

    FEBS JOURNAL   280 ( 18 )   4560 - 4571   2013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  

    Proteins belonging to glycoside hydrolase family63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing -glucosidaseI, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichiacoli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with -d-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O--d-glucopyranosyl--d-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK.
    DatabaseThe coordinates and structure factors for E727A-Glc12Gal and D324N-melibiose have been deposited in the Protein Data Bank under accession numbers 3W7W and 3W7X, respectively

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  • Lacto-N-biosidase Encoded by a Novel Gene of Bifidobacterium longum Subspecies longum Shows Unique Substrate Specificity and Requires a Designated Chaperone for Its Active Expression

    Haruko Sakurama, Masashi Kiyohara, Jun Wada, Yuji Honda, Masanori Yamaguchi, Satoru Fukiya, Atsushi Yokota, Hisashi Ashida, Hidehiko Kumagai, Motomitsu Kitaoka, Kenji Yamamoto, Takane Katayama

    JOURNAL OF BIOLOGICAL CHEMISTRY   288 ( 35 )   25194 - 25206   2013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:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N- tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Gal beta 1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAc beta 1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), and sialyllacto-N-tetraose a (Neu5Ac alpha 2-3Gal beta 1-3GlcNAc beta 1-3-Gal beta 1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-beta-lacto-N-bioside I (Gal beta 1-3-GlcNAc beta-pNP) and GalNAc beta 1-3GlcNAc beta-pNP.GalNAc beta 1-3-GlcNAc beta linkage has been found in O-mannosyl glycans of alpha-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

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  • Characterization of the cytosolic β-N-acetylglucosaminidase from Bifidobacterium longum subsp. longum 査読

    Yuji Honda, Mamoru Nishimoto, Takane Katayama, Motomitsu Kitaoka

    Journal of Applied Glycoscience   60 ( 3 )   141 - 146   2013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:The Japanese Society of Applied Glycoscience  

    The BLLJ_1391 protein from <i>Bifidobacterium longum</i> subsp. <i>longum</i> JCM1217, a cytosolic β-<i>N</i>-acetylglucosaminidase belonging to glycoside hydrolase family (GH) 20, hydrolyzed lacto-<i>N</i>-triose II (LNTri) as well as chitin oligosaccharides. Its reaction was found to follow a substrate-assisted mechanism with anomeric retention, which is common for GH 20 enzymes. Homologous enzymes are found in genomic sequences of <i>B. longum</i> subsp. <i>infantis</i>, <i>Bifidobacterium bifidum</i>, and <i>Bifidobacteium breve</i>, all of which are infant gut-associated species of <i>Bifidobacterium</i>. The distribution resembles that of 1,3-β-galactosyl-<i>N</i>-acetylhexosamine phosphorylase, suggesting that the enzyme plays a role in metabolism of human milk oligosaccharides by hydrolyzing LNTri generated via the cytosolic hydrolysis of lacto-<i>N</i>-tetraose (LNT) by LNT 1,3-β-galactosidase.

    DOI: 10.5458/jag.jag.JAG-2013_001

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

    Takanori Nihira, Erika Suzuki, Motomitsu Kitaoka, Mamoru Nishimoto, Ken’ichi Ohtsubo, Hiroyuki Nakai

    Journal of Applied Glycoscience   60 ( 2 )   137 - 139   2013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)   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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  • Recent development of phosphorylases possessing large potential for oligosaccharide synthesis

    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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    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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  • In vitro comparative evaluation of the impact of lacto-N-biose I, a major building block of human milk oligosaccharides, on the fecal microbiota of infants

    Takumi Satoh, Toshitaka Odamaki, Mariko Namura, Takashi Shimizu, Keiji Iwatsuki, Mamoru Nishimoto, Motomitsu Kitaoka, Jin-zhong Xiao

    ANAEROBE   19   50 - 57   2013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:ELSEVIER SCI LTD  

    Lacto-N-biose I (LNB) is a potential factor for the selective growth of bifidobacteria. We previously reported that the species of bifidobacteria predominant in infant intestines might use LNB. We aimed to assess the prebiotic properties of LNB in comparison to other oligosaccharides using an in vitro fermentation system. Stool samples from formula-fed infants were inoculated with media containing a sole carbon source of 1% LNB, lactulose, raffinose, galactooligosaccharide, or mannanoligosaccharides. LNB significantly increased the total bifidobacterial population similarly to other oligosaccharides, but induced a significantly higher level of Bifidobacterium bifidum in comparison to other oligosaccharides. Furthermore, significantly lower concentrations of lactic acid and significantly higher concentrations of acetic acid were produced in cultures containing LNB in comparison to cultures that contained other oligosaccharides. In conclusion, LNB might have a beneficial effect on the fecal microbiota of infants and is a potential prebiotic for application in infant foods or supplements. (C) 2013 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.anaerobe.2012.12.007

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

    Takanori Nihira, Yuka Saito, Motomitsu Kitaoka, Mamoru Nishimoto, Ken'Ichi Otsubo, Hiroyuki Nakai

    Carbohydrate Research   361   49 - 54   2012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)  

    We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-β-d-glucobiose) phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing α-d-glucose 1-phosphate and d-glucose. Laminaritriose (1,3-β-d- glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-β-d-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from α-d-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (d-glucose, 2-deoxy-d-arabino-hexopyranose, d-xylose, d-glucuronic acid, 1,5-anhydro-d-glucitol, and d-mannose) with C-3 and C-4 equatorial hydroxyl groups. The d-glucose and 2-deoxy-d-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction. © 2012 Elsevier Ltd. All rights reserved.

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

    Takanori Nihira, Yuka Saito, Motomitsu Kitaoka, Ken'Ichi Otsubo, Hiroyuki Nakai

    Carbohydrate Research   360   25 - 30   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)  

    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 flexibility 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. These results suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of d-glucose. © 2012 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.carres.2012.07.014

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  • A glycosynthase derived from an inverting GH19 chitinase from the moss Bryum coronatum

    Takayuki Ohnuma, Tatsuya Fukuda, Satoshi Dozen, Yuji Honda, Motomitsu Kitaoka, Tanno Fukamizo

    BIOCHEMICAL JOURNAL   444   437 - 443   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:PORTLAND PRESS LTD  

    BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (G1cNAc)(n) [a beta-1,4-linked polysaccharide of G1cNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with alpha-(G1cNAc)(2)-F [alpha-(G1cNAc)(2) fluoride] in the absence or presence of (G1cNAc)(2), (G1cNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Seri(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, 5102D, S102G, S102H, S102T, E7OG and E70Q. all of the mutated enzymes, except S102T, hydrolytic activity towards (G1cNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S1020G and E70G were found to successfully synthesize (G1cNAc)4 as a major product from alpha-(G1eNAc)(2)-F in the presence of (G1cNAc)(2). The SIO2A mutant showed the greatest glycosynthase activity owing to its enhanced F-releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.

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  • 1,3-1,4-α-L-Fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 chains

    Haruko Sakurama, Shinya Fushinobu, Masafumi Hidaka, Erina Yoshida, Yuji Honda, Hisashi Ashida, Motomitsu Kitaoka, Hidehiko Kumagai, Kenji Yamamoto, Takane Katayama

    Journal of Biological Chemistry   287 ( 20 )   16709 - 16719   2012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)  

    α-L-Fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially α-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an α-L-fucosynthase that specifically introduces Lea and Lex antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Galβ1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an α-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-α-L-fucosidase (EC 3.2.1.111) and α-L-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Lea/x epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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  • Bifidobacterial Enzymes Involved in the Metabolism of Human Milk Oligosaccharides

    Motomitsu Kitaoka

    ADVANCES IN NUTRITION   3 ( 3 )   422S - 429S   2012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)   出版者・発行元:AMER SOC NUTRITION-ASN  

    Intestinal colonization of bifidobacteria is important for the health of infants. Human milk oligosaccharides (HMO) have been identified as growth factors for bifidobacteria. Recently, a bifidobacterial enzymatic system to metabolize HMO was identified. 1,3-beta-Galactosyl-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which catalyzes the reversible phosphorolysis of galacto-N-biose (GNB) (Gal beta 1,-&gt; 3GalNAc)] and lacto-N-biose I (LNB) (Gal beta 1 -&gt; 3GlcNAc), is a key enzyme to explain the metabolism of HMO. Infant-type bifidobacteria possess the intracellular pathway to specifically metabolize GNB and LNB (GNB/LNB pathway). Bifidobacterium bifidum possesses extracellular enzymes to liberate [NB from HMO. However, Bindobactenum longum subsp. infantis imports intact HMO to be hydrolyzed by intracellular enzymes. Bifidobacterial enzymes related to the metabolism of HMO are useful tools for preparing compounds related to HMO. For instance, LNB and GNB were produced from sucrose and GlcNAc/GalNAc in 1 pot using 4 bifidobacterial enzymes, including GLNBP. [NB is expected to be a selective bifidus factor for infant-type strains. Adv. Nutr. 3: 422S-429S, 2012.

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  • In vitro effect of lacto-N-biose I on the antigen-specific immune responses of naïve splenocytes 査読

    Masao Goto, Yuko Takano-Ishikawa, Mamoru Nishimoto, Motomitsu Kitaoka

    Bioscience of Microbiota, Food and Health   31 ( 2 )   47 - 50   2012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)  

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  • 3-O-α-d-glucopyranosyl-L-rhamnose phosphorylase from Clostridium phytofermentans

    Takanori Nihira, Takanori Nihira, Hiroyuki Nakai, Hiroyuki Nakai, Hiroyuki Nakai, Motomitsu Kitaoka

    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)  

    We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 produced in Escherichia coli did not phosphorolyze α-linked glucobioses, such as trehalose (α1-α1), kojibiose (α1-2), nigerose (α1-3), and maltose (α1-4), which are typical substrates for GH65 enzymes. In reverse phosphorolysis, Cphy1019 utilized only l-rhamnose as the acceptor among various sugars examined with β-d-glucose 1-phosphate as the donor. The reaction product was determined to be 3-O-α-d-glucopyranosyl-l-rhamnose, indicating strict α1-3 regioselectivity. We propose 3-O-α-d-glucopyranosyl-l-rhamnose: phosphate β-d-glucosyltransferase as the systematic name and 3-O-α-d- glucopyranosyl-l-rhamnose phosphorylase as the short name for this novel GH65 phosphorylase. © 2011 Elsevier Ltd. All rights reserved.

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  • Bifidobacterium longum subsp infantis uses two different beta-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides

    Erina Yoshida, Haruko Sakurama, Masashi Kiyohara, Masahiro Nakajima, Motomitsu Kitaoka, Hisashi Ashida, Junko Hirose, Takane Katayama, Kenji Yamamoto, Hidehiko Kumagai

    GLYCOBIOLOGY   22 ( 3 )   361 - 368   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)   出版者・発行元:OXFORD UNIV PRESS INC  

    The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized beta-galactosidases of this subspecies to understand how the organism degrades type-1 (Gal beta 1-3GlcNAc) and type-2 (Gal beta 1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel beta-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) than for lacto-N-biose I (Gal beta 1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT beta-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a beta-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different beta-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of beta-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans.

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  • Characterization of a Bacterial Laminaribiose Phosphorylase

    Motomitsu Kitaoka, Yasuyuki Matsuoka, Kiyotaka Mori, Mamoru Nishimoto, Kiyoshi Hayashi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   76 ( 2 )   343 - 348   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:TAYLOR & FRANCIS LTD  

    Bacterial laminaribiose phosphorylase (LBPbac) was first identified and purified from cell-free extract of Paenibacillus sp. YM-1. It phosphorolyzed laminaribiose into a-glucose 1-phosphate and glucose, but did not phosphorolyze other glucobioses. It slightly phosphorolyzed laminaritriose and higher laminarioligosaccharides. The specificity of the degree of polymerization of the substrate was clearly different from that of the enzyme of Euglena gracilis (LBPEug): LBPbac was more specific to laminaribiose than LBPEug. It showed acceptor specificity in reverse phosphorolysis similar to LBPEug. Cloning of the gene encoding LBPbac (IbpA) has revealed that LBPbac is a member of the glucoside hydrolase family 94, which includes cellobiose phosphorylase, cellodextrin phosphorylase, and N,N'-diacetylchitobiose phosphorylase. The genes that encode the components of an ATP-binding cassette sugar transporter specific to laminarioligosaccharides were identified upstream of IbpA, suggesting that the role of LBPbac, is to utilize laminaribiose generated outside the cell. This role is different from that of LBPEug, which participates in the utilization of paramylon, the intracellular storage 1,3-beta-glucan.

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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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  • Identification of amino acid residues that determine the substrate preference of 1,3-β-galactosyl-N-acetylhexosamine phosphorylase

    Mamoru Nishimoto, Masafumi Hidaka, Masafumi Hidaka, Masahiro Nakajima, Masahiro Nakajima, Shinya Fushinobu, Motomitsu Kitaoka

    Journal of Molecular Catalysis B: Enzymatic   74 ( 1-2 )   97 - 102   2012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)  

    Three amino acid residues of 1,3-β-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) were assigned as the determinants of substrate preference for galacto-N-biose (GNB) and lacto-N-biose I (LNB) based on the three-dimensional structure of the protein. Mutants of GalHexNAcP from Bifidobacterium longum, which acts similarly on both GNB and LNB, were constructed and characterized. V162T mutation led to an increase in the selectivity on GNB. P161S and S336A mutations independently enhanced the selectivity on LNB. The alignment of amino acid sequences suggests that the activities of most homologous sequences are predictable by comparing the corresponding three residues. .© 2011 Elsevier B.V. All rights reserved.

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  • α-N-acetylgalactosaminidase from infant-associated bifidobacteria belonging to novel glycoside hydrolase family 129 is implicated in alternative mucin degradation pathway

    Masashi Kiyohara, Masashi Kiyohara, Takashi Nakatomi, Shin Kurihara, Shinya Fushinobu, Hideyuki Suzuki, Tomonari Tanaka, Shin Ichiro Shoda, Motomitsu Kitaoka, Takane Katayama, Kenji Yamamoto, Kenji Yamamoto, Hisashi Ashida

    Journal of Biological Chemistry   287 ( 1 )   693 - 700   2012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)  

    Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria.Wepreviously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo- α-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Galβ1- 3GalNAcα1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core- 3-type O-glycans (GlcNAcβ1- 3GalNAcα1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel α-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAcα1-Ser/Thr). Sialyl and galactosyl core-3 (Galβ1-3/ 4GlcNAcβ1-3(Neu5Acα2-6)GalNAcα1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as α-sialidase (SiaBb2), lacto-N-biosidase (LnbB), β-galactosidase (BbgIII), and β-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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  • Glycoside Hydrolases

    Motomitsu Kitaoka

    Carbohydrate-Modifying Biocatalysts   183 - 203   2011年11月

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Pan Stanford Publishing Pte. Ltd.  

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  • One-pot enzymatic production of 2-acetamido-2-deoxy-D-galactose (GalNAc) from 2-acetamido-2-deoxy-D-glucose (GlcNAc)

    Kousuke Inoue, Mamoru Nishimoto, Motomitsu Kitaoka

    CARBOHYDRATE RESEARCH   346 ( 15 )   2432 - 2436   2011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:ELSEVIER SCI LTD  

    2-Acetamido-2-deoxy-D-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-D-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin. (C) 2011 Elsevier Ltd. All rights reserved.

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  • Self-transferring product inhibition observed during the hydrolysis of aryl-β-glucopyranosides by a β-glucosidase from Agrobacterium tumefaciens 査読

    Motomitsu Kitaoka, Tomoya Takahashi, Ying Li, Kiyoshi Hayashi

    Journal of Applied Glycoscience   58 ( 4 )   129 - 132   2011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:The Japanese Society of Applied Glycoscience  

    The time profile of an enzymatic reaction at an early stage is generally considered to be linear. We observed that the time profile of the hydrolysis of 1 mM <i>p</i>-nitrophenyl-β-D-glucoside (pNP-Glc) by a β-glucosidase obtained from <i>Agrobacterium tumefaciens</i> (Cbg1) was not linear before 5% of the substrate was consumed, even though the initial concentration of the substrate was much higher than its <i>K</i><small>m</small> value. The time profiles obtained with higher concentrations of pNP-Glc suggested that the time profile was the function of the absolute concentration of <i>p</i>-nitrophenol (pNP). The addition of various alcohols made the time profile linear. A self-transferring product inhibition model was constructed in which the pNP generated during the hydrolysis acts as an acceptor substrate to inhibit the hydrolysis. The theoretical curve agreed well with the experimental data.

    DOI: 10.5458/jag.jag.JAG-2011_003

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  • Physiology of Consumption of Human Milk Oligosaccharides by Infant Gut-associated Bifidobacteria

    Sadaki Asakuma, Emi Hatakeyama, Tadasu Urashima, Erina Yoshida, Takane Katayama, Kenji Yamamoto, Hidehiko Kumagai, Hisashi Ashida, Junko Hirose, Motomitsu Kitaoka

    JOURNAL OF BIOLOGICAL CHEMISTRY   286 ( 40 )   34583 - 34592   2011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:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono-and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

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  • p-Nitrophenyl β-glycosides of β-1,4-gluco/xylo-disaccharides for the characterization of submits in endo-xylanases. 査読

    Mamoru Nishimoto, Atsushi Kobayashi, Yuji Honda, Motomitsu Kitaoka, Kiyoshi Hayashi

    Journal of Applied Glycoscience   58   115 - 118   2011年9月

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  • Interactions between glycoside hydrolase family 94 cellobiose phosphorylase and glucosidase inhibitors 査読

    Shinya Fushinobu, Masafumi Hidaka, Andressa M. Hayashi, Takayoshi Wakagi, Hirofumi Shoun, Motomitsu Kitaoka

    Journal of Applied Glycoscience   58 ( 3 )   107 - 114   2011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)  

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  • An enzymatic colorimetric quantification of orthophosphate 査読

    Bingxue Li, Takanori Nihira, Hiroyuki Nakai, Mamoru Nishimoto, Motomitsu Kitaoka

    Journal of Applied Glycoscience   58 ( 3 )   125 - 127   2011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: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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  • Mutational analysis of fungal family 11 xylanases on the pH optimum determination 査読

    Shinya Fushinobu, Takeo Uno, Motomitsu Kitaoka, Kiyoshi Hayashi, Hiroshi Matsuzawa, Takayoshi Wakagi

    Journal of Applied Glycoscience   58 ( 3 )   107 - 114   2011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)  

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  • Lactose and Oligosaccharides: Indigenous Oligosaccharides in Milk 査読

    T. Urashima, S. Asakuma, M. Kitaoka, M. Messer

    Encyclopedia of Dairy Sciences: Second Edition   241 - 273   2011年1月

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Elsevier Inc.  

    Mammalian milk/colostrum contains from trace to ∼13% of carbohydrate, of which lactose (Gal(β1-4)Glc) usually constitutes more than 80%. Milk/colostrum of most mammals also contains a variety of oligosaccharides, many of which have N-acetylglucosamine, galactose, fucose, and/or sialic acid residues attached to lactose, which is always located at the reducing end. The ratio of milk oligosaccharides to free lactose in milk/colostrum varies, depending on the mammalian species. Most human milk oligosaccharides (HMOs) cannot be digested within the small intestine, and reach the colon where they stimulate the growth of bifidobacteria and act as possible antiadhesion factors against pathogenic microorganisms in the human infant. The biological functions of milk oligosaccharides have recently been an active field of research. In this article, we describe the chemical structures and methods for the structural analysis of milk oligosaccharides, quantitative aspects and methods for their quantification, their gastrointestinal digestion and biosynthesis, and their biological significance as prebiotics, anti-infection factors, receptor analogues, and immunomodulators, with special reference to the oligosaccharides of human milk. We also describe the milk oligosaccharides of domestic farm animals and discuss comparative aspects of milk oligosaccharides among mammalian species, as well as future trends and the industrial utilization of milk oligosaccharides.

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  • Milk oligosaccharides

    Tadasu Urashima, Motomitsu Kitaoka, Takashi Terabayashi, Kenji Fukuda, Masao Ohnishi, Akira Kobata

    Oligosaccharides: Sources, Properties and Applications   1 - 58   2011年

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  • Crystallography of enzymes in the unique sugar metabolism of Bifidobacteria

    Masafumi Hidaka, Ryuichiro Suzuki, Takane Katayama, Motomitsu Kitaoka, Takayoshi Wakagi, Hirofumi Shoun, Hisashi Ashida, Kenji Yamamoto, Shinya Fushinobu

    Photon Factory Activity Report 2009   27 ( B )   250 - 250   2010年12月

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  • Cooperation of β-galactosidase and β-N-acetylhexosaminidase from bifidobacteria in assimilation of human milk oligosaccharides with type 2 structure

    Mika Miwa, Tomohiro Horimoto, Masashi Kiyohara, Masashi Kiyohara, Takane Katayama, Motomitsu Kitaoka, Hisashi Ashida, Kenji Yamamoto

    Glycobiology   20 ( 11 )   1402 - 1409   2010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)  

    Bifidobacteria are predominant in the intestines of breast-fed infants and offer health benefits to the host. Human milk oligosaccharides (HMOs) are considered to be one of the most important growth factors for intestinal bifidobacteria. HMOs contain two major structures of core tetrasaccharide: lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc; type 1 chain) and lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc; type 2 chain). We previously identified the unique metabolic pathway for lacto-N-tetraose in Bifidobacterium bifidum. Here, we clarified the degradation pathway for lacto-N-neotetraose in the same bifidobacteria. We cloned one β-galactosidase (BbgIII) and two β-N-acetylhexosaminidases (BbhI and BbhII), all of which are extracellular membrane-bound enzymes. The recombinant BbgIII hydrolyzed lacto-N-neotetraose into Gal and lacto-N-triose II, and furthermore the recombinant BbhI, but not BbhII, catalyzed the hydrolysis of lacto-N-triose II to GlcNAc and lactose. Since BbgIII and BbhI were highly specific for lacto-N-neotetraose and lacto-N-triose II, respectively, they may play essential roles in degrading the type 2 oligosaccharides in HMOs. © The Author 2010. Published by Oxford University Press. All rights reserved.

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  • Crystal structure of an exo-1,5-α-L-arabinofuranosidase from Streptomyces avermitilis provides insights into the mechanism of substrate discrimination between exo- and endo-type enzymes in glycoside hydrolase family 43

    Zui Fujimoto, Hitomi Ichinose, Tomoko Maehara, Mariko Honda, Motomitsu Kitaoka, Satoshi Kaneko

    Journal of Biological Chemistry   285 ( 44 )   34134 - 34143   2010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)  

    Exo-1,5-α-L-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-L- arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-L-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-L-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite -1, formed by the flexible loop region Tyr-281-Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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  • Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus

    Erina Yoshida, Masafumi Hidaka, Masafumi Hidaka, Shinya Fushinobu, Takashi Koyanagi, Hiromichi Minami, Hisanori Tamaki, Motomitsu Kitaoka, Takane Katayama, Hidehiko Kumagai

    Biochemical Journal   431   39 - 49   2010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)  

    β-Glucosidase from Kluyveromyces marxianus (KmBglI) belongs to the GH3 (glycoside hydrolase family 3). The enzyme is particularly unusual in that a PA14 domain (pf07691), for which a carbohydrate-binding role has been claimed, is inserted into the catalytic core sequence. In the present study, we determined the enzymatic properties and crystal structure of KmBglI in complex with glucose at a 2.55 Å (1 Å=0.1 nm) resolution. A striking characteristic of KmBglI was that the enzyme activity is essentially limited to disaccharides, and when trisaccharides were used as the substrates the activity was drastically decreased. This chain-length specificity is in sharp contrast with the preferred action on oligosaccharides of barley β-D-glucan glucohydrolase (ExoI), which does not have a PA14 domain insertion. The structure of subsite (-1) of KmBglI is almost identical with that of Thermotoga neapolitana β-glucosidase and is also similar to that of ExoI, however, the structures of subsite (+1) significantly differ among them. In KmBglI, the loops extending from the PA14 domain cover the catalytic pocket to form subsite (+1), and hence simultaneously become a steric hindrance that could limit the chain length of the substrates to be accommodated. Mutational studies demonstrated the critical role of the loop regions in determining the substrate specificity. The active-site formation mediated by the PA14 domain of KmBglI invokes α-complementation of β-galactosidase exerted by its N-terminal domain, to which the PA14 domain shows structural resemblance. The present study is the first which reveals the structural basis of the interaction between the PA14 domain and a carbohydrate. © The Authors.

