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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
Author(s) -
Fujishima Masaki,
Sakai Hideki,
Ueno Keiji,
Takahashi Natsuko,
Onodera Shuichi,
Benkeblia Noureddine,
Shiomi Norio
Publication year - 2005
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/j.1469-8137.2004.01231.x
Subject(s) - fructan , sephadex , chemistry , inulin , oligosaccharide , biochemistry , size exclusion chromatography , sucrose , enzyme , chromatography , residue (chemistry)
Summary• A fructosyltransferase that transfers the terminal (2 → 1)‐β‐linked d ‐fructosyl group of fructo‐oligosaccharides (1 F (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 (1 F (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 Hg 2+ , 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 6 G ‐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.