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

Abstract

asterisk inside a circle sign 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). asterisk inside a circle sign 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. asterisk inside a circle sign 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. asterisk inside a circle sign 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. © New Phytologist (2004).