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  • Thermal decomposition of β-d-galactopyranosyl-(1→3)-2-acetamido- 2-deoxy-d-hexopyranoses under neutral conditions

    Kazuhiro Chiku, Mamoru Nishimoto, Motomitsu Kitaoka

    Carbohydrate Research   345 ( 13 )   1901 - 1908   2010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)  

    β-d-Galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-glucose (LNB) and β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-galactose (GNB) decompose rapidly upon heating into d-galactose and mono-dehydrated derivatives of the corresponding 2-acetamido-2-deoxy-d-hexoses, including 2-acetamido-2,3-dideoxy-hex-2-enofuranoses and bicyclic 2-acetamido-3,6-anhydro- 2-deoxy-hexofuranoses. The decomposition is conducted under neutral conditions where glycosyl linkages are generally believed to be stable. The half-lives of LNB and GNB were 8.1 min and 20 min, respectively, at 90 °C and pH 7.5. The pH dependency of decomposition rates suggests that the instabilities are an extension of the conditions for the peeling reaction, often observed with glycans of O-linked glycoproteins under alkaline conditions. Such decomposition under the neutral conditions is commonly observed with 3-O-linked reducing aldoses. © 2010 Elsevier Ltd. All rights reserved.

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  • Practical preparation of D-galactosyl-β1!4-l-rhamnose employing the combined action of Phosphorylases

    Masahiro Nakajima, Mamoru Nishimoto, Motomitsu Kitaoka

    Bioscience, Biotechnology and Biochemistry   74 ( 8 )   1652 - 1655   2010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)  

    D-Galactosyl-β1!4-L-rhamnose (GalRha) was produced enzymatically from 1.1M sucrose and 1.0M L-rhamnose by the concomitant actions of four enzymes (sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, and D-galactosyl-β1!4-L-rhamnose phosphorylase) in the presence of 1.0mM UDP-glucose and 30mM inorganic phosphate. The accumulation of GalRha in 1 liter of the reaction mixture reached 230 g (the reaction yield was 71% from L-rhamnose). Sucrose and fructose in the reaction mixture were removed by yeast treatment, but isolation of GalRha by crystallization after yeast treatment was unsuccessful. Finally, 49 g of GalRha was isolated from part of the reaction mixture with yeast treatment by gel-filtration chromatography.

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  • Glycosynthases from Inverting Hydrolases

    Motomitsu Kitaoka

    Biocatalysis and Biomolecular Engineering   361 - 376   2010年7月

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:John Wiley and Sons  

    DOI: 10.1002/9780470608524.ch23

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  • Improving Enzyme Character by Molecular Breeding: Preparation of Chimeric Genes

    Kiyoshi Hayashi, Motomitsu Kitaoka, Mamoru Nishimoto

    Biocatalysis and Biomolecular Engineering   31 - 42   2010年7月

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:John Wiley and Sons  

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  • Characterization of d-galactosyl-β1→4-l-rhamnose phosphorylase from Opitutus terrae

    Masahiro Nakajima, Mamoru Nishimoto, Motomitsu Kitaoka

    Enzyme and Microbial Technology   46 ( 3-4 )   315 - 319   2010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)  

    We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed d-galactosyl-β1→4-l-rhamnose (GalRha) and also showed phosphorolytic activity on d-galactosyl-β1→3-d-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on l-rhamnose derivatives than on d-glucose derivatives. The enzyme was stable up to 45 °C and at pH 6.0-7.0. The values of kcat and Km of the phosphorolytic activity of the enzyme on GalRha were 60 s-1 and 2.1 mM, respectively. Thus, Oter_1377 protein was identified as d-galactosyl-β1→4-l-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms. © 2009 Elsevier Inc. All rights reserved.

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  • Effect of Growth Temperature, Induction, and Molecular Chaperones on the Solubilization of Over-expressed Cellobiose Phosphorylase from Cellvibrio Gilvus under in vivo Conditions

    S. P. Singh, M. K. Purohit, C. Aoyagi, M. Kitaoka, K. Hayashi

    BIOTECHNOLOGY AND BIOPROCESS ENGINEERING   15 ( 2 )   273 - 276   2010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)   出版者・発行元:KOREAN SOC BIOTECHNOLOGY & BIOENGINEERING  

    In vivo folding of many proteins can be facilitated by growth temperature, extent of induction, and molecular chaperones, which prevent over-expressed protein from being trapped into insoluble inclusion bodies. In the present report, we describe the role of molecular chaperones and growth temperature on the solubilization of over-expressed Cellobiose Phosphorylase (CBP) in Escherichia coli. The growth of host at low temperature enhanced enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yielded higher enzyme in soluble fraction. The synergistic effect of low temperature and induction on the prevention of inclusion bodies was also evident from our results. In addition, co-expression of the target gene with two types of molecular chaperones (GroESL and KODHsp) was also attempted. However, none of these chaperones enhanced the solubilization under in vivo conditions. Nevertheless, effective role of low growth temperature coupled with low level of induction appeared to be an attractive feature for producing recombinant protein.

    DOI: 10.1007/s12257-009-0023-1

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  • Structural explanation for the acquisition of glycosynthase activity

    Masafumi Hidaka, Shinya Fushinobu, Yuji Honda, Takayoshi Wakagi, Hirofumi Shoun, Motomitsu Kitaoka

    JOURNAL OF BIOCHEMISTRY   147 ( 2 )   237 - 244   2010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:OXFORD UNIV PRESS  

    Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0-3.0 A from the NW in the wild type. Their positions exhibited a strong correlation with the strength of glycosynthase activity. Here, we propose that a structural prerequisite for the sufficient glycosynthase reaction is the presence of a water molecule at the NW position, and mutation at the NW holder provides a general strategy for inverting GHs. The idea on the position of a water molecule may also be applicable to the design of efficient glycosynthases from retaining GHs.

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  • Analyses of Bifidobacterial Glycosidases Involved in the Metabolism of Oligosaccharides

    YAMAMOTO Kenji, KATAYAMA Takane, KITAOKA Motomitsu, FUSHINOBU Shinya

    Bioscience and microflora   29 ( 1 )   23 - 30   2010年1月

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    記述言語:英語   出版者・発行元:JAPAN BIFIDUS FOUNDATION  

    Many bifidobacteria produce an endo-α- <i>N</i>-acetylgalactosaminidase that liberates the <i>O</i>-linked galactosyl β-1,3<i>N-</i>acetylgalactosamine (GNB) from intestinal mucin glycoproteins. The molecular cloning of the <i>Bifidobacterium</i> <i>longum</i> enzyme was completed using information in public databases. The enzyme constitutes a novel glycoside hydrolase (GH) family 101 member. The gene encoding a specific 1,2-α -L-fucosidase was cloned from <i>B.bifidum</i>. The recombinant enzyme specifically hydrolyzes the terminal α-1,2-fucosidic linkages of various oligosaccharides, including human milk oligosaccharides and blood group substances. Analysis of its primary structure revealed that this enzyme constitutes a novel GH family 95 member. We also solved the crystal structure of its catalytic domain. We assumed that these bifidobacterial enzymes are involved in the metabolism of oligosaccharides in mucin glycoproteins that are abundant in the intestine. Some bifidobacteria strains produce a <b>lacto-N-</b>biosidase that releases galactosyl β-1,3<i>N</i>-acetylglucosamine (LNB) from human milk oligosaccharides, but the other enteric bacteria do not. This disaccharide is one of the building blocks in human milk oligosaccharides and is rarely found in other mammalian milks. The <b>lacto-N-</b>biosidase gene was cloned from <i>B.bifidum</i> and we hypothesized that this enzyme is crucially involved in the degradation of human milk oligosaccharides. The genes encoding sialidase and α-1,3/4-L-fucosidase were also cloned from <i>B.bifidum.</i> These enzymes release modified sialic acid and L-fucose from human milk oligosaccharides, respectively. A solute-binding protein of a putative ABC transporter specific for GNB and LNB was also discovered, and its gene was cloned from <i>B.longum</i>. We named it GNB/LNB-binding protein and crystallized it. Isothermal titration calorimetry measurements revealed that this protein specifically binds GNB and LNB. We speculate that bifidobacteria have a novel GNB/LNB metabolic pathway.<br>

    DOI: 10.12938/bifidus.29.23

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  • Distribution of In Vitro Fermentation Ability of Lacto-N-Biose I, a Major Building Block of Human Milk Oligosaccharides, in Bifidobacterial Strains

    Jin-zhong Xiao, Sachiko Takahashi, Mamoru Nishimoto, Toshitaka Odamaki, Tomoko Yaeshima, Keiji Iwatsuki, Motomitsu Kitaoka

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   76 ( 1 )   54 - 59   2010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:AMER SOC MICROBIOLOGY  

    This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudo-catenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.

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  • 合目的変異導入によるラクトースホスホリラーゼの作出

    仁平 高則, 小川 徳之, 日高 将文, 伏信 進矢, 片山 高嶺, 北岡 本光

    Journal of Applied Glycoscience Supplement   2010 ( 0 )   81 - 81   2010年

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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  

    DOI: 10.11541/jsag.2010.0.81.0

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  • 還元末端に存在する1,3結合の中性条件下での熱分解

    知久 和寛, 西本 完, 北岡 本光

    Journal of Applied Glycoscience Supplement   2010   120 - 120   2010年

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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  

    DOI: 10.11541/jsag.2010.0.120.0

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  • 環状イソマルトオリゴ糖グルカノトランスフェラーゼの分子解析とBacillus circulansにおける環状イソマルトオリゴ糖の新規な生合成経路の発見

    舟根 和美, 川端 康之, 鈴木 龍一郎, 藤本 瑞, 北岡 本光, 木村 淳夫, 小林 幹彦

    Journal of Applied Glycoscience Supplement   2010   131 - 131   2010年

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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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  • One-pot enzymatic production of β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-galactose (galacto-N-biose) from sucrose and 2-acetamido-2-deoxy-d-galactose (N-acetylgalactosamine)

    Mamoru Nishimoto, Motomitsu Kitaoka

    Carbohydrate Research   344 ( 18 )   2573 - 2576   2009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)  

    β-d-Galactopyranosyl-(1→3)-2-acetamido-2-deoxy-d-galactose (galacto-N-biose, GNB) is an important core structure in functional sugar chains such as T-antigen disaccharide and the core 1 sugar chain in mucin glycoproteins. We successfully developed a one-pot enzymatic production of GNB from sucrose and GalNAc by the concomitant action of four enzymes: sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, and galacto-N-biose/lacto-N-biose I phosphorylase in the presence of UDP-glucose and phosphate, by modifying the method of lacto-N-biose I production [Nishimoto, M.; Kitaoka, M., Biosci. Biotechnol. Biochem., 2007, 71, 2101-2104]. The reaction yield of GNB was 88% from GalNAc. GNB was isolated from the reaction mixture by crystallization after yeast treatment to obtain approximately 45 g of GNB in 95% purity from a 280-mL reaction mixture. © 2009 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.carres.2009.09.031

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  • Synthesis of highly ordered cellulose II in vitro using cellodextrin phosphorylase

    Masao Hiraishi, Kiyohiko Igarashi, Satoshi Kimura, Masahisa Wada, Motomitsu Kitaoka, Masahiro Samejima

    CARBOHYDRATE RESEARCH   344 ( 18 )   2468 - 2473   2009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 SCI LTD  

    Synthesis of cellulose in vitro is expected to afford tailor-made cellulosic materials with highly homogeneous structure compared to natural cellulosic materials. Here we report the enzymatic synthesis of cellulose II with high crystallinity from glucose and alpha-glucose 1-phosphate (alpha G1P) by cellodextrin phosphorylase (CDP). Although glucose had been believed not to act as a glucosyl acceptor of CDP, a significant amount of insoluble cellulose was precipitated without accumulation of soluble cello oligosaccharides when glucose was mixed with alpha G1P and CDP. This phenomenon can be explained in terms of the large difference in acceptor reactivity between glucose and cello oligosaccharides. (1)H NMR spectrometric analysis revealed that this insoluble cellulose had an average degree of polymerization (DP) of nine. TEM observation, together with electron and X-ray diffraction studies, indicated that the insoluble cellulose formed platelet-shaped single lamellar crystals of cellulose II, several mu m in length and several hundred nm in width; this is large compared to reported cellulose crystals. The thickness of the lamellar crystal is 4.5 nm, which is equivalent to a chain length of a cello oligosaccharide with DP nine and is consistent with the (1)H NMR spectroscopic results. These results suggest that cello oligosaccharides having an average DP of nine are synthesized in vitro by CDP when glucose is used as an acceptor, and the product forms highly crystalline cellulose II when it precipitates. (C) 2009 Elsevier Ltd. All rights reserved.

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  • Purification, crystallization and preliminary X-ray analysis of β-glucosidase from Kluyveromyces marxianus NBRC1777

    Erina Yoshida, Masafumi Hidaka, Masafumi Hidaka, Shinya Fushinobu, Takashi Koyanagi, Hiromichi Minami, Hisanori Tamaki, Motomitsu Kitaoka, Takane Katayama, Hidehiko Kumagai

    Acta Crystallographica Section F: Structural Biology and Crystallization Communications   65   1190 - 1192   2009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)  

    The intracellular β-glucosidase from Kluyveromyces marxianus NBRC1777 (KmBglI) belongs to glycoside hydrolase family 3 and has a unique domain architecture. Selenomethionine-labelled KmBglI was purified and crystallized by the hanging-drop vapour-diffusion method using the purified enzyme at 30 mg ml-1, 0.04 M potassium dihydrogen phosphate pH 5.1, 16%(w/v) PEG 8000 and 20%(v/v) glycerol. The crystal belonged to space group C2, with unit-cell parameters a = 245.8, b = 148.7, c = 119.9 Å, β = 112.9°. Multiple-wavelength anomalous dispersion data were collected at 2.4 and 2.5 Å resolution. A tetramer was assumed to be present in the asymmetric unit, which gave a Matthews coefficient of 2.6 Å3 Da-1. © 2009 International Union of Crystallography. All rights reserved.

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  • Bifidobacterium bifidum Lacto-N-Biosidase, a Critical Enzyme for the Degradation of Human Milk Oligosaccharides with a Type 1 Structure (vol 74, pg 3996, 2009) 査読

    Jun Wada, Takuro Ando, Masashi Kiyohara, Hisashi Ashida, Motomitsu Kitaoka, Masanori Yamaguchi, Hidehiko Kumagai, Takane Katayama, Kenji Yamamoto

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   75 ( 19 )   6414 - 6414   2009年10月

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    記述言語:英語   出版者・発行元:AMER SOC MICROBIOLOGY  

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  • Synthesis of cellobiose from starch by the successive actions of two phosphorylases

    Masayuki Suzuki, Kyoko Kaneda, Yukiko Nakai, Motomitsu Kitaoka, Hajime Taniguchi

    NEW BIOTECHNOLOGY   26 ( 3-4 )   137 - 142   2009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:ELSEVIER SCIENCE BV  

    Cellobiose was enzymatically synthesized from starch using two phosphorylases. Under the presence of 1 m Pi (inorganic phosphate), glucan phosphorylase converted 40% of glucose residues in the starch molecule into G1P (glucose-1-phosphate). By electrodialysis fitted with an ion exchange membrane having molecular weight cutoff of 100, Pi was effectively dialyzed out and GIP was recovered with 80% yield. G1P and glucose were incubated with cellobiose phosphorylase in the presence of magnesium acetate at an alkaline condition. Inorganic phosphate coformed with cellobiose was immediately removed as insoluble magnesium ammonium phosphate and 85% of added GIP was converted into cellobiose. On the whole, cellobiose was produced with 60% yield from G1P and, at least, 23.7% yield from starch.

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  • Substrate specificity of N-acetylhexosamine kinase towards N-acetylgalactosamine derivatives

    Li Cai, Wanyi Guan, Wenjun Wang, Wei Zhao, Motomitsu Kitaoka, Jie Shen, Crystal O&apos;Neil, Peng George Wang

    BIOORGANIC & MEDICINAL CHEMISTRY LETTERS   19 ( 18 )   5433 - 5435   2009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:PERGAMON-ELSEVIER SCIENCE LTD  

    We report herein a bacterial N-acetylhexosamine kinase, NahK, with broad substrate specificity towards structurally modified GalNAc analogues, and the production of a GalNAc-1-phosphate library using this kinase. (C) 2009 Published by Elsevier Ltd.

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  • Crystallographic and Mutational Analyses of Substrate Recognition of Endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum 査読

    Ryuichiro Suzuki, Takane Katayama, Motomitsu Kitaoka, Hidehiko Kumagai, Takayoshi Wakagi, Hirofumi Shoun, Hisashi Ashida, Kenji Yamamoto, Shinya Fushinobu

    JOURNAL OF BIOCHEMISTRY   146 ( 3 )   389 - 398   2009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:OXFORD UNIV PRESS  

    Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyses the O-glycosidic bonds in mucin-type O-glycan between alpha-GalNAc and Ser/Thr. Endo-alpha-GalNAc-ase from Bifidobacterium longum JCM1217 (EngBF) is highly specific for the core 1-type O-glycan to release the disaccharide Gal beta 1-3GalNAc (GNB), whereas endo-alpha-GalNAe-ase from Clostridium perfringens (EngCP) exhibits. broader substrate specificity. We determined the crystal structure of EngBF at 2.0 angstrom resolution and performed automated docking analysis to investigate possible binding modes of GNB. Mutational analysis revealed important residues for substrate binding, and two Trp residues (Trp748 and Trp750) appeared to form stacking interactions with the beta-faces of sugar rings of GNB by substrate-induced fit. The difference in substrate specificities between EngBF and EngCP is attributed to the variations in amino acid sequences in the regions forming the substrate-binding pocket. Our results provide a structural basis for substrate recognition by GH101 endo-alpha-GalNAe-ases and will help structure-based engineering of these enzymes to produce various kinds of neo-glycoconjugates.

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  • 2-Acetamido-2-de-oxy-3-O-Β-d-galactopyranosyl-d-glucose dihydrate

    Masahisa Wada, Kayoko Kobayashi, Mamoru Nishimoto, Motomitsu Kitaoka, Keiichi Noguchi

    Acta Crystallographica Section E: Structure Reports Online   65   O1781 - U2293   2009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)  

    In the title compound, C14H25NO11· 2H2O, the primary hydroxyl group connected to the anomeric C atom of the N-acetyl-Β-d-glucopyran-ose residue exhibits positional disorder, with occupancy factors for the and Β anomers of 0.77 and 0.23, respectively. The two torsion angles ( and ) and the bridge angle () that describe conformation of the glycosidic linkage between the galactopyran-ose and glucopyran-ose rings are = -81.6 (3)°, = 118.1 (2)° and = 115.2 (2)°. Two water mol-ecules stabilize the mol-ecular packing by forming hydrogen bonds with the saccharide residues. © 2009 Wada et al.

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  • Characterization of three β-galactoside phosphorylases from Clostridium phytofermetans. Discovery of D-galactosyl-β1→4-L-rhmanose phosphorylase

    Masahiro Nakajima, Mamoru Nishimoto, Motomitsu Kitaoka

    Journal of Biological Chemistry   284 ( 29 )   19220 - 19227   2009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)  

    We characterized three D-galactosyl-β1→3-N-acetyl-D-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; D-Gal- β13-D-GalNAc) and lacto-N-biose I (LNB; D-Gal-β→3-D-GlcNAc), thus indicating that they are D-galactosyl-β→3-N- acetyl-D-hexosamine phosphorylases, subclassified as GNB/LNB phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with L-rhamnose as the acceptor in the reverse reaction using α-D-galactose 1-phosphate as the donor. The reaction product was D-galactosyl-β1→4-L-rhamnose. The enzyme also showed activity on L-mannose, L-lyxose, D-glucose, 2-deoxy-D-glucose, and D-galactose in this order. When D-glucose derivatives were used as acceptors, reaction products were β-1,3-galactosides. Kinetic parameters of phosphorolytic activity on D-galactosyl- β1→4-L-rhamnose were kcat = 45 s-1 and Km = 7.9 mM, thus indicating that these values are common among other phosphorylases. We propose D-galactosyl-β1→4-L-rhamnose phosphorylase as the name for Cphy1920 protein. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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  • Characterization of β-1,3-galactosyl-N-acetylhexosamine phosphorylase from Propionibacterium acnes

    Masahiro Nakajima, Mamoru Nishimoto, Motomitsu Kitaoka

    Applied Microbiology and Biotechnology   83 ( 1 )   109 - 115   2009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)  

    Homologs of the β-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) gene (gnpA) were cloned from the genomic DNA of Propionibacterium acnes JCM6425 and P. acnes JCM6473, showing 99.9% and 97.9% nucleotide sequence identity, respectively, with the ppa0083 gene from the genome-sequenced P. acnes KPA171202. No gnpA gene was detected in the genomic DNA of type strain P. acnes ATCC25746. The recombinant enzyme from P. acnes JCM6425 (GnpA) showed approximately 70 times higher specific activity of phosphorolysis on galacto-N-biose (Galβ1→3GalNAc, GNB) than that on lacto-N-biose I (Galβ1→3GlcNAc). K m value for GnpA on GNB was high, but GnpA did not exhibit activity on any derivatives of GNB examined. These results indicate that GnpA is GalHexNAcP which should be classified as galacto-N-biose phosphorylase. The large k cat value of GnpA on GalNAc suggests that GnpA would be a useful catalyst for the synthesis of GNB. © 2009 Springer-Verlag.

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  • Prebiotic Effect of Lacto-N-biose I on Bifidobacterial Growth

    Masashi Kiyohara, Asaki Tachizawa, Mamoru Nishimoto, Motomitsu Kitaoka, Hisashi Ashida, Kenji Yamamoto

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   73 ( 5 )   1175 - 1179   2009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)   出版者・発行元:TAYLOR & FRANCIS LTD  

    We demonstrated the prebiotic effect of lacto-N-biose I (Gal beta 1-3GlcNAc) on bifidobacteria in vitro. Lacto-N-biose I, a building unit of the type-I milk oligosaccharides, enhanced the growth of many bifidobacteria, especially Bifidobacterium bifidum, B. breve, and B. longum, which are predominant in the intestines of breast-fed infants. It might be a substantial, natural prebiotic in human colostrums.

