Gene Analysis and Enzymatic Properties of Thermostable β-Glycosidase from Pyrococcus kodakaraensis KODI
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概要
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A β-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene enconding β-glycosidase(Pk-gly)consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other β-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity of β-mannosidase from P.furiosus(55.7%), β-glycosidase from Sulfolobus solfataricus(42.7%)and β-glucosidase from P.furiosus(41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The β-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100℃, and had a half-life of 18 h at 90℃. The β-glycosidase hydrolyzed various pNp-β-glycopyranosides, with k_<cat>/K_m values in the order of pNp-β-glucopyranoside=⃥pNp-β-mannopyranoside>pNp-β-galactopyranoside>pNp-β-xylopyranoside. pNp-β-mannopyranoside was the substrate exhibiting the lowest K_m value[0.254 mM]with a k_<cat>/K_m ratio comparable to that of pNp-β-glucopyranoside. This substrate specificity was distinct from previously reported β-glycosidases. We observed that the region in Pk-Gly corresponding to the fifth α-helix and β-strand region of β-glycosidase from S.solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to β-glycosidases and the C-terminal region similar to β-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.
- 社団法人日本生物工学会の論文
- 1999-08-25
著者
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Takagi Masahiro
School Of Materials Science Japan Advanced Institute Of Science And Technology
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Takagi M
School Of Materials Science Japan Advanced Institute Of Science And Technology
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Takagi Mutsumi
International Center For Biotechnology Osaka University:(present Address)division Of Biotechnology A
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FUJIWARA SHINSUKE
Department of Bioscience, School of Science and Technology, Kwansei Gakuin University
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IMANAKA Tadayuki
Department of Synthetic and Biological Chemistry, Graduate School of Engineering, Kyoto University
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TAKAGI MASAHIRO
Department of Biotechnology, Graduate School of Engineering, Osaka University
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EZAKI SATOSHI
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto Un
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Tanaka Takeshi
Department Of Cardiology The Heart Institute Of Japan
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Fujiwara S
School Of Science And Engineering Department Of Life Science Kwansei-gakuin University
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Fujiwara Shinsuke
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Fujiwara Shinsuke
Department Of Bioscience School Of Science And Technology Kwansei Gakuin University
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MIYAOKU KOHEI
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto Un
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NISHI KEN-ICHI
Fine Chemicals Research Laboratories, Kaneka Corporation
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ATOMI HARUYUKI
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto Un
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Takagi M
Nagasaki Univ. Nagasaki Jpn
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Miyaoku Kohei
Department Of Synthetic Chemistry And Biological Chemistry Graduate School Of Engineering Kyoto Univ
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Atomi H
Department Of Synthetic Chemistry And Biological Chemistry Graduate School Of Engineering Kyoto Univ
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Atomi Haruyuki
Laboratory Of Applied Biological Chemistry Department Of Synthetic Chemistry And Biological Chemistr
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Atomi Haruyuki
Department Of Synthetic Chemistry And Biological Chemistry Graduate School Of Engineering Kyoto Univ
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Nishi Ken-ichi
Fine Chemicals Research Laboratories Kaneka Corporation
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Ezaki S
Department Of Synthetic Chemistry And Biological Chemistry Graduate School Of Engineering Kyoto Univ
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Ezaki Satoshi
Department Of Synthetic Chemistry And Biological Chemistry Graduate School Of Engineering Kyoto Univ
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Ezaki Satoshi
Laboratory Of Molecular Gene Technics Graduate School Of Genetic Resources Technology Kyushu Univers
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Imanaka Tadayuki
Department Of Applied Biotechnology Faculty Of Engineering Osaka University
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Miyaoku Kohei
Department Of Environmental Bioengineering Laboratory Graduate School Of Pharmaceutical Sciences Osa
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Fujiwara Shinsuke
School Of Science And Engineering Department Of Life Science Kwansei-gakuin University
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Miyamoto Kazuhisa
Environmental Bioengineering Laboratory Graduated School Of Pharmaceutical Sciences Osaka University
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Miyamoto Kazuhisa
Laboratory Of Biological Defenoe Department Of Insect Physiology And Behavior National Institute Of
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Atomi Haruyuki
Dept. Synth. Chem. Biol. Chem. Grad. Sch. Eng. Kyoto Univ.:crest Imanaka Project
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Miyamoto K
Environmental Biotechnology Laboratory Graduate School Of Pharmaceutical Sciences Osaka University
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Miyamoto K
Faculty Of Pharmaceutical Sciences Osaka University
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Imanaka T
Dep. Of Biotechnology Coll. Of Life Sciences Ritsumeikan Univ.
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Takagi Masahiro
Department Of Biotechnology Faculty Of Engineering Osaka University
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Tanaka Takeshi
Department Of Biomedical Chemistry Graduate School Of Medicine The University Of Tokyo
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Fujiwara Shinsuke
Department Of Bioscience Graduate School Of Science And Technology Kwansei-gakuin University:researc
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TANAKA TAKESHI
Department of Biotechnology, Graduate School of Engineering, Osaka University
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