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Purification and characterization of a fructosyltransferase from onion bulbs and its key role in the synthesis of fructo-oligosaccharides in vivo

Authors

  • Masaki Fujishima,

    1. Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069-8501, Japan.
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  • Hideki Sakai,

    1. Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069-8501, Japan.
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  • Keiji Ueno,

    1. Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069-8501, Japan.
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  • Natsuko Takahashi,

    1. Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069-8501, Japan.
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  • Shuichi Onodera,

    1. Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069-8501, Japan.
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  • Noureddine Benkeblia,

    1. Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069-8501, Japan.
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  • Norio Shiomi

    Corresponding author
    1. Department of Food and Nutrition Sciences, Graduate School of Dairy Science Research, Rakuno Gakuen University, 582 Bunkyodai, Midorimachi, Ebetsu, Hokkaido 069-8501, Japan.
      Author for correspondence: Norio Shiomi Tel: +81 11 3884754 Fax: +81 11 3884754 Email: n-shiomi@rakuno.ac.jp
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Author for correspondence: Norio Shiomi Tel: +81 11 3884754 Fax: +81 11 3884754 Email: n-shiomi@rakuno.ac.jp

Summary

  • • A fructosyltransferase that transfers the terminal (2 → 1)-β-linked d-fructosyl group of fructo-oligosaccharides (1F(1-β-d-fructofuranosyl)n sucrose, n  1) to HO-6 of the glucosyl residue and HO-1 of the fructosyl residue of similar saccharides (1F(1-β-d-fructofuranosyl)m sucrose, m  0) has been purified from an extract of the bulbs of onion (Allium cepa).
  • • Successive column chromatography using DEAE-Sepharose CL-6B, Toyopearl HW65, Toyopearl HW55, DEAE-Sepharose CL-6B (2nd time), Sephadex G-100, Concanavalin A Sepharose, and Toyopearl HW-65 (2nd time) were applied for protein purification.
  • • The general properties of the enzyme, were as follows: molecular masses of 66 kDa (gel filtration chromatography), and of 52 kDa and 25 kDa (SDS-PAGE); optimum pH of c. 5.68, stable at 20–40°C for 15 min; stable in a range of pH 5.30–6.31 at 30°C for 30 min, inhibited by Hg2+, Ag+, p-chloromercuribenzoic acid (p-CMB) and sodium dodecyl sulfate (SDS), activated by sodium deoxycholate, Triton X-100 and Tween-80. The amino acid sequence of the N-terminus moiety of the 52-kDa polypeptide was ADNEFPWTNDMLAWQRCGFHFRTVRNYMNDPSGPMYYKGWYHLFYQHNKDFAYXG and the amino acid sequence from the N-terminus of the 25-kDa polypeptide was ADVGYXCSTSGGAATRGTLGPFGLL VLANQDLTENTATYFYVSKGTDGALRTHFCQDET.
  • • The enzyme tentatively classified as fructan: fructan 6G-fructosyltransferase (6G-FFT). The enzyme is proposed to play an important role in the synthesis of inulin and inulinneo-series fructo-oligosaccharides in onion bulbs.

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