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  • アノマー反転型糖質加水分解酵素のグライコシンターゼ化

    本多 裕司, 伏信 進矢, 日高 將文, 若木 高善, 祥雲 弘文, 谷口 肇, 北岡 本光

    Journal of applied glycoscience   56 ( 2 )   119 - 124   2009年4月

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    出版者・発行元:日本応用糖質科学会  

    還元末端オリゴキシラナーゼ[EC 3.2.1.156](REX)はキシロオリゴ糖を還元末端側から加水分解するアノマー反転型酵素である。野生型REXにα-X2-FとX1を作用させると、いわゆる&quot;Hehre resynthesis-hydrolysis&quot;と呼ばれる反応を触媒した。REXをグライコシンターゼに変換するために、REXの触媒塩基であるD263にsaturation mutagenesisによって種々のアミノ酸に置換した変異型酵素を作製し、α-X2-FとX1を作用させてグライコシンターゼ反応を調べた。9種のD263変異型酵素がグライコシンターゼ反応産物であるX3を生成したが、これらの酵素の中でD263Cが良いグライコシンターゼであった。しかし、これらの変異型酵素は、X3が生成するとともに加水分解産物であるX2を生成した。REXの活性部位構造を詳細にみると、求核試薬として作用する水分子がD263およびY198と水素結合を形成していることが判明した。次にY198に変異を導入したY198FとD263C/Y198FおよびD263N/Y198Fを作製した。これらの変異型酵素によるX3加水分解活性は、野生型酵素と比較して大幅に減少した。またY198Fのα-X2-F消費能は野生型の1.5倍であったが、D263N/Y198Fは1/10以下であり、D263C/Y198Fは微弱な活性しか示さなかった。次にY198FとD263N/Y198Fによるグライコシンターゼ反応の経時変化を調べると、X3が効率良く蓄積され、分解産物であるX2がほとんど検出されなかった。さらに、両酵素によるα-X2-F消費能の比活性を比較すると、Y198FがD263N/Y198Fより約20倍程度高かったので、Y198Fが最も良いグライコシンターゼであると総合的に判断した。

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  • Crystal structure of glycoside hydrolase family 55-β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium

    Takuya Ishida, Shinya Fushinobu, Rie Kawai, Motomitsu Kitaoka, Kiyohiko Igarashi, Masahiro Samejima

    Journal of Biological Chemistry   284 ( 15 )   10100 - 10109   2009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)  

    Glycoside hydrolase family 55 consists of β-1,3-glucanases mainly from filamentous fungi. A β-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes β-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from β-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has two β-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two β-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various β-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the twoβ-helical domains, demonstrating an unexpected variety of carbohydrate binding modes. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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  • The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase A LARGE DEFORMATION OF A TIM BARREL SCAFFOLD

    Masafumi Hidaka, Mamoru Nishimoto, Motomitsu Kitaoka, Takayoshi Wakagi, Hirofumi Shoun, Shinya Fushinobu

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 11 )   7273 - 7283   2009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)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) from Bifidobacterium longum, a key enzyme for intestinal growth, phosphorolyses galacto-N-biose and lacto-N-biose I with anomeric inversion. GLNBP homologues are often found in human pathogenic and commensal bacteria, and their substrate specificities potentially define the nutritional acquisition ability of these microbes in their habitat. We report the crystal structures of GLNBP in five different ligand-binding forms. This is the first three-dimensional structure of glycoside hydrolase (GH) family 112. The GlcNAc-and GalNAc-bound forms provide structural insights into distinct substrate preferences of GLNBP and its homologues from pathogens. The catalytic domain consists of a partially broken TIM barrel fold that is structurally similar to a thermophilic beta-galactosidase, strongly supporting the current classification of GLNBP homologues as one of the GH families. Anion binding induces a large conformational change by rotating a half-unit of the barrel. This is an unusual example of molecular adaptation of a TIM barrel scaffold to substrates.

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  • Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy

    Motomitsu Kitaoka, Yuji Honda, Shinya Fushinobu, Masafumi Hidaka, Takane Katayama, Kenji Yamamoto

    TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY   21 ( 117 )   23 - 39   2009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:GAKUSHIN PUBL CO  

    Reducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156) is an inverting xylanolytic enzyme, belonging to the glycoside hydrolase (GH) family 8, which hydrolyzes xylooligosaccharides to release xylose (X-1) from its reducing end. Rex hydrolyzes alpha-xylobiosyl fluoride (alpha-X2F) to yield xylobiose (X-2) only in the presence of X-1, confirming the Hehre resynthesis-hydrolysis mechanism. A library of mutant Rex at the catalytic base (D263) was constructed by saturation mutagenesis, in which D263C accumulated the highest level of xylotriose (X-3) from alpha-X2F and X-1. However, F-releasing activities of the mutants were much less than that of the wild type. Next, Y198 residue of Rex that forms a hydrogen bond with nucleophilic water was substituted with phenylalanine, causing a marked decrease in hydrolytic activity and a small increase in the F- releasing activity from alpha-X2F in the presence of X-1. Y198F of Rex accumulated more product during the glycosynthase reaction than D263C. Recently, an inverting alpha-1,2-fucosidase belonging to GH95 was converted into glycosynthase by mutating a catalytic base residue. In both cases, the catalytic base should be intact.

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  • Conversion of an Inverting Glycoside Hydrolase into Glycosynthase 査読

    Honda Y, Fushinobu S, Hidaka M, Wakagi T, Shoun H, Taniguchi H, Kitaoka M

    Journal of Applied Glycoscience   291 ( 3 )   883 - 125   2009年

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    Honda, Y, Fushinobu, S, Hidaka, M, Wakagi, T, Shoun, H, Taniguchi, H &amp; Kitaoka, M, 2009, &#039;Conversion of an Inverting Glycoside Hydrolase into Glycosynthase&#039;, &lt;i&gt;Journal of Applied Glycoscience&lt;/i&gt;, vol. 291, no. 3, pp. 883-125.

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  • Milk oligosaccharides

    T. Urashima, M. Kitaoka, S. Asakuma, M. Messer

    Advanced Dairy Chemistry   3   295 - 349   2009年

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Springer New York  

    Mammalian milk contains up to 10% carbohydrate, of which the disaccharide, lactose (Gal(β1-4)Glc), is usually a prominent component. Milk and colostrum also contain lesser amounts of other saccharides, referred to as milk oligosaccharides, nearly all of which have a lactose unit at their reducing end to which GlcNAc, Gal, Fuc and/or Neu5Ac or Neu5Gc residues can be attached (Jenness et al., 1964
    Newburg and Neubauer, 1995
    Boehm and Stahl, 2003
    Urashima et al., 2001
    Messer and Urashima, 2002). Pronounced heterogeneity as well as homology of milk oligosaccharide structures among different mammalian species has been documented (Urashima et al., 2001
    Messer and Urashima, 2002). © 2009 Springer Science+Business Media, LLC.

    DOI: 10.1007/978-0-387-84865-5_8

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  • A chemoenzymatic route to N-acetylglucosamine-1-phosphate analogues: substrate specificity investigations of N-acetylhexosamine 1-kinase

    Li Cai, Wanyi Guan, Motomitsu Kitaoka, Jie Shen, Chengfeng Xia, Wenlan Chen, Peng George Wang

    CHEMICAL COMMUNICATIONS   ( 20 )   2944 - 2946   2009年

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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:ROYAL SOC CHEMISTRY  

    Reports an efficient chemoenzymatic production of an N-acetylhexosamine 1-phophate analogues library by N-acetylhexosamine 1-kinase (NahK) and describes the respective substrate specificity on this enzyme.

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  • 生育環境の異なるバクテリア由来のα-1,2-L-フコシダーゼの基質特異性の違い

    仁平 高則, 片山 高嶺, 芦田 久, 山本 憲二, 矢追 克郎, 三石 安, 北岡 本光

    Journal of Applied Glycoscience Supplement   2009   84 - 84   2009年

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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  

    DOI: 10.11541/jsag.2009.0.84.0

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  • ホスホリラーゼを活用したデンプンからの効率的なセロビオース合成法の開発

    鈴木 雅之, 北岡 本光, 谷口 肇

    Journal of Applied Glycoscience Supplement   2009   131 - 131   2009年

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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  

    DOI: 10.11541/jsag.2009.0.131.0

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  • ビフィズス菌由来エンド-α-<I>N</I>-アセチルガラクトサミニダーゼの構造機能解析

    鈴木 龍一郎, 片山 高嶺, 芦田 久, 山本 憲二, 北岡 本光, 熊谷 英彦, 若木 高善, 祥雲 弘文, 伏信 進矢

    Journal of Applied Glycoscience Supplement   2009   71 - 71   2009年

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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  

    DOI: 10.11541/jsag.2009.0.71.0

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  • ビフィズス菌由来ヒトミルクオリゴ糖加水分解酵素ラクト<I>N</I>ビオシダーゼのX線結晶構造解析

    伊藤 佑, 鈴木 龍一郎, 片山 高嶺, 若木 高善, 祥雲 弘文, 芦田 久, 山本 憲二, 北岡 本光, 伏信 進矢

    Journal of Applied Glycoscience Supplement   2009   72 - 72   2009年

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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  

    DOI: 10.11541/jsag.2009.0.72.0

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  • Bifidobacterium bifidum JCM1254由来GNB/LNBホスホリラーゼアイソザイムの遺伝子構造解析

    西本 完, 北岡 本光

    Journal of Applied Glycoscience Supplement   2009   53 - 53   2009年

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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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  • ホスホリラーゼを用いたβ-1,4-ガラクトシル-L-ラムノースの大量調製

    中島 将博, 西本 完, 北岡 本光

    Journal of Applied Glycoscience Supplement   2009   52 - 52   2009年

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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  

    DOI: 10.11541/jsag.2009.0.52.0

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  • Crystallographic analysis of novel sugar metabolic enzymes from Bifidobacteria

    Ryuichiro Suzuki, Jun Wada, Takane Katayama, Shinya Fushinobu, Takayoshi Wakagi, Hirofumi Shoun, Motomitsu Kitaoka, Hidehiko Kumagai, Hisashi Ashida, Kenji Yamamoto

    Photon Factory Activity Report 2007   25 ( B )   235 - 235   2008年12月

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    記述言語:英語  

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  • 1,2-α-l-Fucosynthase: A glycosynthase derived from an inverting α-glycosidase with an unusual reaction mechanism

    Jun Wada, Jun Wada, Yuji Honda, Masamichi Nagae, Masamichi Nagae, Ryuichi Kato, Soichi Wakatsuki, Takane Katayama, Hajime Taniguchi, Hidehiko Kumagai, Motomitsu Kitaoka, Kenji Yamamoto

    FEBS Letters   582 ( 27 )   3739 - 3743   2008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)  

    Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucα1,2Gal linkage by introducing glycosynthase technology into 1,2-α-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using β-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2′-fucosyllactose. 1,2-α-l-Fucosynthase is the first glycosynthase derived from an inverting α-glycosidase and from a glycosidase with an unusual reaction mechanism. © 2008 Federation of European Biochemical Societies.

    DOI: 10.1016/j.febslet.2008.09.054

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  • Diversity and Similarity of Microbial Communities in Petroleum Crude Oils Produced in Asia 査読

    Kunio Yamane, Hideaki Maki, Tsuyoshi Nakayama, Toshiaki Nakajima, Nobuhiko Nomura, Hiroo Uchiyama, Motomitsu Kitaoka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   72 ( 11 )   2831 - 2839   2008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:TAYLOR & FRANCIS LTD  

    To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.

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  • Enzymatic hydrolysis of 1,3-1,4-β-glucosyl oligosaccharides by 1,3-1,4-β-glucanase from Synechocystis PCC6803: A comparison with assays using polymer and chromophoric oligosaccharide substrates

    Tamo Fukamizo, Kanako Hayashi, Masahiro Tamoi, Yusuke Fujimura, Hideki Kurotaki, Anna Kulminskaya, Motomitsu Kitaoka

    Archives of Biochemistry and Biophysics   478 ( 2 )   187 - 194   2008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)  

    The specificity of 1,3-1,4-β-glucanase from Synechocystis PCC6803 (SsGlc) was investigated using novel substrates 1,3-1,4-β-glucosyl oligosaccharides, in which 1,3- and 1,4-linkages are located in various arrangements. After the enzymatic reaction, the reaction products were separated and determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As a result, SsGlc was found to hydrolyze the pentasaccharides, which possess three contiguous 1,4-β-glycosidic linkages (cellotetraose sequence) adjacent to 1,3-β-linkage, but none of the other oligosaccharides were hydrolyzed. To further analyze the specificity, kinetic measurements were performed using polymeric substrates and 4-methylumbelliferyl derivatives of laminaribiose and cellobiose (1,3-β-(Glc)2-MU and 1,4-β-(Glc)2-MU). The kcat/Km value obtained for barley β-glucan was considerably larger than that for lichenan, indicating that SsGlc prefers 1,3-1,4-β-glucan possessing a larger amount of cellotetraose sequence. This is consistent with the data obtained for 1,3-1,4-β-glucosyl oligosaccharides. However, the kcat/Km value obtained for 1,4-β-(Glc)2-MU was considerably lower than that for 1,3-β-(Glc)2-MU, suggesting inconsistency with the data obtained from the other natural substrates. It is likely that the kinetic data obtained from such chromophoric substrates do not always reflect the true enzymatic properties. © 2008 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.abb.2008.07.019

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  • Identification of lacto-N-biose I phosphorylase from Vibrio vulnificus CMCP6

    Masahiro Nakajima, Motomitsu Kitaoka

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   74 ( 20 )   6333 - 6337   2008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:AMER SOC MICROBIOLOGY  

    A beta-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalGlyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GlcNAc and GalNAc as acceptors in the presence of alpha-D-galactose-1-phosphate as a donor to form lacto-N-biose I (LNB) (Gal beta 1 -&gt; 3GlcNAc) and galacto-N-biose (GNB) (Gal beta 1 -&gt; 3GalNAc), respectively. GlcNAc was a much better acceptor than GalNAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the k(cat)/K(m) for LNB was approximately 60 times higher than the k(cat)/K(m) for GNB. This result indicated that the enzyme was remarkably different from GalGlyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GalGlyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated lacto-N-biose I phosphorylase. The discovery of an LNB-specific GalGlyNAcP resulted in recategorization of bifidobacterial GalGlyNAcPs as galacto-N-biose/lacto-N-biose I phosphorylases.

    DOI: 10.1128/AEM.02846-07

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  • Characterization of Bacillus halodurans α-galactosidase Mel4A encoded by the mel4A gene (BH2228)

    Andian Ari Anggraeni, Andian Ari Anggraeni, Makiko Sakka, Tetsuya Kimura, Khanok Ratanakhaokchai, Motomitsu Kitaoka, Kazuo Sakka

    Bioscience, Biotechnology and Biochemistry   72 ( 9 )   2459 - 2462   2008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)  

    A family-4 α-galactosidase Mel4A of Bacillus halodurans was expressed in Escherichia coli and characterized. Recombinant enzyme rMel4A depended on NAD +, some divalent cations such as Mn 2+, and reducing reagents such as dithiothreitol. rMel4A was active on small saccharides such as raffinose but not on highly polymerized galactomannan. Immunological analysis indicated that raffinose induced the production of Mel4A in B. halodurans.

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  • Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure

    Jun Wada, Takuro Ando, Masashi Kiyohara, Hisashi Ashida, Motomitsu Kitaoka, Masanori Yamaguchi, Hidehiko Kumagai, Takane Katayama, Kenji Yamamoto

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   74 ( 13 )   3996 - 4004   2008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 MICROBIOLOGY  

    Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Gal beta 1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Gal beta 1,3GlcNAc beta 1,3Gal beta 1,4G1c), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified P-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type I chain.

    DOI: 10.1128/AEM.00149-08

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  • Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase

    Shinya Fushinobu, Blake Mertz, Anthony D. Hill, Masafurni Hidaka, Motomitsu Kitaoka, Peter J. Reilly

    CARBOHYDRATE RESEARCH   343 ( 6 )   1023 - 1033   2008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)   出版者・発行元:ELSEVIER SCI LTD  

    GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate (G1P) and D-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites -1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. AUTODOCK was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy C-4(1) conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from S-1(3) as the pre-transition state conformer (Michaelis complex) via E-3 as the transition state candidate to C-4(1) as the GIP product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The S-1(3) pre-transition state conformer is highly stable compared with other conformers, and a conformational change from C-4(1) to B-1,B-4 was observed. (c) 2008 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.carres.2008.02.026

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  • Structural and thermodynamic analyses of solute-binding protein from Bifidobacterium longum specific for core 1 disaccharide and lacto-N-biose I

    Ryuichiro Suzuki, Jun Wada, Takane Katayama, Shinya Fushinobu, Takayoshi Wakagi, Hirofumi Shoun, Hayuki Sugimoto, Akiyoshi Tanaka, Hidehiko Kumagai, Hisashi Ashida, Motomitsu Kitaoka, Kenji Yamamoto

    JOURNAL OF BIOLOGICAL CHEMISTRY   283 ( 19 )   13165 - 13173   2008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)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Gal beta 1-3GlcNAc) and galacto-N-biose (GNB; Gal beta 1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K-d values of 0.087 and 0.010 mu M, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose ( Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.

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  • Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase

    Yuji Honda, Shinya Fushinobu, Masafumi Hidaka, Takayoshi Wakagi, Hirofumi Shoun, Hajime Taniguchi, Motomitsu Kitaoka

    GLYCOBIOLOGY   18 ( 4 )   325 - 330   2008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)   出版者・発行元:OXFORD UNIV PRESS INC  

    The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F-releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F-releasing activity. As a result of the high F-releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

    DOI: 10.1093/glycob/cwn011

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  • A reducing-end-acting chitinase from Vibrio proteolyticus belonging to glycoside hydrolase family 19

    Yuji Honda, Hajime Taniguchi, Motomitsu Kitaoka

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   78 ( 4 )   627 - 634   2008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)   出版者・発行元:SPRINGER  

    A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus (chi19) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N-acetylated chitooligosaccharides, (GlcNAc)(n) , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc) (n) at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)(2) from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.

    DOI: 10.1007/s00253-008-1352-2

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  • Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124

    Masahiro Nakajima, Takanori Nihira, Mamoru Nishimoto, Motomitsu Kitaoka

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   78 ( 3 )   465 - 471   2008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)   出版者・発行元:SPRINGER  

    Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Gal beta 1 -&gt; 3GlcNAc, LNB) and galacto-N-biose (Gal beta 1 -&gt; 3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of alpha-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k (cat)/K-m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar k(cat)/K-m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-alpha-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.

    DOI: 10.1007/s00253-007-1319-8

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  • アノマー反転型糖質加水分解酵素のグライコシンターゼ化

    本多 裕司, 伏信 進矢, 日高 將文, 若木 高善, 祥雲 弘文, 谷口 肇, 北岡 本光

    Journal of Applied Glycoscience Supplement   2008 ( 0 )   149 - 149   2008年

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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  

    DOI: 10.11541/jsag.2008.0.149.0

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  • Strategy for Converting an Inverting Glycoside Hydrolase into a Glycosynthase

    Motomitsu Kitaoka, Yuji Honda, Masafumi Hidaka, Shinya Fushinobu

    Carbohydrate-Active Enzymes: Structure, Function and Applications   193 - 205   2008年

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    記述言語:英語   掲載種別:論文集(書籍)内論文   出版者・発行元:Elsevier Ltd.  

    We found a novel inverting xylanolytic enzyme belonging to GH8, reducing end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156), that hydrolyzed xylooligosaccharides (X3 or larger) to release X1 at their reducing end. Rex hydrolyzed α-X2F into X2 only in the presence of X1, clearly proving the Hehre-resynthesis hydrolysis mechanism. A library of mutant Rex at the catalytic base (Asp263) was constructed by saturation mutagenesis. Among them, D263C showed the highest level of X3 production, and D263N exhibited the fastest consumption of α-X2F. However, F- releasing activities of the mutants were much less than that of wild type. Next, Y198 of Rex that forms a hydrogen bond with the nucleophilic water was substituted with phenylalanine, causing a drastic decrease in the hydrolytic activity and a small increase in F- releasing activity from α-xylobiosyl fluoride in the presence of xylose. Y198F of Rex accumulates much more product during the glycosynthase reaction than D263C. We here conclude that an inverting glycosidase is effectively converted into glycosynthase by mutating a residue holding the nucleophilic water molecule with the general base residue while keeping the general base residue intact. © 2008 Woodhead Publishing Limited. All rights reserved.

    DOI: 10.1533/9781845695750.2.193

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  • &beta;-1,3-ガラクトシル-<I>N</I>-アセチルヘキソサミンホスホリラーゼの基質特異性決定部位の同定

    西本 完, 中島 将博, 日高 將文, 伏信 進矢, 北岡 本光

    Journal of Applied Glycoscience Supplement   2008 ( 0 )   105 - 105   2008年

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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  

    DOI: 10.11541/jsag.2008.0.105.0

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  • GH94セロビオースホスホリラーゼとグルコシダーゼ阻害剤の相互作用

    伏信 進矢, 日高 將文, 林 Andressa真奈美, 北岡 本光

    Journal of Applied Glycoscience Supplement   2008 ( 0 )   117 - 117   2008年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:日本応用糖質科学会  

    DOI: 10.5458/jag.jag.JAG-2010_022

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  • Clostridium phytofermentans由来β-1,4-D-ガラクトシル-L-ラムノースホスホリラーゼの同定

    中島 将博, 西本 完, 北岡 本光

    Journal of Applied Glycoscience Supplement   2008   107 - 107   2008年

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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  

    DOI: 10.11541/jsag.2008.0.107.0

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  • <I>Clostridium phytofermentans</I>由来 α-L-フコシダーゼのクローニングおよび発現

    仁平 高則, 中島 将博, 北岡 本光

    Journal of Applied Glycoscience Supplement   2008   137 - 137   2008年

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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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  • <I>Clostridium phytofermentans</I>由来GH112酵素の特異性解析

    北岡 本光, 中島 将博, 西本 完

    Journal of Applied Glycoscience Supplement   2008   106 - 106   2008年

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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  

    DOI: 10.11541/jsag.2008.0.106.0

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  • ビフィズス菌由来endo-α-<I>N</I>-acetylgalactosaminidaseのX線結晶構造解析

    鈴木 龍一郎, 伏信 進矢, 片山 高嶺, 芦田 久, 熊谷 英彦, 山本 憲二, 北岡 本光, 若木 高善, 祥雲 弘文

    Journal of Applied Glycoscience Supplement   2008   147 - 147   2008年

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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  

    DOI: 10.11541/jsag.2008.0.147.0

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  • セロビトールの製造・利用に関する研究 (第4報)反応系からのリン酸除去によるセロビオース生産の最適化

    鈴木 雅之, 裏地 達哉, 北岡 本光, 中井 由起子, 谷口 肇

    Journal of Applied Glycoscience Supplement   2008   19 - 19   2008年

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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  

    DOI: 10.11541/jsag.2008.0.19.0

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  • Colorimetric quantification of α-d-galactose 1-phosphate

    Takanori Nihira, Masahiro Nakajima, Kousuke Inoue, Mamoru Nishimoto, Motomitsu Kitaoka

    Analytical Biochemistry   371 ( 2 )   259 - 261   2007年12月

  • Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/Galacto-N-Biose metabolic pathway in Bifidobactetium longum

    Mamoru Nishimoto, Motomitsu Kitaoka

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   73 ( 20 )   6444 - 6449   2007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:AMER SOC MICROBIOLOGY  

    We have determined the functions of the enzymes encoded by the lnpB, InpC, and InpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine I-phosphate; the InpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine I-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDPgalactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.

    DOI: 10.1128/AEM.01425-07

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  • Purification, crystallization and preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from Bifidobacterium longum JCM1217

    Jun Wada, Ryuichiro Suzuki, Shinya Fushinobu, Motomitsu Kitaoka, Takayoshi Wakagi, Hirofumi Shoun, Hisashi Ashida, Hidehiko Kumagai, Takane Katayama, Kenji Yamamoto

    Acta Crystallographica Section F: Structural Biology and Crystallization Communications   63 ( 9 )   751 - 753   2007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)  

    A recombinant galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) from Bifidobacterium longum JCM1217 has been prepared and crystallized by the hanging-drop vapour-diffusion method using 10 mg ml-1 purified enzyme, 0.01 M zinc sulfate, 0.1 M MES buffer pH 5.9-6.4 and 20-22%(v/v) PEG MME 550 in the presence of 5 mM disaccharide ligands. Suitable crystals grew after 10 d incubation at 293 K. The crystals belong to space group C2221, with unit-cell parameters a = 106.3, b = 143.6, c = 114.6 Å for the lacto-N-biose I complex and a = 106.4, b = 143.4, c = 115.5 Å for the galacto-N-biose complex, and diffracted to 1.85 and 1.99 Å resolution, respectively. © International Union of Crystallography 2007.

    DOI: 10.1107/S1744309107036263

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  • Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk

    Mamoru Nishimoto, Motomitsu Kitaoka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   71 ( 8 )   2101 - 2104   2007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:TAYLOR & FRANCIS LTD  

    A one-pot enzymatic reaction to produce lacto-N-biose I (LNB), which is supposed to represent the bifidus factor in human milk oligosaccharides, was demonstrated. Approximately 500 mM of LNB was generated in 10-liter of reaction mixture initially containing 660 m,mM of sucrose and 600 mm of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose-hexose-1-phospate uridylyltransferase, UDPglucose 4-epimerase, and lacto-N-biose phosphorylase, in the presence of UDP-Glc and phosphate, indicating a reaction yield of 83%. LNB was isolated from the mixture by crystallization after yeast treatment. Finally, 1.4 kg of LNB of 99.6% purity was recovered after recrystallization.

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  • A unique thermostable lichenase from Thermotoga maritima MSB8 with divergent substrate specificity

    Mohammed Abdul, Sattar Khan, Mohammed Abdul, Sattar Khan, Mohammed Akbar, Motomitsu Kitaoka, Kiyoshi Hayashi

    Indian Journal of Biotechnology   6   315 - 320   2007年7月

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    A putative endoglucanase gene corresponding to locus TM 1752 (AAD36817.1, Q9X274) of Thermotoga maritima MSB8 was cloned and expressed in Escherichia coli. The enzyme contains a NEP (Asn-Glu-Pro) motif but lacks a PCD (proposed catalytic domain) block. This endoglucanase is grouped under family 5 of the glycosyl hydrolases members of which predominantly hydrolyze β-1,4-linkages of carbohydrate polymers. The enzyme efficiently hydrolyzes the glycosidic bonds of mixed β-1,3-1,4 linkages in lichenan and barley β-glucan, but did not display any activity on crystalline cellulose or laminarin. However, negligible amount of hydrolysis was observed with CM-cellulose. This enzyme produced glucosyl β-1,3 glucosyl β-1,4 glucose as the major end product and glucosyl β-1,4 glucosyl β-1,3 glucose was not detected. The putative endoglucanase of T. maritima appears to be a unique endoglucanase being the first lichenase producing glucosyl β-1,3 glucosyl β-1,4 glucose to be placed into family 5 of the glucosyl hydrolases.

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  • Molecular anatomy of the alkaliphilic xylanase from Bacillus halodurans C-125

    Mamoru Nishimoto, Shinya Fushinobu, Akimasa Miyanaga, Motomitsu Kitaoka, Kiyoshi Hayashi

    Journal of Biochemistry   141 ( 5 )   709 - 717   2007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)  

    Two regions in xylanase A from Bacillus halodurans C-125 (XynA), an alkaliphilic xylanase, were identified to be responsible for its activity at basic pH by comparing the dissociation constants of the XynA proton donor Glu residue (pKe2 and pKes2) with those of xylanase B from Clostridium stercorarium F9 (XynB) and their mutants constructed by substituting either Ser137/Asn127 of XynA/XynB or the 4th loop, designed based on the structural difference close to the proton donor. The substitution of XynB at Asn127 into Ser increased pKe2 by 0.37. The effect is explained that the positive charge of His126 likely affects the proton donor via Asnl27 and a water molecule in XynB, resulting in a decrease in pKe2, whereas such interactions were not observed with Ser. The substitution of XynB at the 4th loop into XynA (XynB Loop4A) increased the pKe2 and pKes2 values by 0.29 and 0.62, respectively. The effect of the 4th loop in XynA is likely due to a hydrogen bond between Asp 199 in the loop and Tyr239, which interacts with both the proton donors Glu195 and Arg204, with flexibility of the loop. Both the mutations independently affected the increases in pK e2. © 2007 The Japanese Biochemical Society.

    DOI: 10.1093/jb/mvm072

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  • Characterization of raffinose synthase from rice (Oryza sativa L. var. Nipponbare)

    Suhong Li, Tuoping Li, Wook-Dong Kim, Motomitsu Kitaoka, Shigeki Yoshida, Mitsutoshi Nakajima, Hideyuki Kobayashi

    BIOTECHNOLOGY LETTERS   29 ( 4 )   635 - 640   2007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)   出版者・発行元:SPRINGER  

    The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45 degrees C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-alpha-D-galactoside as galactosyl donors, and sucrose, lactose, 4-beta-galactobiose, N-acetyl-D-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.

    DOI: 10.1007/s10529-006-9268-3

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  • Phosphorylases in the Production of Oligosaccharides

    Motomitsu Kitaoka

    INDUSTRIAL APPLICATION OF ENZYMES ON CARBOHYDRATE-BASED MATERIAL   972   195 - 206   2007年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:AMER CHEMICAL SOC  

    Phosphorylases are one of the three main types of enzymes involved in the formation and cleavage of glycosyl linkages. They are expected to function as unique catalysts in the production of oligosaccharides, owing to their reactions being reversible and highly regio-specific. However, they have not been studied as extensively as the other two types, the hydrolases and synthases. Pairs of phosphorylases have been used to prepare oligosaccharides, including trehalose, cellobiose, and laminaribiose. New processes using phosphorylases to produce oligosaccharides with alpha-1,2 glucosyl linkage and amylose are reported.

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  • Identification of the putative proton donor residue of lacto-N-biose phosphorylase (EC 2.4.1.211)

    Mamoru Nishimoto, Motomitsu Kitaoka

    Bioscience, Biotechnology and Biochemistry   71 ( 6 )   1587 - 1591   2007年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Two lacto-N-biose phosphorylase (LNBP) isozyme genes were cloned from Bifidobacterium bifidum JCM1254. Alignment of the amino acid sequences of LNBP and its homologs identified 24 completely conserved acidic amino acid residues. All single mutants of Bifidobacterium longum LNBP at residues other than D313N retained considerable activity, suggesting that Asp313 is the putative proton donor residue in LNBP.

    DOI: 10.1271/bbb.70064

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  • Erratum: Error-prone rolling circle amplification: The simplest random mutagenesis protocol (Nature Protocols (2007))

    Ryoto Fujii, Motomitsu Kitaoka, Kiyoshi Hayashi

    Nature Protocols   1   2006年12月

  • A-12 原油由来DNAの解析 : カタール産と日本産に共通する塩基配列から原核・真核生物を推定する(群集構造解析,(2)口頭発表会,研究発表会)

    山根, 國男, 野村, 暢彦, 内山, 裕夫, 北岡, 本光

    日本微生物生態学会講演要旨集   0 ( 22 )   212 - 212   2006年10月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:日本微生物生態学会  

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  • Structural dissection of the reaction mechanism of cellobiose phosphorylase

    Masafumi Hidaka, Motomitsu Kitaoka, Kiyoshi Hayashi, Takayoshi Wakagi, Hirofumi Shoun, Shinya Fushinobu

    Biochemical Journal   398 ( 1 )   37 - 43   2006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)  

    Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into α-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 Å (1 Å = 0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established. © 2006 Biochemical Society.

    DOI: 10.1042/BJ20060274

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  • Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium

    Rie Kawai, Kiyohiko Igarashi, Makoto Yoshida, Makoto Yoshida, Motomitsu Kitaoka, Masahiro Samejima

    Applied Microbiology and Biotechnology   71 ( 6 )   898 - 906   2006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)  

    When Phanerochaete chrysosporium was grown with laminarin (a β-1,3/1,6-glucan) as the sole carbon source, a β-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear β-1,3-glucan, branched β-1,3/1,6-glucan, and β-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (β-d-Glcp-(1-&gt;6)-β-d-Glcp-(1- &gt;3)-β-d-Glcp-(1-&gt;3)-d-Glc) and 4-O-glucosyl-laminaribiose (β-d-Glcp-(1-&gt;4)-β-d-Glcp-(1-&gt;3)-d-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes β-d-Glcp-(1-&gt;3)-d-Glcp at subsites -2 and -1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a β-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched β-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular β-1,3-glucanases. © Springer-Verlag 2005.

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  • Characterization of glycosynthase mutants derived from glycoside hydrolase family 10 xylanases

    Masahiro Sugimura, Mamoru Nishimoto, Motomitsu Kitaoka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   70 ( 5 )   1210 - 1217   2006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)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Four xylanases belonging to glycoside hydrolase family 10-Thermotoga maritima XylB (TM), Clostridium stercorarium XynB (CS), Bacillus halodurans XynA (BH), and Cellulonumas fimi Cex (CF)-were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric beta-1,4-linked xylopyranose as a precipitate during reaction with alpha-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X-2 as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.

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  • RAISE: A simple and novel method of generating random insertion and deletion mutations

    Ryota Fujii, Ryota Fujii, Motomitsu Kitaoka, Kiyoshi Hayashi

    Nucleic Acids Research   34   2006年3月

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    Although proteins may be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult, and the mutations introduced are no more variable than those introduced by the introduction of random point mutations. We describe here a three-step method called RAISE, which is based on gene shuffling and can introduce a wide variety of insertions, deletions and substitutions. To test the efficacy of this method, we used it to mutate TEM β-lactamase to generate improved antibiotic resistance. Some unique insertion or deletion mutations were observed in the improved mutants, some of which caused higher activities than point mutations. Our findings indicate that the RAISE method can yield unique mutants and may be a powerful technique of protein engineering. © 2006 Oxford University Press.

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  • Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-β-D-arabino-hexopyranosyl-(1→4)-D-glucose (2<sup>II</sup>-deoxycellobiose)

    Motomitsu Kitaoka, Satoru Nomura, Satoru Nomura, Michiteru Yoshida, Kiyoshi Hayashi

    Carbohydrate Research   341 ( 4 )   545 - 549   2006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)  

    Four derivatives of 2II-deoxycellobiose were synthesized from d-glucal and acceptor sugars (D-glucose, D-xylose, D-mannose, and 2-deoxy-D-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the β-(1→4) linkage of 2-deoxy-D-arabino-hexopyranoside. The acceptor specificity for the D-glucal reaction was identical to that for the α-D-glucose 1-phosphate reaction, but the activity of D-glucal was approximately 500 times less than that of α-D-glucose 1-phosphate, using 10 mM substrates. © 2006 Elsevier Ltd. All rights reserved.

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  • The first glycosynthase derived from an inverting glycoside hydrolase

    Y Honda, M Kitaoka

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 3 )   1426 - 1431   2006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:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Reducing end xylose-releasing exooligoxylanase (Rex, EC 3.2.1.156) is an inverting GH that hydrolyzes xylooligosaccharides (&gt;= X-3) to release X-1 at their reducing end. The wild-type enzyme exhibited the Hehre resynthesis hydrolysis mechanism, in which alpha-X2F was hydrolyzed to X-2 and HF in the presence of X-1 as an acceptor molecule. However, the transglycosidation product (X-3) was not detectable in the reaction. To convert reducing end xylose-releasing exooligoxylanase to glycosynthase, derivatives with mutations in the catalytic base (Asp-263) were constructed by saturation random mutagenesis. Nine amino acid residue mutants (Asp-263 to Gly, Ala, Val, Thr, Leu, Asn, Cys, Pro, or Ser) were found to possess glycosynthase activity forming X-3 from alpha-X2F and X-1. Among them, D263C showed the highest level of X-3 production, and D263N exhibited the fastest consumption of alpha-X2F. The D263C mutant showed 10-fold lower hydrolytic activity than D263N, resulting in the highest yield of X-3. X-2 was formed from the early stage of the reaction of the D263C mutant, indicating that a portion of the X-3 formed by condensation was hydrolyzed before its release from the enzyme. To acquire glycosynthase activity from inverting enzymes, it is important to minimize the decrease in F--releasing activity while maximizing the decrease in the hydrolytic activity. The present study expands the possibility of conversion of glycosynthases from inverting enzymes.

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  • Error-prone rolling circle amplification: the simplest random mutagenesis protocol

    Ryota Fujii, Motomitsu Kitaoka, Kiyoshi Hayashi

    NATURE PROTOCOLS   1 ( 5 )   2493 - 2497   2006年

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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:NATURE PUBLISHING GROUP  

    A simple protocol to introduce random mutations, named error-prone rolling circle amplification (RCA), is described. A template plasmid is amplified by RCA in the presence of MnCl(2) and used for transformation of a host strain to give a mutant library with three to four random point mutations per kilobase throughout the entire plasmid. The prime advantage of this method is its simplicity. This protocol requires neither the design of specific primers nor the exploration of thermal cycling conditions. It takes just 10 min to prepare the reaction mixture, followed by overnight incubation and transformation of a host strain. This method permits rapid preparation of randomly mutated plasmid libraries, and will enable the wider adoption of random mutagenesis.

    DOI: 10.1038/nprot.2006.403

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  • A new method of carbohydrate synthesis in both solution and solid phases using a special hydroxy protecting group

    S Komba, M Kitaoka, T Kasumi

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY   ( 24 )   5313 - 5329   2005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-V C H VERLAG GMBH  

    A new carbohydrate synthesis method using a special hydroxy protecting group (uni-chemo protection = UCP) in both solution and solid phases was developed. The UCP group was comprised of polymerized amino acid derivatives. Each hydroxyl group was protected by a UCP group with a different degree of polymerization, which allowed them to be uniquely identified. To deprotect the UCP group, one cycle of Edman degradation was performed as follows: 1) removal of the amino protecting group; 2) phenyl isothiocyanate coupling; 3) removal of the N-terminal phenyl thiocarbamoyl mono-amino acid derivative by treatment with trifluoroacetic acid; and 4) re-protection of the newly exposed amino group with a Boc group. The hydroxyl groups were deprotected successively from the UCP group with the lowest to the highest degree of polymerization by repeating this Edman degradation cycle. First, commercially available N-alpha-t-Boc-sarcosine, N-alpha-t-Boc-N-alpha-methyl-L-alanine, and N-alpha-t-Boc-L-phenylglycine were examined as UCP groups. Despite successfully protecting and selectively deprotecting the hydroxyl groups, there were problems with stability or reactivity. To address these problems, N-alpha-1-ethylpropylglycine was chosen as the UCP group, and we successfully synthesized two sialyl-T antigen analogues. Tri-UCP and mono-UCP were attached to the 6- and 3-positions of the D-galactosamine (GalN) derivative, respectively, using cyanuric chloride. To selectively deprotect the mono-UCP group on the 3-position of the GalN derivative, one cycle of Edman degradation was performed. As a result of this cycle, the tri-UCP on the 6-position of the GalN derivative was degraded to di-UCP, and the mono-UCP on the 3-position was selectively deprotected to yield a GalN derivative with a free 3-position. Glycosylation of this selectively deprotected free 3-position GalN derivative with a suitably protected D-galactose (Gal) derivative in which the 3-position was protected by a mono-UCP group using N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) in dichloromethane yielded the desired disaccharide in high yield in both a position- and stereoselective manner. By repeating one cycle of Edman degradation, the X-position-free disaccharide in which the 6-position was protected by a mono-UCP group was prepared selectively. Subsequent acetylation and a final cycle of Edman degradation (except the third and fourth steps) yielded the 6-position-free disaccharide selectively. Two disaccharides, one with a free X-position and another with a free 6-position, were coupled with a sialic acid donor using NIS and TfOH in acetonitrile to obtain sialyl (2 -&gt; 3) T and sialyl (2 -&gt; 6) T antigen derivatives, respectively. Solid-phase synthesis was demonstrated using polystyrene-type beads and a new linker, 2-{4 - (hydroxymethyl)benzamido) acetic acid (HMBA-Gly), to synthesize Galp (1 -&gt; 3) Gal from a suitably protected Gal derivative in which the 3-position was protected by mono-UCR Solid-phase glycosylation was successfully monitored by measuring the removal of the Fmoc group, which protected the amino group on UCP, similar to the method for monitoring solid-phase Fmoc peptide synthesis. This UCP hydroxy protecting group was suitable for solid-phase synthesis, and will be the key technique in both automated oligosaccharide synthesis and oligosaccharide library synthesis. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).

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  • Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum

    M Kitaoka, JS Tian, M Nishimoto

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   71 ( 6 )   3158 - 3162   2005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 MICROBIOLOGY  

    A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract of Bifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein of Bifidobacterium longum NCC2705 encoded by the BL1641 gene. The homologous gene of the type strain B. longum JCM1217, lnpA, was expressed in Escherichia coli to confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. The lnpA gene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.

    DOI: 10.1128/AEM.71.6.3158-3162.2005

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  • The role of conserved arginine residue in loop 4 of glycoside hydrolase family 10 xylanases

    M Nishimoto, M Kitaoka, S Fushinobu, K Hayashi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   69 ( 5 )   904 - 910   2005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)   出版者・発行元:TAYLOR & FRANCIS LTD  

    An arginine residue in loop 4 connecting beta strand 4 and et-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg(204) in xylanase A from Bacillus halodurans C-125 (XynA) and Arg(196) in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k(cat)/K-m and the pH-k(cat) relationships of these mutant enzymes were measured. The pK(e2) and pK(es2) values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK(es2) value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK(a) value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.

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  • Structural basis for the specificity of the reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 査読

    S Fushinobu, M Hidaka, Y Honda, T Wakagi, H Shoun, M Kitaoka

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 17 )   17180 - 17186   2005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)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the beta-anomeric hydroxyl configuration from the alpha-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35 - 2.20-angstrom resolution and revealed the structural aspects of its three subsites ranging from - 2 to + 1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite + 2 is blocked by a barrier formed by a kink in the loop before helix alpha(10). His-319 in this loop forms a direct hydrogen bond with the beta-hydroxyl of xylose at subsite + 1, contributing to the specific recognition of anomers at the reducing end.

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  • Novel PCR-mediated mutagenesis employing DNA containing a natural abasic site as a template and translesional Taq DNA polymerase 査読

    A Kobayashi, M Kitaoka, K Hayashi

    JOURNAL OF BIOTECHNOLOGY   116 ( 3 )   227 - 232   2005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)   出版者・発行元:ELSEVIER SCIENCE BV  

    We describe a novel method of PCR-mediated mutagenesis employing DNA containing a natural abasic site and translesional Taq DNA polymerase. This method incorporated an adenine (80.8%) or guanine (7.7%) residue or led to a base deletion mutation (11.2%) opposite the abasic site. We conclude that the combination of DNA containing an abasic site and translesional Taq DNA polymerase is an easy and useful technique for PCR-mediated mutagenesis, having advantages different from those of conventional error-prone PCR. (C) 2004 Elsevier B.V. All rights reserved.

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  • Crystallization and preliminary X-ray analysis of reducing-end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 査読

    Y Honda, S Fushinobu, M Hidaka, T Wakagi, H Shoun, M Kitaoka

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   61 ( Pt 3 )   291 - 292   2005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)   出版者・発行元:WILEY-BLACKWELL  

    The reducing-end xylose-releasing wxo-oligoxylanase (Rex) from Bacillus halodurans C-125, a novel family GH8 glycoside hydrolase, was crystallized by the hanging-drop vapour-diffusion method using 13.6 mg ml(-1) purified Rex, 5.6% (v/v) polyethylene glycol 4000, 70 mM sodium acetate pH 4.6 and 30% (v/v) glycerol. Suitable crystals grew after incubation for 5 d at 293 K. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.69, b = 86.02, c = 87.92 angstrom. X-ray diffraction data were collected at a resolution of 1.35 angstrom.

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  • A family 8 glycoside hydrolase from bacillus halodurans C-125 (BH2105) is a reducing end xylose-releasing exo-oligoxylanase 査読

    Y Honda, M Kitaoka

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 53 )   55097 - 55103   2004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:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The gene encoding family 8 glycoside hydrolases from Bacillus halodurans C-125 (BH2105), an alkalophilic bacterium with a known genomic sequence, was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it did not possess a signal peptide and that it was an intracellular enzyme. The recombinant enzyme showed no hydrolytic activity on xylan, whereas it had been annotated as xylanase Y. It hydrolyzed xylooligosaccharide whose degree of polymerization is greater than or equal to 3 in an exosplitting manner with anomeric inversion, releasing the xylose unit at the reducing end. Judging from its substrate specificity and reaction mechanism, we named the enzyme reducing end xylose-releasing exo-oligoxylanase (Rex). Rex was found to utilize only the beta-anomer of the substrate to form beta-xylose and alpha-xylooligosaccharide. The optimum pH of the enzymatic reaction (6.2-7.3) was found in the neutral range, a range beneficial for intracellular enzymes. The genomic sequence suggests that B. halodurans secretes two endoxylanases and possesses two alpha-arabinofuranosidases, one alpha-glucuronidase, and three beta-xylosidases intracellularly in addition to Rex. The extracellular enzymes supposedly hydrolyze xylan into arabino/glucurono-xylooligosaccharides that are then transported into the cells. Rex may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides.

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  • Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1 -&gt; 3)-beta-glucosidase from the white-rot fungus Planerochaete chrysosporium 査読

    R Kawai, K Igarashi, M Kitaoka, T Ishii, M Samejima

    CARBOHYDRATE RESEARCH   339 ( 18 )   2851 - 2857   2004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 SCI LTD  

    To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1--&gt;3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1--&gt;6)-beta-D-Glcp-(1 --&gt; 3)-D-Glc) by H-1 NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K-m, K-m2, k(cat), k(cat2)). The rate of 6-0-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K-m k(cat), and affinity for glucosyl-enzyme intermediates (K-m2) were estimated for laminarioligosaccharides (DP = 3-5), the K-m value of laminaribiose was approximately 5 9 times higher than those of the other oligosaccharides (DP = 3-5), whereas the Km2 values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K-m and k(cat)), and a possible mechanism of transglycosylation is proposed. (C) 2004 Elsevier Ltd. All rights reserved.

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  • Crystallization and preliminary X-ray analysis of cellobiose phosphorylase from Cellvibrio gilvus 査読

    M Hidaka, M Kitaoka, K Hayashi, T Wakagi, H Shoun, S Fushinobu

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   60 ( Pt 10 )   1877 - 1878   2004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:BLACKWELL MUNKSGAARD  

    A recombinant cellobiose phosphorylase from Cellvibrio gilvus has been prepared and crystallized by the sitting-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 1.5 M ammonium sulfate, 0.1 M MES buffer pH 7.0 and 5 mM glucose. A suitable crystal was obtained after 10 d incubation at 298 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 84.77, b = 98.31, c = 104.04 Angstrom, beta = 102.73degrees. X-ray diffraction data to 2.1 Angstrom resolution have been collected at KEK-PF BL-5A.

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  • Kinetic evidence related to substrate-assisted catalysis of family 18 chitinases 査読

    Y Honda, M Kitaoka, K Hayashi

    FEBS LETTERS   567 ( 2-3 )   307 - 310   2004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:ELSEVIER SCIENCE BV  

    The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAC)(2)-UMB (1). The k(cat) of 2 was 3.4% of 1, but the K-m of 2 was 10-fold that of 1. In contrast, the k(cat) of 3 was only 0.3%, of that of 1, and the two reactions had an identical K-m. The drastic decreases in k(cat) were probably due to the weak nucleophilic activity of the C2-N-trifluoroacetamide and N-thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N-acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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  • Chitobiose phosphorylase from vibrio Proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold 査読

    M Hidaka, Y Honda, M Kitaoka, S Nirasawa, K Hayashi, T Wakagi, H Shoun, S Fushinobu

    STRUCTURE   12 ( 6 )   937 - 947   2004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:CELL PRESS  

    Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.

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  • Effects of truncation at the non-homologous region of a family 3 p-glucosidase from Agrobacterium tumefaciens 査読

    L Ying, M Kitaoka, K Hayashi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   68 ( 5 )   1113 - 1118   2004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)   出版者・発行元:TAYLOR & FRANCIS LTD  

    The function of the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region. Five truncated mutants named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 having deletions of 4, 31, 62, 89, and 119 amino acid residues starting from Phe417, respectively, were expressed in Escherichia coli and purified. All the mutants exhibited beta-glucosidase activity, indicating that the non-homologous region was not essential for the activity. The truncation caused thermal instability, decrease in pK(a) of the proton donor residue (Glu616), and deficient transglycosylation activity. The thermal stability and the pKa of Glu616 were partially recovered with longer truncation, suggesting that the truncation perturbed the structure and that their presence in the region was not essential. The main role of the non-homologous region could be formation of a hydrophobic atmosphere at the acceptor site to make the enzyme suitable for hydrolyzing hydrophobic glucosides.

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  • Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp NRRL B-21195 査読

    YK Kim, M Kitaoka, K Hayashi, CH Kim, GL Cote

    CARBOHYDRATE RESEARCH   339 ( 6 )   1179 - 1184   2004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)   出版者・発行元:ELSEVIER SCI LTD  

    A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0. and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35degreesC and the enzyme exhibited stability up to 50degreesC. The enzyme hydrolyzed cycloalternan [CA; cyclo{--&gt; 6)-alpha-D-Glcp-(1 --&gt; 3)-alpha-D-Glcp-(1--&gt;6)-alpha-D-Glcp-(--&gt;3)alpha-D-Glcp-(1--&gt;}] as the best substrate. to produce only isomaltose via an intermediate, alpha-isomaltosyl-(l --&gt; 3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates. such as panose, alpha-isomaltosyl-(1--&gt;4)-maltooligosaccharides, alpha-isomaltosyl-(1--&gt;3)-glucose, and alpha-isomaltosyl-(1 --&gt; 3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1--&gt;4)- or (1--&gt;3)-linkages. The K-m value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The k(cat) value on panose (14.4 s(-1)) was not significantly different from that of cycloalternan (10.8s(-1)). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195. its role being to hydrolyze cycloalternan inside the cells. (C) 2004 Elsevier Ltd. All rights reserved.

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  • A new oligosaccharide synthesis using special hydroxy protecting group 査読

    S Komba, M Kitaoka, T Kasumi

    TETRAHEDRON LETTERS   45 ( 13 )   2759 - 2762   2004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)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    A new hydroxy protecting group for convenient preparation of oligosaccharide was developed using uni-chemo Protection (UCP) concept. The UCP group was comprised of polymerized amino acid derivatives and protecting each hydroxyl groups by ester linkage. Depending on the polymerization degree, the hydroxyl groups were characterized and controlled. Using this protecting group, two kinds of sialyl-T analogues were Successfully synthesized from same sugar parts merely by repeating Edman degradation and coupling. (C) 2004 Elsevier Ltd. All rights reserved.

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  • Crystallization and preliminary X-ray analysis of xylanase B from Clostridium stercorarium 査読

    M Nishimoto, S Fushinobu, A Miyanaga, T Wakagi, H Shoun, K Sakka, K Ohmiya, S Nirasawa, M Kitaoka, K Hayashi

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   60 ( Pt 2 )   342 - 343   2004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:BLACKWELL MUNKSGAARD  

    Recombinant mature xylanase B from Clostridium stercorarium has been prepared and crystallized by the sitting-drop vapour-diffusion method using 4 mg ml(-1) purified enzyme, 10.3%(w/v) polyethylene glycol 1500, 8.6%(v/v) glycerol and 0.34 M non-detergent sulfobetaine 195. A suitable crystal grew after incubation for ten weeks at 293 K. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.76, b = 96.60, c = 138.44 Angstrom. X-ray diffraction data were collected to 1.80 Angstrom resolution.

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  • Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: Identification of family 36 glycosyltransferase in Vibrio 査読

    Yuji Honda, Motomitsu Kitaoka, Kiyoshi Hayashi

    Biochemical Journal   377 ( 1 )   225 - 232   2004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)  

    A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc) 2 (chitobiose) to form 2-acetamide-2-deoxy-α-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with α-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce β-D-glucosyl-(1 → 4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-β-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k0 = 5.5 s-1, Km = 2. 0 mM
    synthetic reaction, k0 = 10 s-1, Km = 14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.

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  • Reclassification of inverting phosphorylases based on structure determination

    M. Hidaka, Y. Honda, M. Kitaoka, T. Wakagi, H. Shoun, S. Fushinobu

    Photon Factory Activity Report   22   43 - 44   2004年

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  • One-step random mutagenesis by error-prone rolling circle amplification 査読

    R Fujii, M Kitaoka, K Hayashi

    NUCLEIC ACIDS RESEARCH   32 ( 19 )   e145   2004年

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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  

    In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3-4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique.

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  • Analyses of PCR products using DNA templates containing a consecutive deoxyinosine sequence. 査読

    Kobayashi A, Kitaoka M, Hayashi K

    Nucleic acids symposium series (2004)   ( 48 )   225 - 226   2004年

  • A thermostable non-xylanolytic alpha-glucuronidase of Thermotoga maritima MSB8 査読

    C Suresh, M Kitaoka, K Hayashi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   67 ( 11 )   2359 - 2364   2003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:TAYLOR & FRANCIS LTD  

    A putative alpha-glucosidase belonging to glycosyl hydrolase family 4 of Thermotoga maritima (TM0752) was expressed in Escherichia coli and it was found that the recombinant protein (Agu4B) was a p-nitrophenyl alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase, not alpha-glucosidase. It did not hydrolyze 4-O-methyl-D-glucuronoxylan or its fragment oligosaccharides. Agu4B was thermostable with an optimum temperature of 80degreesC. It strictly required Mn2+ and thiol compounds for its activity. The presence of NAD(+) slightly activated the enzyme. The amino acid sequence of Agu4B showed higher identity with Agu4A (another alpha-glucuronidase of T. maritima, 61%) than with AglA (alpha-glucosidase of T. maritima, 48%).

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  • A synergistic reaction mechanism of a cycloalternan-forming enzyme and a D-glucosyltransferase for the production of cycloalternan in Bacillus sp NRRL B-21195 査読

    YK Kim, M Kitaoka, HA Kiyoshi, CH Kim, GL Cote

    CARBOHYDRATE RESEARCH   338 ( 21 )   2213 - 2220   2003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:ELSEVIER SCI LTD  

    Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan. In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so. In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein. The latter was found to be a disproportionating enzyme (DE) that catalyzes transfer of a D-glucopyranosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing end. CAFE then transfers the isomaltosyl residue to the non-reducing end of another isomaltosyl maltooligosaccharide, to form an alpha-isomaltosyl-(1--&gt;3)-alpha-isomaltosyl-(1--&gt;4)-maltooligosaccharide, and subsequently catalyzes a cyclization to produce cycloalternan. Thus, DE and CAFE act synergistically to produce cycloalternan directly from maltodextrin or starch. (C) 2003 Elsevier Ltd. All rights reserved.

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  • Enzymatic synthesis of a library of beta-(1 -&gt; 4) hetero-D-glucose and D-xylose-based oligosaccharides employing cellodextrin phosphorylase 査読

    K Shintate, M Kitaoka, YK Kim, K Hayashi

    CARBOHYDRATE RESEARCH   338 ( 19 )   1981 - 1990   2003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:ELSEVIER SCI LTD  

    Enzymatic synthesis was attempted of six trisaccharides and 14 tetrasaccharides comprising beta-(1--&gt;4)-linked D-glucose and D-xylose residues, using cellodextrin phosphorylase (CDP, EC 2.4.1.49) as the enzyme catalyst, with alpha-D-glucose 1-phosphate (1) alpha-D-xylose 1-phosphate (2) as the donor substrates, and cellobiose (3), xylobiose (4), betaGlc-(l--&gt;4)-Xyl (5), or betaXyl-(1--&gt;4)-Glc (6) as the acceptor substrates. All enzymatic reactions were performed at pH 7.0 and the products purified by gel-filtration chromatography. We successfully synthesized all six hetero-trisaccharides and 10 of the 14 possible hetero-tetrasaccharides. It was not found possible to synthesize the four tetrasaccharides with a Xyl--&gt;Glc sequence at their non-reducing ends employing this method. The stereochemistries of the isolated products were assessed by analysis of their 2D NMR spectra (DQF-COSY, TOCSY, HSQC, HMBC). confirming that all of the glycosidic bonds in the products were beta-(1--&gt;4) linkages. (C) 2003 Elsevier Ltd. All rights reserved.

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  • Fusion of family 2b carbohydrate-binding module increases the catalytic activity of a xylanase from Thermotoga maritima to soluble xylan 査読

    FS Kittur, SL Mangala, A Abu Rus&apos;d, M Kitaoka, H Tsujibo, K Hayashi

    FEBS LETTERS   549 ( 1-3 )   147 - 151   2003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:ELSEVIER SCIENCE BV  

    A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90degreesC. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-p-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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  • Synthesis of a cellobiosylated dimer and trimer and of cellobiose-coated polyamidoamine (PAMAM) dendrimers to study accessibility of an enzyme, cellodextrin phosphorylase 査読

    AK Choudhury, M Kitaoka, K Hayashi

    EUROPEAN JOURNAL OF ORGANIC CHEMISTRY   2003 ( 13 )   2462 - 2470   2003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:WILEY-V C H VERLAG GMBH  

    To examine the accessibility of the enzyme cellodextrin phosphorylase (CDP) towards multivalent cluster carbohydrates, the cellobiosylated dimer 10 and trimer 12, as well as the cellobiose-coated PAMAM dendrimers 14, 16, 18, 20 and 22, with four, eight, sixteen, thirty-two and sixty-four cellobiose units at the outer surface of PAMAM dendrimers, respectively, have been synthesized for the first time and used as acceptor substrates for the enzyme CDP. It was found that CDP was able to transfer a glucosyl moiety from glucose-1-phosphate (Glc-1-P) into these synthesized cluster cellobiosylated glycoconjugates and cellobiose-coated PAMAM dendrimers, which were thus acceptor substrates for CDP. It was found that the ability of CDP to interact with smaller cellobiosylated glyconjugates and with PAMAM dendrimers containing up to eight cellobiose units was similar to that seen with cellobiose. However, this capability of CDP was somewhat lessened with the PAMAM dendrimer containing sixteen cellobiose moieties and dramatically decreased towards PAMAM dendrimers with thirty-two and sixty-four cellobiose units. This might be due to their steric bulk, CDP enzyme no longer being able to hold them properly on its active site. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

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  • Substrate specificity of the N,6-O-diacetylmuramidase from Streptomyces globisporus 査読

    HJ Seo, M Kitaoka, K Ohmiya, K Hayashi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   95 ( 3 )   313 - 316   2003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)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    We found that the N,6-O-diacetylmuramidase from Streptomyces globisporus (M-1) hydrolyzed the cell walls from Micrococcus lysodeikticus and Staphylococcus aureus. In contrast, hen egg white lysozyme (HEWL) was only able to hydrolyze the cell walls from M. lysodeikticus. 6-O-Acetylation of the muramoyl moieties, as found in the S. aureus cell walls, did not inhibit the activity of the M-1 enzyme whereas it was sufficient to inhibit HEWL. The disaccharide GlcNAc-MurNAc was not observed in the M. lysodeikticus cell wall hydrolyzate produced by the M-1, indicating that M-1 acts on the MurNAc moiety which are linked by peptides at the lactyl groups of the MurNAc moiety. M-1 displays both N-acetylmuramidase and N,6-O-diacetylmuramidase activity and has a different substrate specificity from HEWL.

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  • General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease 査読

    B Tang, S Nirasawa, M Kitaoka, C Marie-Claire, K Hayashi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   301 ( 4 )   1093 - 1098   2003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:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T64. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme.. Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC). In order to investigate the structural determinants within the N-terminal propeptide that play a role in the folding, processing, and enzyme inhibition of PA protease, we constructed a chimeric pro-PA protease by replacing the N-terminal propeptide with that of vibriolysin, a homologue of PA protease. Our results indicated that, although the N-terminal propeptide of vibriolysin shares only 36% identity with that of PA protease, it assists the refolding of MC, inhibits the folded MC to process its C-terminal propeptide, and shows a stronger inhibitory activity toward the mature PA protease than that of PA protease. These results suggest that the N-terminal propeptide domains in these thermolysin-like proteases may have similar functions, in spite of their primary sequence diversity. In addition, the conserved regions in the N-terminal propeptides of PA protease and vibriolysin may be essential for the functions of the N-terminal propeptide. (C) 2003 Elsevier Science (USA). All rights reserved.

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  • Kinetic studies on the hydrolysis of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside by the family 18 chitinases ChiA and ChiB from Serratia marcescens 査読

    Y Honda, M Kitaoka, K Tokuyasu, C Sasaki, T Fukamizo, K Hayashi

    JOURNAL OF BIOCHEMISTRY   133 ( 2 )   253 - 258   2003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:JAPANESE BIOCHEMICAL SOC  

    Kinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(GlcNAc)(2)-UMB (1), GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)] by ChiA and ChiB from Serratia marcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of I by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another. However, both curve shapes were explained by assuming some of the enzyme present formed complexes with multiple molecules of the substrate. Conversely, the S-v curves generated in the cleavage of 2 and 3 by ChiA exhibited typical Michaelis-Menten profiles. Both enzymes hydrolysed 2 with an approximately 14-fold higher K-m value relative to 1, indicating that the N-acetyl group was recognised at the -2 subsite. The k(cat) value obtained with ChiA was identical to the k(cat) value observed for 1. However, the k(cat) value for ChiB was one-fourth that of 1, suggesting that the removal of the N-acetyl group caused an increase in the formation of a non-productive ES-complex. ChiA and ChiB hydrolysed 3 with 5- and 20-fold greater K-m values relative to 1, respectively, and 60- and 30-fold smaller k(cat) values relative to 1, respectively. The reaction mechanism of family 18 chitinases is discussed based upon the results obtained from the hydrolysis of these compounds.

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  • Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8 査読

    E Rajashekhara, M Kitaoka, YK Kim, K Hayashi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   66 ( 12 )   2578 - 2586   2002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  

    The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80degreesC. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70degreesC in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The K-m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k(cat) was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) k(cat) followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the k(cat) of 8.0 s(-1) had the highest (1.1x10(4) M-1 s(-1)) k(cat)/K-m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.

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  • Enhancement of transglycosylation activity by construction of chimeras between mesophilic and thermophilic beta-glucosidase 査読

    K Goyal, BJ Kim, JD Kim, YK Kim, M Kitaoka, K Hayashi

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   407 ( 1 )   125 - 134   2002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:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    The family 3 beta-glucosidase from Thermotoga maritima is a highly thermostable enzyme (85degreesC) that displays transglycosylation activity. In contrast, the beta-glucosidase from Cellvibrio gilvus is mesophilic (35degreesC) and displays no such transglycosylation activity. Both enzymes consist of two domains, an N-terminal and a C-terminal domain, and the amino acid identities between the two enzymes in these domains are 32.4 and 36.4%, respectively. In an attempt to identify the molecular basis underpinning the display of transglycosylation activity and the requirements for thermal stability, eight chimeric genes were constructed by shuffling the two parental beta-glucosidase genes at four selected borders, two in the N-terminal domain and two in the C-terminal domain. Of the eight chimeric genes constructed, only two chimeric enzymes (Tm578/606Cg and Tm638/666Cg) gave catalytically active forms and these were the ones shuffled in the C-terminal domain. For these active chimeric enzymes, 80%, (Tm578/606Cg) and 88% (Tm638/666Cg) of their amino acid sequences originated from T. maritima. With regard to their thermal profiles, the two active chimeric enzymes, Tm578/606Cg and Tm638/666Cg, displayed profiles intermediate to those of the two parental enzymes as they were optimally active at 65 and 70degreesC, respectively. These two chimeric enzymes were optimally active at pH 4.1 and 3.9, which is closer to that observed for the T. maritima enzyme (pH 3.2-3.5) than that for the C. gilvus enzyme (pH 6.2-6.5). Kinetic parameters for the chimeric enzymes were investigated with five different substrates including pNP-beta-D-glucopyranoside. The kinetic parameters obtained for the chimeric enzymes were closer to those of the T. maritima enzyme than to those of the C. gilvus enzyme. Transglycosylation activity was observed for both chimeric enzymes and the activity of the Tm578/606Cg chimera was at a level twice that observed with the T. maritima enzyme. This study is an effective demonstration of the usefulness of chimeric enzymes in altering the characteristics of an enzyme. (C) 2002 Elsevier Science (USA). All rights reserved.

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  • Employing chimeric xylanases to identify regions of an alkaline xylanase participating in enzyme activity at basic pH 査読

    M Nishimoto, M Kitaoka, K Hayashi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   94 ( 5 )   395 - 400   2002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:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    The xylanase A (XynA) from the alkaliphilic Bacillus halodurans C-125 and the xylanase B (XynB) from Clostridium stercorarium F9 were subdivided into four fragments at highly homologous regions present in their primary structures: an amino-terminal region (A or a), a region containing the putative proton donor (P or p), a region containing the putative catalytic nucleophile (N or n), and a carboxyl-terminal region (C or c). Six chimeric xylanases were constructed by the selective substitution of the four fragments using an overlapping PCR technique. Two of the six xylanases, APnc and Apnc (regions originating from XynA are denoted by upper case letters and those from XynB are denoted by lower case letters), were produced in Escherichia coli while the other four xylanases were obtained only as inclusion bodies. The APnc and Apnc chimeric enzymes were purified by column chromatography using Ni-NTA agarose and DEAE-Toyopearl. The respective pH and temperature stabilities of the purified enzymes were observed from pH 5.6 to 11.6 and up to 45degreesC for APnc, and from pH 5.6 to 11.2 and up to 45degreesC for Apnc. Thus, these enzymes were slightly less stable than the parental xylanases. An assessment of the pH-activity relationships for the chimeric xylanases employed p-nitrophenyl-beta-D-xylobioside as the substrate in determinations of the k(cat) values. The pK(a1) values for the APnc and Apnc chimeric enzymes were 4.3 and 4.2, respectively, which were almost identical to those for the parental xylanases. In contrast, the pK(a2) values obtained for APnc and Apnc were 9.1 and 8.5, respectively; these values fall between those for the parental xylanases, XynA (9.4) and XynB (7.8). These results indicate that the main regions necessary to maintain the high pK(a2) value of XynA locate in the A and P sections.

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  • Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli 査読

    YK Kim, M Kitaoka, M Krishnareddy, Y Mori, K Hayashi

    JOURNAL OF BIOCHEMISTRY   132 ( 2 )   197 - 203   2002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:JAPANESE BIOCHEMICAL SOC  

    A cellobiose phosphorylase (CBP) cloned from the Clostridium thermocellum YM4 strain was purified to homogeneity, and the reaction mechanisms of both the phosphorolytic and synthetic reactions were studied in detail. The enzyme reaction proceeded via an ordered bi bi mechanism, in which P-i bound to the enzyme prior to D-cellobiose and then G 1-P was released after D-glucose. The order of substrate binding was different from that of CBP from Cellvibrio gilvus, which bound to cellobiose prior to P-i. In the synthetic reaction, the enzyme showed three times higher activity with beta-D-glucose than with alpha-D-glucose, and also showed weak activity with 1,5-anhydro-D-glucitol, indicating that the beta-anomeric hydroxyl group of D-glucose is highly required. However, even when it is removed enzyme activity remains. The substrate specificity and kinetic studies revealed that the configurations of the C3 and C4 hydroxyl groups were strictly required for the enzyme activity, whereas those of C2 and C6 could be substituted or deleted. The mechanism of substrate inhibition by D-glucose was studied in detail and it was concluded that D-glucose competed with G 1-P for its binding site in the synthetic reaction.

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  • The role of the N-terminal propeptide of the pro-aminopeptidase processing protease: refolding, processing, and enzyme inhibition 査読

    B Tang, S Nirasawa, M Kitaoka, K Hayashi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   296 ( 1 )   78 - 84   2002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:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Pro-aminopeptidase processing protease (PA protease) is an extracellular zinc metalloprotease produced by Aeromonas caviae T-64 and it is classified as M04.016 according to the MEROPS database. The precursor of PA protease consists of four regions; a signal peptide, an N-terminal propeptide, a C-terminal propeptide, and the mature PA protease. The in vitro refolding of the intermediate pro-PA protease containing the C-terminal propeptide (MC) was investigated in the presence and absence of the N-terminal propeptide. The results indicate that the noncovalently linked N-terminal propeptide is able to assist in the refolding of MC. In the absence of the N-terminal propeptide, MC is trapped into a folding competent state that is converted into the active form by the addition of the N-terminal propeptide. Moreover, the N-terminal propeptide was found to form a complex with the folded MC and inhibit further processing of MC into the mature PA protease. Inhibitory activity of the purified N-terminal propeptide toward mature PA protease was also observed, and the mode of this inhibition was determined to be a mixed, noncompetitive inhibition with an associated allosteric effect. (C) 2002 Elsevier Science (USA). All rights reserved.

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  • Characterization of Cellobiose Phosphorylase and Cellodextrin Phosphorylase (糖質関連酵素化学シンポジウム)

    Kitaoka Motomitsu, Taniguchi Hajime, Hayashi Kiyoshi

    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience   49 ( 2 )   221 - 227   2002年4月

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

    Two intracellular enzymes, cellobiose phosphorylase (CBP) and cellodextrin phosphorylase (CDP) are involved in the phosphorolytic pathway in cellulose degradation. Those enzymes are considered to be useful in syntheses of oligosaccharides because the reactions are reversible. CBP from Cellvibrio gilvus and Clostridium thermocellum YM4, and CDP from C. thermocellum YM4 were cloned and over-expressed in Escherichia coli. All the three enzymes showed ordered bi bi mechanism. However the orders of the substrate binding of the CBPs were different. It was found that CBP from C. gilvus strictly recognized the hydroxyl groups at positions β-1, 3, and 4 of the acceptor molecule in the reverse reaction. On the other hand, the recognition of the hydroxyl groups at positions 2 and 6 was not so strict. Three branched β-1, 4-glucosyl trisaccharides were synthesized by using the reverse reaction of C. gilvus CBP. A new substrate inhibition pattern, competitive substrate inhibition, was also found in the reverse reaction of CBP using glucose as the acceptor. Specific colorimetric quantification of cellobiose was designed by using the reaction of CBP. Cellobiose was produced from sucrose at 90% yield by a combined action of three enzymes including CBP.

    DOI: 10.5458/jag.49.221

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  • Evidence that the putative alpha-glucosidase of Thermotoga maritima MSB8 is a pNP alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase 査読

    C Suresh, A Abu Rus'd, M Kitaoka, K Hayashi

    FEBS LETTERS   517 ( 1-3 )   159 - 162   2002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)   出版者・発行元:ELSEVIER SCIENCE BV  

    The gene (agu) encoding p-nitrophenyl alpha-D-glucuronopyranoside (pNP-GUA) hydrolyzing alpha-glucuronidase of the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified and characterized. The gene previously designated as putative alpha-glucosidase was found to code for a protein that had no alpha-glucosidase activity. It showed a rare activity profile with its ability to hydrolyze pNP-GUA, an activity not known in the alpha-glucuronidases from microbial sources. This is the first report on the occurrence of an alpha-glucuronidase which belongs to the family 4 of glycosyl hydrolases. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S0014-5793(02)02611-X

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  • A kinetic study on pH-activity relationship of XynA from alkaliphilic Bacillus halodurans C-125 using aryl-xylobiosides 査読

    M Nishimoto, Y Honda, M Kitaoka, K Hayashi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   93 ( 4 )   428 - 430   2002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)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    Xylanase A from alkaliphilic Bacillus halodurans C-125 was expressed in Escherichia coli and purified by affinity and anion exchange chromatographies. It exhibited a strong substrate inhibition using xylan as the substrate. Its k(i) value increased with an increase in pH. The effect of pH on the enzyme activity was determined using two aryl-xylobiosides as substrates, and it was found that the enzyme had a flat k(cat)-pH curve in the pH range of 5.8-8.8. This range was different from that obtained with 0.45% xylan as previously reported (Honda, H. et. al., Agric. Biol. Chem., 49, 3165-3169, 1985). The substrate inhibition was presumed to cause the difference. It has been clarified that the use of aryl-xylobiosides as substrates yields more accurate kinetic results than that of xylan.

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  • In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64. 査読

    Tang B, Nirasawa S, Kitaoka M, Hayashi K

    Biochimica et biophysica acta   1596 ( 1 )   16 - 27   2002年4月

  • Adsorption of bisphenol A by cross-linked β-cyclodextrin polymer 査読

    Motomitsu Kitaoka, Kiyoshi Hayashi

    Journal of Inclusion Phenomena   44 ( 1-4 )   429 - 431   2002年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)  

    An insolubilized β-cyclodextrin derivative (polyCD) was prepared by polymerizing cyclodextrin with epichlrohydrine. By stirring 5 mg/ml of polyCD in 0.2 mM bisphenol A (BPA) solution at pH 7.0 for 2 h, more than 98% of BPA was adsorbed on polyCD. The capacity of the adsorption was determined to be 84 mg-BPA/g-polyCD, meaning 0.65 mol-BPA/mol-CD (in polyCD). The effect of pH on the adsorption was studied and found that BPA was effectively adsorbed on polyCD at pH between pH 2.2-9.1, but the efficiency decreased at pH 10.8, suggesting that it did not adsorb the BPA anion. The polyCD did not adsorb aromatic amino acids, indicating that BPA could selectively be removed from a solution containing amino acids.

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  • Syntheses of 4-methylumbelliferyl-β-d-xylobioside and 5-bromo-3-indolyl-β-d-xylobioside for sensitive detection of xylanase activity on agar plates 査読

    Satoshi Kaneko, Motomitsu Kitaoka, Atsushi Kuno, Kiyoshi Hayashi

    Bioscience, Biotechnology and Biochemistry   64 ( 4 )   741 - 745   2000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)  

    4-Methylumbelliferyl-β-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-β-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate. © 2000 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

    DOI: 10.1271/bbb.64.741

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  • KINETIC-STUDIES ON P-NITROPHENYL-CELLOBIOSIDE HYDROLYZING XYLANASE FROM CELLVIBRIO-GILVUS 査読

    M KITAOKA, K HAGA, Y KASHIWAGI, T SASAKI, H TANIGUCHI, KUSAKABE, I

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   57 ( 12 )   1987 - 1989   1993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  

    Reactions of the p-nitrophenyl-cellobioside (G2-pNP) hydrolyzing xylanase from Cellvibrio gilvus (XCEL) were investigated kinetically in detail. XCEL hydrolyzed only aglyconic bonds in various arylcellobiosides with close kinetic paramters together. Its kinetic parameters toward various p-nitrophenyl cellooligosaccharides were also close. Two more xylanases, from Streptomyces sp. E86 (XSTR) and from Aspergillus japonicus (XASP), were found to hydrolyze G2-pNP at a lower rate compared with XCEL. The V(max) of XSTR and XASP were comparable to that of XCEL, suggesting that the high G2-pNP-hydrolyzing activity of XCEL was due to its small K(m). A xylanse from Robillarda sp. Y-20 (XROB) did not have any activity on G2-pNP. p-Nitrophenyl-xylobioside (X2-pNP) and p-nitrophenyl-glucosyl-xyloside (GX-pNP) were examined as substrates to the four xylanases. Three of the four xylanases hydrolyzed these substrates, only at their aglyconic bonds, rather faster than xylan, but XROB hydrolyzed them with a very small rate. Classification of xylanases based on their activity on the aryl-glycosides is discussed. The advantage of using X2-pNP or GX-pNP for xylanase assay is also discussed.

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  • PURIFICATION AND PROPERTIES OF LAMINARIBIOSE PHOSPHORYLASE (EC-2.4. 1.31) FROM EUGLENA-GRACILIS Z 査読

    M KITAOKA, T SASAKI, H TANIGUCHI

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   304 ( 2 )   508 - 514   1993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:ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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  • スクロ-スホスホリラ-ゼ,キシロ-スイソメラ-ゼおよびラミナリビオ-スホスホリラ-ゼを用いたスクロ-スのラミナリビオ-スへの変換〔英文〕

    北岡 本光, 佐々木 堯, 谷口 肇

    澱粉科学   40 ( 3 )   311 - 314   1993年

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

    ラミナリビオースを,スクロースを原料として触媒量のリン酸存在下でスクロースホスホリラーゼ,キシロースイソメラーゼおよびラミナリビオースホスホリラーゼを同時に作用させることにより合成した.50mMイミダゾールー塩酸緩衝液(pH7.0)中,20mMリン酸存在下において,200mMスクロースに0.47U/mlスクロースホスホリラーゼ,0.072U/mlキシロスイソメラーゼ,0.37U/m1ラミナリビオースボスホリラーゼの混合酵素系を37℃ で作用させたところ,反応48時間後に反応混液中のラミナリビオース濃度は110mM(収率55%)に達した.反応15時間後からラミナリトリオースの生成がみられ,反応48時間後には22mMに達した.また微量のラミナリテトラオースの生成も確認された.セロビオースボスホリラーゼを用いた同様なセロピオースの合成実験では,三糖以上のオリゴ糖は生成しなかった.この違いはセロビオースホスホリラーゼが三糖以上にはまったく作用しないのに対して,ラミナリビオースボスホリラーゼは弱いながらも三糖以上に作用するため生成されるものと考察された.

    DOI: 10.5458/jag1972.40.311

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  • SYNTHETIC REACTION OF CELLVIBRIO-GILVUS CELLOBIOSE PHOSPHORYLASE

    M KITAOKA, T SASAKI, H TANIGUCHI

    JOURNAL OF BIOCHEMISTRY   112 ( 1 )   40 - 44   1992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:JAPANESE BIOCHEMICAL SOC  

    The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail.It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with beta-D-glucose than with the alpha-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. beta-D-Cellobiose was also phosphorolyzed faster than the alpha-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with alpha-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model.

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  • PHOSPHOROLYTIC REACTION OF CELLVIBRIO-GILVUS CELLOBIOSE PHOSPHORYLASE

    M KITAOKA, T SASAKI, H TANIGUCHI

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   56 ( 4 )   652 - 655   1992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)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Cellobiose phosphorylase was purified from Cellvibrio gilvus cells by the method reported previously with some modifications, and its kinetic properties were studied in detail. The initial velocity of the synthetic reaction was 1.4 times as fast as that of the phosphorolytic one. The equilibrium constant of the phosphorolysis was 0.32 at 37-degrees-C and pH 7.0. NO D-[U-C-14]glucose exchange reaction was observed in the absence of Pi. Kinetic studies on the phosphorolytic reaction showed that the reaction follows an ordered bi bi mechanism. These results make a sharp contrast to those of sucrose phosphorylase, which catalyzes fructose exchange reaction and follows a ping pong bi bi mechanism. Kinetic parameters were calculated as K(mA) = 2.6mM, K(mB) = 0.61 mM, and K(iA) = 6.8 mM (A, D-cellobiose; B, Pi).

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  • Synthesis of laminarioligosaccharides using crude extract of Euglena gracilis z cells

    Motomitsu Kitaoka, Takashi Sasaki, Hajime Taniguchi

    Agricultural and Biological Chemistry   55 ( 5 )   1431 - 1432   1991年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1080/00021369.1991.10870772

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  • Purification and properties of a Xylanase from Cellvibrio gilvus that hydrolyzes p-Nitrophenyl Cellooligosaccharides

    Keiko Haga, Motomitsu Kitaoka, Yutaka Kashiwagi, Takashi Sasaki, Hajime Taniguchi

    Agricultural and Biological Chemistry   55 ( 8 )   1959 - 1967   1991年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    An enzyme component that hydrolyzes pNP-G2but not CMC has been isolated from a culture broth of Cellvibrio gilvus by a multi-step procedure involving Butyl-Toyopearl, DEAE-Toyopearl, and CM-Toyopearl chromatographies. The purified enzyme gave a single protein band on native, SDS-, and IEF-PAGE. The enzyme had a molecular weight of 40, 000, an isoelectric point of 5.0, an optimum pH of 6.5, and an optimum temperature of 55°C. It was stable from pH 4.0 to 9.0 at 37°C for 1 hr and below 50°C for 30min. It hydrolyzed agluconic bonds not only of pNP-G2but also of pNP-G3, pNP-G4, and pNP-G5. Cellooligosaccharides with D.P. of 3 to 5 were not hydrolyzed at all. Instead, the enzyme hydrolyzed xylan 4 times as fast as pNP-G2. Both HgCl2and p-chloromercuribenzoic acid inhibited the two activities completely. Xylan inhibited the hydrolysis of pNP-G2competitively. From these results, the purified enzyme was considered to be a unique xylanase that hydrolyzed the agluconic bonds of pNP-Gn. © 1991 by the Japan Society for Bioscience, Biotechnology, and Agrochemistry.

    DOI: 10.1080/00021369.1991.10870897

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  • PRODUCTION OF GLUCOSYL-XYLOSE USING CELLVIBRIO-GILVUS CELLS AND ITS PROPERTIES

    M KITAOKA, H TANIGUCHI, T SASAKI

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   34 ( 2 )   178 - 182   1990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 VERLAG  

    4-O-\-d-Glucopyranosyl-d-xylose (GX) was synthesized from equimolar amounts of d-xylose and α-d-glucose-1-phosphate (G-1-P) using acetone-treated cells of Cellvibrio gilvus. It was found that ethanol treatment of acetone-treated cells selectively removed phosphoglucomutase activity, which competes with cellobiose phosphorylase for G-1-P in the synthetic reaction. The yield of synthesis was 60%, based on d-xylose used. GX was purified by charcoal column chromatography with a 32% yield based on d-xylose. Nuclear magnetic resonance and fast atm bombardment mass data of GX are presented. The possibility for this saccharide to be used as a new foodstuff is also discussed. © 1990 Springer-Verlag.

    DOI: 10.1007/BF00166776

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  • A SIMPLE METHOD OF CELLULASE IMMOBILIZATION ON A MODIFIED SILICA SUPPORT

    M KITAOKA, H TANIGUCHI, T SASAKI

    JOURNAL OF FERMENTATION AND BIOENGINEERING   67 ( 3 )   182 - 185   1989年

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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:SOC FERMENTATION BIOENGINEERING, JAPAN  

    Two types of cellulase (Robillarda sp. Y-20 and Trichoderma reesei) were immobilized on Aminosilica-1 by physical adsorption. Enzymes were quickly immobilized and stable on the support. Specific activities of two types of immobilized cellulase were 0.24 U/mg support with Robillarda cellulase and 0.15 U/mg support with the Trichoderma one as CMCase. The pH-activity curves of both cellulase shifted slightly to lower pH on immobilization. Both immobilized cellulases showed essentially the same pH stabilities as their free forms. However, the immobilized enzymes were less stable than the free forms at temperatures higher than 50°C. © 1989.

    DOI: 10.1016/0922-338X(89)90119-0

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MISC

  • Bifidobacterium属細菌のムチン型糖鎖資化経路の解明

    CHOI Moonhak, 後藤愛那, 阪中幹祥, 加藤紀彦, 西本完, 杉山友太, 栗原新, 吹谷智, 横田篤, 北岡本光, 片山高嶺

    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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  • ビフィズス菌とヒトミルクオリゴ糖

    北岡本光

    日本調理科学会誌   52 ( 5 )   352‐357(J‐STAGE)   2019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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  • ビフィズス菌のフコシルラクトーストランスポーターに見られる適応進化が母乳栄養児腸管でのビフィズスフローラ形成に関与する

    阪中幹祥, 阪中幹祥, 栗原新, 栗原新, 片山高嶺, 片山高嶺, HANSEN Morten Ejby, 後藤愛那, 加藤紀彦, 吉田圭佑, 小田巻俊孝, 杉山友太, 廣瀬潤子, 浦島匡, 清水(肖)金忠, 北岡本光, 吹谷智, 横田篤, LEGGIO Leila Lo, HACHEM Maher Abou

    Institute for Fermentation, Osaka. Research Communications   ( 33 )   2019年

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  • 糖質関連酵素高度活用技術の開発

    北岡本光

    応用糖質科学   8 ( 1 )   20‐32   2018年2月

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    記述言語:日本語  

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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

    Web of Science

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  • オリゴ糖実用的調製に有用なホスホリラーゼ

    北岡本光

    応用糖質科学   7 ( 2 )   104‐108   2017年5月

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    記述言語:日本語  

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  • 酵素工学を活用した糖質資源高度利用プラットフォームの構築

    北岡本光

    JATAFジャーナル   5 ( 4 )   45 - 45   2017年4月

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    記述言語:日本語   掲載種別:記事・総説・解説・論説等(その他)  

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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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  • 酵素耐熱化の実例―ミルクオリゴ糖合成酵素の耐熱化―

    北岡本光

    New Food Ind   58 ( 4 )   33 - 37   2016年4月

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    記述言語:日本語  

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  • Ruminococcus albus由来4‐O‐β‐D‐マンノシル‐D‐グルコースホスホリラーゼのサブサイト+1に保存されるArg92の基質結合への関与

    塩田咲耶子, 尾高伶, 佐分利亘, YE Yuxin, 薦田圭介, 加藤公児, 西本完, 北岡本光, YAO Min, 森春英

    日本農芸化学会大会講演要旨集(Web)   2016   4D009 (WEB ONLY)   2016年3月

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    記述言語:日本語  

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  • ホスホリラーゼを用いたスクロースからのセルロース合成

    久我友大, 砂川直輝, 石田卓也, 竹内美由紀, 五十嵐圭日子, 鮫島正浩, 北岡本光

    セルロース学会年次大会講演要旨集   23rd   2016年

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  • Ba-9 Ruminococcus albus由来4-O-β-D-mannosyl-D-glucose phosphorylase RaMP1の無機リン酸に対するホモトロピック相互作用(ホスホリラーゼ関連,一般講演,一般社団法人日本応用糖質科学会平成27年度大会(第64回))

    尾高 伶, 佐分 利亘, Ye Yuxin, 薦田 圭介, 加藤 公児, 西本 完, 北岡 本光, 姚 閔, 森 春英

    応用糖質科学 : 日本応用糖質科学会誌   5 ( 3 )   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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  • Ruminococcus albus由来4‐O‐β‐D‐mannosyl‐D‐glucose phosphorylase RaMP1の無機リン酸に対するホモトロピック相互作用

    尾高伶, 佐分利亘, YE Yuxin, 薦田圭介, 加藤公児, 西本完, 北岡本光, 姚閔, 森春英

    応用糖質科学   5 ( 3 )   42   2015年8月

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    記述言語:日本語  

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  • Ba-10 Bifidobacterium longum由来酢酸キナーゼの機能解析(ホスホリラーゼ関連,一般講演,一般社団法人日本応用糖質科学会平成27年度大会(第64回))

    劉 遠, 西本 完, 北岡 本光

    応用糖質科学 : 日本応用糖質科学会誌   5 ( 3 )   B42   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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  • 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  

    CiNii Article

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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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    記述言語:日本語  

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  • ホスホリラーゼ及びその利用

    北岡本光

    食品の試験と研究   ( 49 )   34 - 37   2015年2月

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    記述言語:日本語  

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  • ビフィズス菌由来N-アセチルヘキソサミン1-キナーゼのオリゴ糖合成を目指した機能改変

    佐藤真与, 荒川孝俊, 西本完, 北岡本光, 伏信進矢

    日本農芸化学会大会講演要旨集(Web)   2015   2015年

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  • 母乳が優先増殖させる乳児腸内のビフィズス菌―プレバイオティクス研究の原点への回帰によって見えた分子機構―

    北岡本光

    日本農学会シンポジウム講演要旨   2014   3 - 6   2014年10月

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    記述言語:日本語  

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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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    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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  • 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  

    CiNii Article

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  • Cp-16 Bifidobacterium longum JCM1217由来N-アセチルヘキソサミン1-キナーゼの構造・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))

    佐藤 真与, 西本 完, 荒川 孝俊, 北岡 本光, 伏信 進矢

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B52   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-15 Bacteroides thetaiotaomicron由来L-フコキナーゼ/GDP-L-フコースピロ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26年度大会(第63回))

    劉 遠, 仁平 高則, 西本 完, 北岡 本光

    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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    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-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-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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    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-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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    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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    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-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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    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-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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    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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    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-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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    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-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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    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-17 Vibrio proteolyticus由来GH94キトビオースホスホリラーゼの合成反応における基質阻害(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))

    伊藤 佑, 劉 遠, 西本 完, 北岡 本光

    応用糖質科学 : 日本応用糖質科学会誌   4 ( 3 )   B52   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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  • 糖質関連酵素の革新的利用技術・改変技術の開発

    西本完, 北岡本光, 林清

    化学と生物   52 ( 8 )   505 - 511   2014年8月

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  • ビフィズス菌とヒトミルクオリゴ糖

    北岡本光

    精糖技術研究会誌   60   27 - 33   2014年5月

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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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  • ビフィズス菌の機能解明と新たな利用 母乳のビフィズス菌増殖因子研究の最前線

    北岡本光

    Bio Ind   31 ( 5 )   5 - 12   2014年5月

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  • 新規糖質ホスホリラーゼの発見とそれらを利用したオリゴ糖製造技術

    北岡本光, 西本完

    食品試験研究成果情報   ( 26 )   64 - 65   2014年3月

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  • アクセプタ結合部位を改変したRuminococcus albus由来マンノシドホスホリラーゼの基質特異性と反応特性の解析

    尾高伶, 佐分利亘, 福士江里, 西本完, 北岡本光, 松井博和, 森春英

    日本農芸化学会大会講演要旨集(Web)   2014   3D02P09 (WEB ONLY)   2014年3月

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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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  • Aa-3 Family GH19キチナーゼのサブサイト構造に基づいた長鎖キチンオリゴ糖の酵素合成戦略(キチナーゼ・キチン関連酵素,一般講演,日本応用糖質科学会平成25年度大会(第62回))

    道善 聡, 大沼 貴之, 本多 裕司, 北岡 本光, 深溝 慶

    応用糖質科学 : 日本応用糖質科学会誌   3 ( 3 )   B25   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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  • Ap-11 Listeria innocua由来β-グルコシダーゼの機能解析(セルラーゼ,一般講演,日本応用糖質科学会平成25年度大会(第62回))

    吉田 龍太, 中島 将博, 宮永 顕正, 北岡 本光, 田口 速男

    応用糖質科学 : 日本応用糖質科学会誌   3 ( 3 )   B30   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-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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  • この人に聞く : 研・究・最・前・線 ビフィズス菌のヒトミルクオリゴ糖代謝機構 : 分子機構から読み解く 農研機構 食品総合研究所 食品バイオテクノロジー研究領域 酵素研究ユニット長 北岡本光氏に聞く

    北岡 本光

    Food style 21   17 ( 6 )   8 - 11   2013年6月

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    記述言語:日本語   出版者・発行元:食品化学新聞社  

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  • ビフィズス菌とヒトミルクオリゴ糖

    北岡本光

    精糖技術研究会講演要旨   111th   26 - 29   2013年

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  • Cp-10 Listeria innocua由来新規酵素1,2-β-D-オリゴグルカン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))

    豊泉 大幸, 阿部 紘一, 中島 将博, 田口 速男, 平石 正男, 五十嵐 圭日子, 鮫島 正浩, 北岡 本光

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

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    記述言語:日本語   出版者・発行元:一般社団法人 日本応用糖質科学会  

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  • Listeria innocua由来の新規酵素1,2-β-D-オリゴグルカンホスホリラーゼの機能解析

    豊泉大幸, 阿部紘一, 中島将博, 中島将博, 田口速男, 平石正男, 五十嵐圭日子, 鮫島正浩, 北岡本光

    日本農芸化学会大会講演要旨集(Web)   2013   2013年

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  • ビフィズス菌のヒトミルクオリゴ糖代謝に関わる酵素群の同定および利用

    北岡本光

    Actinomycetologica   26 ( 2 )   12 - 13   2012年12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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    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

  • Bp1-9 アミノ酸置換によるガラクトキナーゼの基質特異性改変(ホスホリラーゼおよび関連酵素,一般講演,日本応用糖質科学会平成24年度大会(第61回))

    西本 完, 北岡 本光

    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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  • Ba2-7 Photobacterium profundum SS9由来エキソβ-D-グルコサミニダーゼの糖鎖合成酵素化(バイオマス関連酵素(キチナーゼ・キトサナーゼ他),一般講演,日本応用糖質科学会平成24年度大会(第61回))

    新井 祥子, 鳥本 真理恵, 鈴木 健太郎, 伏信 進矢, 北岡 本光, 本多 裕司

    応用糖質科学 : 日本応用糖質科学会誌   2 ( 3 )   B52   2012年8月

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  • Bp1-8 耐熱性galacoto-N-biose/lacto-N-biose phosphorylase(GLNBP)の探索条件の最適化(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))

    小山 善幸, 西本 完, 北岡 本光

    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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  • 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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  • ヒトミルクオリゴ糖によるビフィズス菌増殖促進作用の分子機構

    北岡本光

    ミルクサイエンス   61 ( 2 )   115 - 124   2012年8月

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    記述言語:日本語   出版者・発行元:日本酪農科学会  

    新生児腸管でのビフィズス菌主体の菌叢形成は乳児の感染防御に重要である。母乳栄養乳児のビフィズス菌の腸内定着が乳児の健康維持に重要であると認識された。母乳にはラクトース以外に三糖以上のオリゴ糖の混合物であるヒトミルクオリゴ糖(HMOs)を10-20g/L含んでいる。牛乳(ウシ常乳)にはラクトース以外の糖はほとんど含まれていないため,牛乳ベースの人工乳にはHMOsに相当する成分が含まれていない。プレバイオティクスオリゴ糖はビフィズス菌や乳酸菌などに選択的に資化されることにより機能を発揮する。現在の人工乳にはプレバイオティックオリゴ糖が添加されており,人工栄養乳児腸管にもビフィズス菌が定着することにより健康上の問題はほぼ解決されている。しかしながら,現在でも母乳栄養乳児と人工栄養乳児での腸内でのビフィズス菌の定着速度には差が見られており,母乳中のビフィズス因子の理解は未だ重要な課題として残されている。最近明らかになったビフィズス菌のHMOsの代謝経路を中心にビフィズス菌増殖促進作用の分子機構についての理解の現状について述べる。

    DOI: 10.11465/milk.61.115

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  • ビフィズス菌酵素を利用したオリゴ糖の実用的調製法

    北岡本光, 西本完, 井上公輔, 知久和寛, 中島将博

    応用糖質科学   2 ( 2 )   136 - 141   2012年5月

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    DOI: 10.5458/bag.2.2_136

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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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  • Bifidobacterium longum subsp.infantis由来1,3-1,4-α-L-フコシダーゼが有するユニークな基質特異性の構造学的基盤

    櫻間晴子, 伏信進矢, 北岡本光, 日高將文, 芦田久, 山本憲二, 熊谷英彦, 片山高嶺

    腸内細菌学雑誌   26 ( 2 )   2012年

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  • 1,3-1,4-α-L-フコシダーゼの結晶構造解析に基づく基質特異性の構造学的基盤

    櫻間晴子, 伏信進矢, 北岡本光, 日高將文, 芦田久, 片山高嶺, 山本憲二, 熊谷英彦

    日本農芸化学会大会講演要旨集(Web)   2012   2012年

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  • 2Cp26 ビフィズス菌由来ガラクトキナーゼのL-アラビノキナーゼへの変換(酵素学,酵素工学,一般講演)

    西本 完, 北岡 本光

    日本生物工学会大会講演要旨集   64   45 - 45   2012年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • オリゴ糖研究の最前線 その1 機能性オリゴ糖の研究 ホスホリラーゼ組み合わせ反応を用いた実用的オリゴ糖調製

    北岡本光

    応用糖質科学   1 ( 4 )   286 - 290   2011年11月

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    記述言語:日本語  

    DOI: 10.5458/bag.1.4_286

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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

  • Glycoside Hydrolase Family 63に属する酵素YgjKのグライコシンターゼ化

    宮崎剛亜, 松田佳奈, 森春英, 北岡本光, 北野克和, 西河淳, 殿塚隆史

    応用糖質科学   1 ( 3 )   (35)   2011年7月

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  • シクロイソマルトオリゴ糖グルカノトランスフェラーゼの分子解析とBacillus circulans T‐3040株における環状イソマルトオリゴ糖の新規な生合成経路の発見

    舟根和美, 川端康之, 鈴木龍一郎, 藤本瑞, 北岡本光, 木村淳夫, 小林幹彦

    応用糖質科学   1 ( 2 )   179 - 185   2011年4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  

    <i>Bacillus circulans</i> T-3040株由来の環状イソマルトオリゴ糖グルカノトランスフェラーゼ (CITase,EC2.4.1.248) は,デキストランから環状イソマルトオリゴ糖および環状イソマルトメガロ糖 (両者あわせてサイクロデキストラン,CI) を合成する酵素であり,中でもグルコース8分子から成るCI-8を最も多く生産する。部位特異的変異導入によりN末端領域 (Ser1-Gly403) に在るAsp145,Asp270,Glu342が触媒活性に重要な役割を果たすことが示唆された。C末端領域を4つ (R1, Tyr404-Tyr492; R2, Glu493-Ser596; R3, Gly597-Met700; R4, Lys701-Ser934) に分け欠失変異導入を行った結果,R2およびR3はCITaseに必須,R1およびR4は糖質結合モジュールであり,R1はCI-8生産特異性と酵素の安定化にも関与することが示唆された。CI-Taseは,生産菌をデキストランを炭素源として培養した場合に生産誘導されると考えられていたが,T-3040株のCITaseはデンプンを炭素源として培養した場合にも生産誘導された。これまでのCI生産にはショ糖からデキストランを生産する菌株を必要としていたが,1菌株でデンプンからCIを生産する系が存在することが示唆された。

    DOI: 10.5458/bag.1.2_179

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  • 澱粉原料からのサイクロデキストラン製造技術の開発

    舟根和美, 木村啓太郎, 鈴木龍一郎, 北岡本光, 儀部茂八, 渡嘉敷唯章, 川端康之, 小林幹彦, 藤本瑞, 木村敦夫

    食品試験研究成果情報   ( 23 )   30 - 31   2011年3月

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    記述言語:日本語  

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  • 母乳によるビフィズス菌増殖の分子機構

    北岡本光

    日本乳酸菌学会誌   22 ( 1 )   15 - 25   2011年3月

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  • 1Ep02 ビフィズス菌のヒトミルクオリゴ糖代謝経路(酵素学・酵素工学/タンパク質工学/糖鎖工学,一般講演)

    北岡 本光, 西本 完, 片山 高嶺, 清原 正志, 和田 潤, 芦田 久, 山本 憲二, 浦島 匡

    日本生物工学会大会講演要旨集   63   39 - 39   2011年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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    その他リンク: http://search.jamas.or.jp/link/ui/2012048534

  • 1Ep03 ビフィズス菌によるヒトミルクオリゴ糖の分解 : 培養上清におけるオリゴ糖濃度の経時変化(酵素学・酵素工学/タンパク質工学/糖鎖工学,一般講演)

    片山 高嶺, 朝隈 貞樹, 畑山 恵美, 浦島 匡, 吉田 永史奈, 熊谷 英彦, 芦田 久, 廣瀬 潤子, 北岡 本光, 山本 憲二

    日本生物工学会大会講演要旨集   63   40 - 40   2011年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • 母乳によるビフィズス菌増殖の分子機構

    北岡本光

    日本乳酸菌学会誌   21 ( 3 )   237 - 238   2010年11月

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  • 選択的ビフィズス菌増殖活性を持つオリゴ糖の設計

    北岡本光

    バイオサイエンスとインダストリー   68 ( 2 )   131 - 132   2010年3月

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  • Enzymatic Production of Cellobiose from Starch and Its Reduction to Cellobiitol

    鈴木雅之, 北岡本光, 谷口肇

    J Appl Glycosci   57 ( 2 )   113-119 (J-STAGE) - 119   2010年

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  • 糖質加水分解酵素ファミリー3(GH3)Kluyveromyces marxianus由来β-グルコシダーゼ(KmBglI)のX線結晶構造解析

    日高將文, 吉田永史奈, 伏信進矢, 北岡本光, 片山高嶺, 熊谷英彦

    日本農芸化学会大会講演要旨集   2010   2010年

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  • 母乳によるビフィズス菌増殖メカニズムの解明および鍵となるヒトミルクオリゴ糖成分の製造技術の開発

    北岡本光

    食品と容器   50 ( 8 )   486 - 491   2009年8月

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    記述言語:日本語   出版者・発行元:缶詰技術研究会  

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  • 食品用酵素と食品開発 食品用酵素利用の現状と動向

    北岡本光

    食品の包装   40 ( 2 )   18 - 20   2009年3月

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  • Crystal Structure of GH101 Endo-.ALPHA.-N-acetylgalactosaminidase from Bifidobacterium longum

    鈴木龍一郎, 片山高嶺, 伏信進矢, 北岡本光, 熊谷英彦, 若木高善, 祥雲弘文, 芦田久, 山本憲二

    J Appl Glycosci   56 ( 2 )   105-110 (J-STAGE)   2009年

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    記述言語:英語  

    DOI: 10.5458/jag.56.105

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  • Conversion of an Inverting Glycoside Hydrolase into Glycosynthase

    本多裕司, 伏信進矢, 日高將文, 若木高善, 祥雲弘文, 谷口肇, 北岡本光

    J Appl Glycosci   56 ( 2 )   119-125 (J-STAGE)   2009年

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    記述言語:英語  

    DOI: 10.5458/jag.56.119

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  • 1,2-α-L-フコシダーゼのグライコシンターゼ化による2′-フコシルラクトースの合成

    和田潤, 本多裕司, 長江雅倫, 加藤龍一, 若槻壮市, 片山高嶺, 谷口肇, 熊谷英彦, 北岡本光, 山本憲二

    日本農芸化学会大会講演要旨集   2009   2009年

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  • セロデキストリンホスホリラーゼを用いた高結晶性セルロースの酵素合成

    平石正男, 五十嵐圭日子, 木村聡, 和田昌久, 鮫島正浩, BRUMER Harry, III, IBATULLIN Farid M., 北岡本光

    セルロース学会年次大会講演要旨集   16th   2009年

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  • 担子菌由来β-1,3-グルカナーゼの結晶構造解析

    石田卓也, 伏信進矢, 川合理恵, 北岡本光, 五十嵐圭日子, 鮫島正浩

    PFシンポジウム要旨集   26th   2009年

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  • アノマー反転型β-キシロシダーゼのグライコシンターゼ化

    本多裕司, 片山高嶺, 谷口肇, 熊谷英彦, 日高將文, 伏信進矢, 北岡本光

    日本農芸化学会大会講演要旨集   2009   2009年

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  • ビフィズス菌由来ガラクト-N-ビオース/ラクト-N-ビオースIホスホリラーゼの結晶構造解析

    日高將文, 日高將文, 西本完, 中島将博, 北岡本光, 若木高善, 祥雲弘文, 伏信進矢

    PFシンポジウム要旨集   26th   2009年

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  • ヒトミルクオリゴ糖に含まれるビフィズス因子にせまる ビフィズス菌によるラクト‐N‐ビオースIの選択的代謝とその製造法

    西本完, 北岡本光

    化学と生物   46 ( 8 )   522 - 524   2008年8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.46.522

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  • Vibrio proteolyticus由来GH19酵素の機能解析

    本多裕司, 谷口肇, 北岡本光

    キチン・キトサン研究   14 ( 2 )   172 - 173   2008年7月

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  • 糖質ホスホリラーゼの立体構造からわかる加水分解酵素との進化的関連 一筋縄ではいかないグリコシダーゼの反応機構と分類

    伏信進矢, 日高將文, 北岡本光

    化学と生物   46 ( 5 )   308 - 309   2008年5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.46.308

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  • 地球温暖化が農林水産業に与える影響の評価及び対策技術の開発―化石燃料に代替する新エネルギー生産実用化技術の開発―第2章 農林系廃棄物のエタノール変換システムの開発 1 糖化・エタノール発酵技術(1)低利用多糖類を分解する酵素の分子レベルでの特性改良

    林清, 北岡本光, 韮澤悟

    農林水産省農林水産技術会議事務局研究成果   ( 443 )   32 - 36   2008年1月

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  • GHファミリー1に属する&beta;-グルコシダーゼBGL1Bのセロビオース認識機構について

    塚田 剛士, 五十嵐 圭日子, 伏信 進矢, 北岡 本光, 鮫島 正浩

    Journal of Applied Glycoscience Supplement   2008 ( 0 )   118 - 118   2008年

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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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  • ビフィズス菌の選択的生育に関わる新規ホスホリラーゼの構造解析

    日高將文, 伏信進矢, 西本完, 北岡本光, 若木高善, 祥雲弘文

    日本農芸化学会関東支部講演要旨集   2008 ( Oct )   2008年

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  • ビフィズス菌由来ガラクト-N-ビオース/ラクト-N-ビオースIホスホリラーゼの構造と機能

    日高將文, 伏信進矢, 西本完, 中島将博, 北岡本光

    日本乳酸菌学会誌   19 ( 2 )   2008年

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  • 3Ga11 固定化酵素によるラクト-N-ビオースIの調製(酵素学,酵素工学,タンパク質工学,糖鎖工学,一般講演)

    西本 完, 北岡 本光

    日本生物工学会大会講演要旨集   20   187 - 187   2008年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • ビフィズス菌由来ガラクト-N-ビオース/ラクト-N-ビオースIホスホリラーゼの構造解析:基質結合によるTIMバレル骨格の構造変化

    日高將文, 西本完, 北岡本光, 伏信進矢

    日本蛋白質科学会年会プログラム・要旨集   8th   2008年

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  • Kinetic analyses of mutagenic hyperthermostable glycogen phosphorylase from Aquifex aeolicus on a 27 MHz quartz-crystal microbalance

    Toshiaki Mori, Takanori Nihira, Motomitsu Kitaoka, Yoshio Okahata

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   48 ( 3-4 )   103 - 103   2007年9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:ELSEVIER SCIENCE BV  

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  • Purification, crystallization and preliminary X-ray analysis of the galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) of the ABC transporter from Bifidobacterium longum JCM1217

    Jun Wada, Ryuichiro Suzuki, Shinya Fushinobu, Motomitsu Kitaoka, Takayoshi Wakagi, Hirofumi Shoun, Hisashi Ashida, Hidehiko Kumagai, Takane Katayama, Kenji Yamamoto

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   63 ( 9 )   751 - 753   2007年9月

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    記述言語:英語   出版者・発行元:INT UNION CRYSTALLOGRAPHY  

    A recombinant galacto-N-biose-/ lacto-N-biose I-binding protein ( GL-BP) from Bifidobacterium longum JCM1217 has been prepared and crystallized by the hanging-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 0.01 M zinc sulfate, 0.1 M MES buffer pH 5.9 -6.4 and 20 -22%( v/ v) PEG MME 550 in the presence of 5 mM disaccharide ligands. Suitable crystals grew after 10 d incubation at 293 K. The crystals belong to space group C222(1), with unit-cell parameters a = 106.3, b = 143.6, c = 114.6 angstrom for the lacto-N-biose I complex and a = 106.4, b = 143.4, c = 115.5 angstrom for the galacto-N-biose complex, and diffracted to 1.85 and 1.99 angstrom resolution, respectively.

    DOI: 10.1107/S1744309107036263

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  • ラン藻由来β-1, 3-1, 4-グルカナーゼの加水分解特異性

    藤村 悠介, 林 加奈子, 田茂井 政宏, 北岡 本光, 深溝 慶

    キチン・キトサン研究 = Chitin and chitosan research   13 ( 2 )   200 - 200   2007年8月

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  • キシラン分解系における新しい酵素の発見と応用:還元末端エキソオリゴキシラーゼ(Rex)

    本多裕司, 伏信進矢, 日高將文, 若木高善, 祥雲弘文, 谷口肇, 北岡本光

    キチン・キトサン研究   13 ( 2 )   80 - 81   2007年7月

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  • ヒトミルクオリゴ糖中のビフィズス因子と推定される二糖であるラクト-N-ビオースI製造技術の開発

    北岡 本光, 西本 完

    腸内細菌学雑誌 = Journal of intestinal microbiology   21 ( 2 )   64 - 64   2007年4月

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  • ビフィズス増殖に効果的なミルクオリゴ糖成分の製造方法

    北岡本光

    食品試験研究成果情報   ( 19 )   42 - 43   2007年3月

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  • ホスホリラーゼ工学による有用オリゴ糖の調製

    北岡本光

    食糧その科学と技術   ( 45 )   19 - 32   2007年3月

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  • ビフィズス菌の新規ガラクトース代謝経路とビフィズス因子の関係

    北岡本光, 西本完

    ミルクサイエンス   55 ( 3 )   171 - 174   2007年1月

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  • セロデキストリンホスホリラーゼを用いたセルロースの酵素合成

    平石正男, 五十嵐圭日子, 木村聡, 和田昌久, 北岡本光, 鮫島正浩

    セルロース学会年次大会講演要旨集   14th   2007年

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  • 1-01 原油から直接抽出されるDNAの解析 : 真正細菌16S rRNA遺伝子の塩基配列のプロファイリングと近縁種の解析(群集構造解析1,口頭発表)

    山根 國男, 中山 剛, 野村 暢彦, 中島 敏明, 内山 裕夫, 北岡 本光

    日本微生物生態学会講演要旨集   ( 23 )   37 - 37   2007年

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    記述言語:日本語   出版者・発行元:日本微生物生態学会  

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  • 3I11-5 酵素法によるガラクト-N-ビオースの合成(ペプチド工学・プロテオーム,糖鎖工学,一般講演)

    西本 完, 北岡 本光

    日本生物工学会大会講演要旨集   19   204 - 204   2007年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • 還元末端オリゴキシラナーゼ(Rex)によるグライコシンターゼ反応の高効率化

    本多裕司, 伏信進矢, 日高將文, 若木高善, 祥雲弘文, 谷口肇, 北岡本光

    Journal of Applied Glycoscience   54 ( Suppl. )   2007年

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  • コンピュータ解析で明らかにするGH94セロビオースホスホリラーゼの反応機構

    伏信進矢, MERTZ Blake, HILL Anthony D., 日高將文, 北岡本光, 若木高善, 祥雲弘文, REILLY Peter J.

    Journal of Applied Glycoscience   54 ( Suppl. )   2007年

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  • 合目的変異導入によるラクトースホスホリラーゼの創出

    新井健司, 伏信進矢, 鈴木龍一郎, 日高将文, 片山高嶺, 北岡本光, 若木高善, 祥雲弘文

    日本農芸化学会大会講演要旨集   2007   2007年

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  • ビフィズス菌の新規ガラクトース代謝経路とビフィズス因子の関係

    北岡本光

    ミルクサイエンス   55 ( 2 )   95 - 96   2006年7月

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  • オリゴ糖ライブラリーの調製と利用

    北岡本光

    食品研究成果情報   ( 18 )   38 - 39   2006年3月

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  • 新規な遺伝子ランダム変異導入技術

    北岡本光, 藤井亮太, 小林厚志, 林清

    ブレインテクノニュース   ( 113 )   5 - 8   2006年1月

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  • 新規オリゴ糖の開発に向けた酵素の遺伝子レベルでの改変

    林清, 北岡本光

    ブレインテクノニュース   ( 113 )   1 - 4   2006年1月

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  • セロデキストリンホスホリラーゼを用いたセルロースIIの酵素合成

    平石正男, 和田昌久, 五十嵐圭日子, 鮫島正浩, 北岡本光

    セルロース学会年次大会講演要旨集   13th   2006年

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  • 1F10-5 ビフィズス菌由来UDP-Glc-Gal 1-P uridylyltransferase遺伝子のクローニングと諸性質(酵素学,酵素工学,タンパク質工学,一般講演)

    西本 完, 北岡 本光

    日本生物工学会大会講演要旨集   18   92 - 92   2006年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • Close Up実験法 Series147 かんたん“ワンステップ”ランダム変異導入法

    藤井亮太, 北岡本光

    実験医学   23 ( 16 )   2493 - 2497   2005年10月

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  • GH-94加リン酸分解酵素の反応機構と基質認識

    日高 將文, 本多 裕司, 北岡 本光, 韮澤 悟, 林 清, 若木 高善, 祥雲 弘文, 伏信 進矢

    Journal of applied glycoscience   52 ( 2 )   191 - 196   2005年4月

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  • ラクトNビオースホスホリラーゼのビフィズス菌腸内定着への関与 : 母乳栄養乳児の腸管定着に関する仮説

    北岡 本光, 西本 完

    腸内細菌学雑誌 = Journal of intestinal microbiology   19 ( 2 )   86 - 86   2005年4月

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  • 還元末端から単糖を遊離するエキソ型キシラナーゼの発見

    北岡本光

    食品研究成果情報   ( 17 )   40 - 41   2005年3月

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  • 生物機能を利用した内分泌かく乱物質分解技術の開発 2.森林生物の機能を活用した内分泌かく乱物質分解技術の開発(2)糖鎖含有ポリマーの活用による内分泌かく乱物質の無毒化技術の開発

    林清, 北岡本光

    農林水産省農林水産技術会議事務局研究成果   ( 433 )   423 - 425   2005年3月

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  • 糖質加リン酸分解酵素研究の新展開

    北岡本光

    バイオサイエンスとインダストリー   63 ( 3 )   171 - 174   2005年3月

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  • テクノ・トレンド ローリングサークル増幅を応用した“ワンステップ”ランダム変異導入

    藤井亮太, 北岡本光

    バイオテクノロジージャーナル   5 ( 2 )   200 - 202   2005年3月

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  • 糖質加水分解酵素のメカニズム決定法

    北岡本光, 本多裕司

    J Appl Glycosci   52 ( 1 )   79 - 80   2005年1月

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  • 担子菌Phanerochaete chrysosporiumが生産するβ-1,3-グルカナーゼの反応特性

    川合理恵, 五十嵐圭日子, 北岡本光, 鮫島正浩

    Journal of Applied Glycoscience   52 ( Suppl. )   2005年

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  • 還元末端エキソオリゴキシラナーゼ(REX)の還元末端β-アノメリック水酸基認識機構

    本多裕司, 伏信進矢, 日高将文, 日高将文, 若木高善, 祥雲弘文, 北岡本光

    Journal of Applied Glycoscience   52 ( Suppl. )   2005年

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  • 還元末端エキソオリゴキシラナーゼ(REX)のX線結晶構造解析

    伏信進矢, 日高将文, 日高将文, 本多裕司, 若木高善, 祥雲弘文, 北岡本光

    Journal of Applied Glycoscience   52 ( Suppl. )   2005年

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  • Cellvibrio gilvus由来セロビオースホスホリラーゼの構造と反応特異性

    日高将文, 日高将文, 伏信進矢, 林清, 祥雲弘文, 若木高善, 北岡本光

    Journal of Applied Glycoscience   52 ( Suppl. )   2005年

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  • Transglycosylation reaction of xylanase B from the hyperthermophilic Thermotoga maritima with the ability of synthesis of tertiary alkyl beta-D-xylobiosides and xylosides

    ZQ Jiang, YP Zhu, L Li, XH Yu, Kusakabe, I, M Kitaoka, K Hayashi

    JOURNAL OF BIOTECHNOLOGY   114 ( 1-2 )   125 - 134   2004年10月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    The recombinant xylanase B (XynB) of Thermotoga maritima MSB8 was characterized and was found to cleave beta-nitrophenyl beta-D-xyloside via the transglycosylation reaction in the previous study. XynB was activated in the presence of alcohols, and XynB activity was increased by iso-propanol (2 M) to 2.1-fold. This type of activation was investigated and was shown to be due to the transglycosylation activity with beta-nitrophenyl beta-D-xylobioside being converted to alkyl beta-D-xylobiosides in the presence of XynB and alcohols. Through the transglycosylation reaction, alkyl beta-xylosides and xylobiosides were simultaneously produced in the presence of xylan and alcohols. Primary alcohols were found to be the best acceptors. The highest yields of alkyl beta-xylosides and xylobiosides were 33% and 50% of the total sugar, respectively. XynB showed a great ability to transfer xylose and xylobiose to secondary alcohol acceptors, and was unique for being able to synthesize the tertiary alkyl beta-xylosides and xylobiosides with high yields of 18.2% and 11.6% of the total sugar, respectively. This is the first report of a xylanase with the ability to synthesize tertiary alkyl beta-xylosides and xylobiosides. The specificity of the beta-linkage was confirmed by the proton nuclear magnetic resonance (H-1 NMR). Thus, XynB of T maritima appears to be an ideal enzyme for the synthesis of useful alkyl beta-xylosides and xylobiosides. (C) 2004 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.jbiotec.2004.05.007

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  • 1pD15-1 GH10キシラナーゼのループ4に保存されたアルギニン残基変異酵素の解析(酵素学・酵素工学・タンパク質工学,一般講演)

    西本 完, 伏信 進矢, 北岡 本光, 林 清

    日本生物工学会大会講演要旨集   16   75 - 75   2004年8月

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    記述言語:日本語   出版者・発行元:公益社団法人日本生物工学会  

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  • Vibrio proteolyticus 由来キトビオースホスホリラーゼの反応機構

    本多 裕司, 北岡 本光, 日高 將文, 伏信 進矢, 林 清

    キチン・キトサン研究 = Chitin and chitosan research   10 ( 2 )   90 - 91   2004年7月

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  • Purification and characterization of an intracellular cycloalternandegrading enzyme from Bacillus sp. NRRL B-21195 (vol 339, pg 1179, 2004)

    YK Kim, M Kitaoka, K Hayashi, CH Kim, GL Cote

    CARBOHYDRATE RESEARCH   339 ( 9 )   1663 - 1663   2004年6月

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    記述言語:英語   出版者・発行元:ELSEVIER SCI LTD  

    DOI: 10.1016/j.carres.2004.05.001

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  • 糖加水分解酵素ファミリー18に属するキチナーゼの反応機構解析

    本多 裕司, 北岡 本光, 徳安 健, 林 清

    Journal of applied glycoscience   51 ( 2 )   155 - 160   2004年4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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  • 1pD14-3 担子菌Phanerochaete chrysosporium由来ラミナリナーゼの機能解析(酵素学・酵素工学・タンパク質工学,一般講演)

    川合 理恵, 吉田 誠, 五十嵐 圭日子, 北岡 本光, 鮫島 正浩

    日本生物工学会大会講演要旨集   16   2004年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • 2B15-4 ローリングサークル型増幅法を利用した簡便なランダム変異導入(酵素学・酵素工学・タンパク質工学,一般講演)

    藤井 亮太, 北岡 志光, 林 清

    日本生物工学会大会講演要旨集   16   126 - 126   2004年

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  • Characterization of a hyperthermostable glycogen phosphorylase from Aquifex aeolicus expressed in Escherichia coli

    SH Bhuiyan, A Abu Rus'd, M Kitaoka, KB Ayashi

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   22 ( 3-4 )   173 - 180   2003年6月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    The glycogen phosphorylase gene (glgP) of Aquifex aeolicus (Aae), a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli and the characteristics of the expressed enzyme were examined. The recombinant enzyme was purified to homogeneity by heat-treatment at 70degreesC for 15 min to denature the contaminating E. coli proteins, followed by Ni-NTA agarose column chromatography to selectively trap the His-tagged enzyme. The purified enzyme gave a single band on SDS-PAGE with a molecular mass of approximately 80 kDa. The enzyme displayed optimal activity at pH 6.5 and was stable in the pH range from 4.0 to 10.0. The temperature at which optimal enzyme activity was observed was 100 degreesC and the enzyme retained 66% of its original activity after heating at 100 degreesC for 30 min. Kinetic studies using the purified enzyme demonstrated that the smallest primer molecule accepted for catalysis in the synthetic direction was maltotriose (G3) and that the smallest effective substrate for the reverse process, phosphorolysis, was maltotetraose (G4). The K-m and k(cat) values were determined for various oligosaccharides (G3-G7) in both synthetic and phosphorolytic reactions and, remarkably, a maximum degree of specificity was observed toward substrates in the phosphorolytic direction. (C) 2003 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(03)00029-8

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  • A cycloamylose-forming hyperthermostable 4-alpha-glucanotransferase of Aquifex aeolicus expressed in Escherichia coli

    SH Bhuiyan, M Kitaoka, K Hayashi

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   22 ( 1-2 )   45 - 53   2003年4月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    The gene (malM) encoding for 4-alpha-glucanotransferase (4alphaGTase) from the hyperthermophilic bacterium Aquifex aeolicus was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity and its behavior towards various substrates was examined. The enzyme was hyperthermostable, exhibiting maximal activity at 90degreesC and it retained 70% of its original activity after incubation at 90degreesC for 30 min. A low affinity was observed for the enzyme towards maltose (Km = 71 mM) and the k(cat) /K-m value for maltotriose was approximately 166 times greater than that observed for maltose but the values did not change significantly with larger maltooligosaccharides. The A. aeolicus 4aGTase produced cycloamylose with a minimum degree of polymerization (DP) of 16, whereas the cycloamylose produced by the amylomaltase from the thermophilic bacterium Thermus aquaticus, which is also a Type II4alphaGTase, produced a cycloamylose with a minimum DP of 22. These findings indicate that the A. aeolicus 4alphaGTase differs from the T. aquaticus amylomaltase and the glucanotransferases produced by other hyperthermophilic organisms. (C) 2003 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(03)00005-5

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  • 超好熱性細菌ゲノム上から発見された新規αグルクロニダーゼ

    北岡本光

    食品研究成果情報   ( 15 )   38 - 39   2003年3月

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  • Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

    SL Mangala, FS Kittur, M Nishimoto, K Sakka, K Ohmiya, M Kitaoka, K Hayashi

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   21 ( 4-6 )   221 - 230   2003年2月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60degreesC and the enzyme was stable up to 50degreesC. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan. (C) 2002 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(02)00226-6

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  • 糖質加リン酸分解酵素に関する基礎および応用研究

    北岡本光

    J Appl Glycosci   50 ( 1 )   83 - 87   2003年1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  

    DOI: 10.5458/jag.50.83

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  • 分子動力学法によるセロビオース脱水素酵素と基質との相互作用の解析

    日比 航介, 寺田 透, 中村 周吾, 五十嵐 圭日子, 北岡 本光, 清水 謙多郎

    生物物理   43 ( 0 )   S35   2003年

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    記述言語:日本語   出版者・発行元:一般社団法人 日本生物物理学会  

    DOI: 10.2142/biophys.43.S35_1

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  • 2B11-4 キシラナーゼ置換酵素におけるアルカリ側での活性発現と温度安定性

    西本 完, 北岡 本光, 林 清

    日本生物工学会大会講演要旨集   15   72 - 72   2003年

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  • β‐1,4オリゴ糖の合成ツールとして有用なセロオリゴ糖を加リン酸分解する酵素

    北岡本光

    Cellul Commun   9 ( 4 )   213 - 215   2002年12月

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  • Adsorption of bisphenol A by cross-linked beta-cyclodextrin polymer

    M Kitaoka, K Hayashi

    JOURNAL OF INCLUSION PHENOMENA AND MACROCYCLIC CHEMISTRY   44 ( 1-4 )   429 - 431   2002年12月

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    記述言語:英語   出版者・発行元:SPRINGER  

    An insolubilized beta-cyclodextrin derivative (polyCD) was prepared by polymerizing cyclodextrin with epichlrohydrine. By stirring 5 mg/ml of polyCD in 0.2 mM bisphenol A (BPA) solution at pH 7.0 for 2 h, more than 98% of BPA was adsorbed on polyCD. The capacity of the adsorption was determined to be 84 mg-BPA/g-polyCD, meaning 0.65 mol-BPA/mol-CD (in polyCD). The effect of pH on the adsorption was studied and found that BPA was effectively adsorbed on polyCD at pH between pH 2.2-9.1, but the efficiency decreased at pH 10.8, suggesting that it did not adsorb the BPA anion. The polyCD did not adsorb aromatic amino acids, indicating that BPA could selectively be removed from a solution containing amino acids.

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  • スクロースからセロビオースを生産する 高純度,高収率,経済的な調製を可能にした“スクロース異性化酵素”とは?

    北岡本光

    化学と生物   40 ( 8 )   498 - 500   2002年8月

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  • オリゴ糖基質を用いた Serratia marcescens キチナーゼ (ChiA, ChiB) の反応機構解析

    本多 裕司, 北岡 本光, 徳安 健, 佐々木 千絵, 深溝 慶, 林 清

    キチン・キトサン研究 = Chitin and chitosan research   8 ( 2 )   160 - 161   2002年7月

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  • In vitro stepwise autoprocessing of the proform of pro-aminopeptidase processing protease from Aeromonas caviae T-64

    B Tang, S Nirasawa, M Kitaoka, K Hayashi

    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY   1596 ( 1 )   16 - 27   2002年4月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    PA protease (pro-aminopeptidase processing protease) is an extracellular zinc metalloprotease produced by the Gram-negative bacterium Aeromonas caviae T-64. The 590-amino-acid precursor of PA protease is composed of a putative 19-amino-acid signal sequence, a 165-amino-acid N-terminal propeptide, a 33 kDa mature protease domain and an 11 kDa C-terminal propeptide. The proform of PA protease, which was produced as inclusion bodies in Escherichia coli, was subjected to in vitro refolding. It was revealed that the processing of the proform involved a stepwise autoprocessing mechanism. Firstly, the N-terminal propeptide was autocatalytically removed on completion of refolding and secondly, the C-terminal propeptide was autoprocessed after the degradation of the N-terminal propeptide. Both the N- and C-terminal propeptides existed as intact peptides after their successive removal, and they were subsequently degraded gradually. The degradation of the N-terminal propeptide appears to be the rate-limiting step in the maturation of the proform of PA protease. (C) 2002 Elsevier Science B.V. All rights reserved.

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  • P-nitrophenyl glycosides of beta-1,4-gluco/xylo-disaccharides for characterization of xylanase activity.

    M Kitaoka, M Nishimoto, A Kobayashi, Y Honda, K Hayashi

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   223   U128 - U128   2002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:AMER CHEMICAL SOC  

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  • Carbohydrate-processing phosphorolytic enzymes

    M Kitaoka, K Hayashi

    TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY   14 ( 75 )   35 - 50   2002年1月

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    記述言語:英語   掲載種別:書評論文,書評,文献紹介等   出版者・発行元:GAKUSHIN PUBL CO  

    Phosphorolytic enzymes that act on carbohydrates are interesting enzymes not only in terms of their basic properties but also in regard to their various applications. However, they have not been studied in as much detail as other types of enzymes such as the hydrolases and the synthases. Phosphorolytic enzymes have attracted attention because of their application in the synthesis of carbohydrate chains, exploiting their strict regio-specificities. In addition, a number of now phosphorolytic enzymes have been discovered recently. In this review, the characteristics and applications of the phosphorolytic enzymes are described in an overview of the current research that has been performed on this interesting class of enzymes.

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  • An investigation of the pH-activity relationships of Cex, a family 10 xylanase from Cellulomonas fimi: Xylan inhibition and the influence of nitro-substituted aryl-β-D-xylobiosides on xylanase activity

    Yuji Honda, Motomitsu Kitaoka, Kazuo Sakka, Kunio Ohmiya, Kiyoshi Hayashi

    Journal of Bioscience and Bioengineering   93 ( 3 )   313 - 317   2002年

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    記述言語:英語  

    The kinetic parameters of Cex, a family 10 xylanase from Cellulomonas fimi, were determined at various pH levels using soluble birchwood xylan (BWX) as a natural polymeric substrate along with three other synthetic aryl-β-D-xylobioside substrates. Using BWX, a high level of substrate inhibition was observed which increased with decreasing pH. In contrast, typical Michaelis-Menten-type profiles were obtained using the three aryl-β-D-xylobiosides as substrates. The kcat values determined using o-nitrophenyl-β-D-xylobioside did not change as the pH increased, whereas the kcat values obtained with BWX, phenyl-β-D-xylobioside and p-nitrophenyl-β-D-xylobioside decreased, suggesting that the presence of an ortho nitro group affects the activity displayed by Cex. These differences were not observed with XynB from Clostridium stercorarium F9, a member of the same family of xylanases as Cex. These results indicate that a careful evaluation is required when employing substituted aryl-β-D-xylobiosides in the characterization of xylanases.

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  • 酵素の特性を分子レベルで改良する技術

    林清, 金子哲, 韮沢悟, 北岡本光

    食品の試験と研究   ( 37 )   12 - 16   2002年

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  • 大腸菌で発現させたClostridium thermocellum YM4株由来セロデキストリンホスホリラーゼ

    Krishnareddy Manem, 金 然桂, 北岡 本光, 森 隆, 林 清

    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience   49 ( 1 )   1 - 8   2002年

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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  

    PCR法を用いて,Clostridium thermocellum YM4株由来セロデキストリンホスホリラーゼ遺伝子のクローニングを行った.セロデキストリンホスホリラーゼ遺伝子は,984アミノ酸残基のポリペプチド鎖をコードする2952bpのオープンリーディングフレームを含んでいた.得られたアミノ酸配列は,C.thermocellum ATCC27405株由来セロデキストリンボスホリラーゼと高い相同性(92%)を示したが,c.stercorarium由来のセロデキストリンホスホリラーゼとは相同性が低かった(20%).組替セロデキストリンホスホリラーゼはセロトリオース以上のセロオリゴ糖を加リン酸分解し,セロビオース以上のセロオリゴ糖をアクセプターとして認識した.温度安定性は60℃以下であり,至適pHは7.5であった.

    DOI: 10.5458/jag.49.1

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  • Escherichia coliで発現したThermotoga maritima由来のエンド-β-1,4-グルカナーゼの特性

    ラーマン Md. マジブル, ブイヤン シャカワットホッサン, 韮澤 悟, 北岡 本光, 林 清

    応用糖質科学 : oyo toshitsu kagaku = Journal of applied glycoscience   49 ( 4 )   487 - 495   2002年

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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  

    Thermotoga maritima由来のエンド-β-1,4-グルカナーゼ遺伝子(TM1751,Swissprot Q9X273,GenBank AAD36816)を,大腸菌で発現し,各種カラムクロマトグラフィーにより収率21%で電気泳動的に単一バンドとなるまで精製し,得られた精製酵素の特性を明らかにした.本酵素の分子量は38kDa,至適反応温度は90。Cであり,至適反応pHは6.6であった.本酵素は,85℃ではpH4-9.5の広範囲で安定であった.PNP-β-D-セロテトラオシド,PNP-β-D-セロペンタオシドに対するKm値は,それぞれ0.25,0.24mMであり,触媒活性(kcat/Km)は,PNP-β-D-セロテトラオシドで最大であることから,本酵素には四つのサブサイトが存在するものと推定された.PNP-β-D-セロオリゴ糖の加水分化物をTLC分析したところ,セロビオース,セロトリオース主産物であり,PNP-β-D-セロビオシドからはセロトリオースが生成することから,本酵素は糖転移作用を有することが明らかとなった.本酵素は,カルボキシメチルセルロースよりも,β-1,3/1,4結合を含むバーレイグルカン,リケナンをよく加水分解した.

    DOI: 10.5458/jag.49.487

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    その他リンク: http://agriknowledge.affrc.go.jp/RN/2010660613

  • 522 Clostridium stercorarium F9株由来キシラナーゼBの分子デザインによるアルカリ領域での活性発現(酵素・酵素工学・タンパク質工学,一般講演)

    西本 完, 北岡 本光, 粟冠 和郎, 大宮 邦雄, 林 清

    日本生物工学会大会講演要旨集   14   78 - 78   2002年

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  • 473 Dissecting Agrobacterium tumefaciens β-glucosidases by splitting the gene into two pieces at non-homologous region :

    Kim Bong-Jo, Nirasawa Satoru, Kitaoka Motomitsu, Hayashi Kiyoshi

    日本生物工学会大会講演要旨集   14   69 - 69   2002年

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  • 647 A putative proline iminopeptidase of Thermotoga maritime with leucine- and lysine-p-nitroanilide hydrolyzing activities :

    Ratnayake Sunil, Selvarkumar Ponniah, Kitaoka Motomitsu, Nirasawa Satoru, Hayashi Kiyoshi

    日本生物工学会大会講演要旨集   14   106 - 106   2002年

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  • 産業用酵素の分子レベルでの特性改良

    林清, 金子哲, 町田幸子, 韮沢悟, 本多祐司, 北岡本光

    ブレインテクノニュース   ( 88 )   23 - 27   2001年11月

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  • Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside

    ZQ Jiang, A Kobayashi, MM Ahsan, L Lite, M Kitaoka, K Hayashi

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   92 ( 5 )   423 - 428   2001年11月

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    記述言語:英語   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    Thermotoga maritima MSB8 possesses two xylanase genes, xynA and xynB. The xynB gene was isolated from the genomic DNA of T maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme, produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70degreesC, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100degreesC from pH 7.0 to pH 8.5. At 50degreesC, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90degreesC. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-beta-D-xylobioside with K-m and k(cat) values of 0.0077 mM and 5.5 s(-1), respectively, at 30degreesC. It was also active towards p-nitrophenyl-beta-D-xyloside. The initial product of the cleavage of p-nitrophenyl-beta-D-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis.

    DOI: 10.1263/jbb.92.423

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  • Construction and characterization of chimeric enzymes of the Agrobacterium tumefaciens and Thermotoga maritima beta-glucosidases

    K Goyal, YK Kim, M Kitaoka, K Hayashi

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   16 ( 1 )   43 - 51   2001年11月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    The beta -glucosidases of Agrobacterium tumefaciens and Thermotoga maritima display quite distinct characteristics, both in terms of their pH optima, temperature optima and substrate specificities. Despite such differences, the encoded enzymes show homologies of approximately 45 and 37% in the amino acid sequences of the N- and C-terminal regions, respectively. Based on the amino acid alignment of these two enzymes, three chimeric beta -glucosidase genes were constructed by shuffling selected DNA regions. The chimeric genes were expressed in Escherichia coli BL 21(DE3) and two catalytically active enzymes were obtained. Unlike other chimeras which display profiles intermediate to those of their respective parents, these chimeric enzymes displayed quite different profiles. The observed thermal stability of the more catalytically active chimeric enzyme was lower than that observed for either parental enzyme, however, the kinetic parameters were more similar to those of the T. maritima beta -glucosidase. (C) 2001 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(01)00043-1

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  • TIMバレル構造を持つβ‐1,4グリカナーゼ

    本多裕司, 北岡本光, 林清

    キチン・キトサン研究   7 ( 3 )   233 - 240   2001年10月

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  • Colorimetric quantification of cellobiose employing cellobiose phosphorylase

    M Kitaoka, C Aoyagi, K Hayashi

    ANALYTICAL BIOCHEMISTRY   292 ( 1 )   163 - 166   2001年5月

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

    DOI: 10.1006/abio.2001.5049

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  • 遺伝子の部分置換による酵素特性の改良

    林清, 韮沢悟, 北岡本光

    バイオサイエンスとインダストリー   59 ( 4 )   235 - 238   2001年4月

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  • 遺伝子の部分置換による酵素特性の改良

    林 清, 韮沢 悟, 北岡 本光

    バイオサイエンスとインダストリー = Bioscience & industry   59 ( 4 )   23 - 26   2001年4月

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    記述言語:日本語   出版者・発行元:バイオインダストリ-協会  

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  • Modification of enzyme character of B-glucosidase, xylanase and aminopeptidase by gene replacement

    K Hayashi, S Nirasawa, S Kaneko, Y Honda, M Kitaoka

    FASEB JOURNAL   15 ( 4 )   A202 - A202   2001年3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:FEDERATION AMER SOC EXP BIOL  

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  • Improving enzyme characteristics by gene shuffling; application to beta-glucosidase

    K Hayashi, L Ying, S Singh, S Kaneko, S Nirasawa, T Shimonishi, Y Kawata, T Imoto, M Kitaoka

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   11 ( 4-6 )   811 - 816   2001年1月

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    記述言語:英語   出版者・発行元:ELSEVIER SCIENCE BV  

    The genes of family 3 beta -glucosidase enzymes consist of five distinct regions; the N-terminal residues, an N-terminal catalytic domain, a nonhomologous region, a C-terminal domain of unknown function and the C-terminal residues. The beta -glucosidase genes derived from Cellvibrio gilvus (CG) and Agrobacterium tumefaciens (AT) have been subjected to gene deletion, truncation and shuffling. The folding information was found to be distributed unevenly across the different regions based on the gene manipulation results. Chimeric enzymes with improved enzyme characteristics were obtained only by gene shuffling at the C-terminal domain. (C) 2001 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1381-1177(00)00161-2

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  • 最近の糖質研究と食品開発 遺伝子シャッフリングによりオリゴ糖を生産する酵素を改良する

    林清, 金子哲, 韮沢悟, 北岡本光

    食品工業   43 ( 10 )   38 - 45   2000年5月

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  • Uneven distribution of the folding information on B-glucosidase.

    K Hayashi, S Kaneko, S Nirasawa, M Kitaoka

    BIOPHYSICAL JOURNAL   78 ( 1 )   293A - 293A   2000年1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:BIOPHYSICAL SOCIETY  

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  • Clostridium thermocellum YM4由来のセロデキストリンフォスフォリラーゼおよびセロビオースフォスフォリラーゼのクローニングと発現

    MANEM Krishnareddy, 北岡 本光, 青柳 千佳, YING Li, 森 隆, 林 清

    日本分子生物学会年会プログラム・講演要旨集   21   326 - 326   1998年12月

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  • A CELLOBIOSE PHOSPHORYLASE FROM CELLVIBRIO-GILVUS RECOGNIZES ONLY THE BETA-DEUTERIUM-FORM OF 5A-CARBA-GLUCOPYRANOSE

    M KITAOKA, S OGAWA, H TANIGUCHI

    CARBOHYDRATE RESEARCH   247   355 - 359   1993年9月

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    記述言語:英語   掲載種別:記事・総説・解説・論説等(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

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  • シュクロ-スホスホリラ-ゼ,キシロ-スイソメラ-ゼおよびセロビオ-スホスホリラ-ゼを用いたシュクロ-スのセロビオ-スへの変換〔英文〕

    北岡 本光, 佐々木 尭, 谷口 肇

    澱粉科学   39 ( 4 )   p281 - 283   1992年

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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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  • シリカ担体への簡易なセルラ-ゼの固定化方法〔英文〕

    北岡 本光, 谷口 肇, 佐々木 尭

    食品総合研究所研究報告   ( 55 )   p111 - 114   1991年3月

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    記述言語:日本語   出版者・発行元:食品総合研究所  

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  • セロビオースホスホリラーゼを用いたヘテロ二糖類の合成(酵素-糖質関連酵素-)

    北岡 本光, 栢木 豊, 谷口 肇, 佐々木 堯

    日本農藝化學會誌   62 ( 3 )   346 - 346   1988年3月

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    記述言語:日本語   出版者・発行元:社団法人日本農芸化学会  

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