Purification, Cloning and Functional Characterization of Fructan: Fructan 1-Fructosyltransferase from Edible Burdock (Arctium lappa L.)

Abstract

A fructan:fructan 1-fructosyltransferase (1-FFT) was purified from edible burdock for the first time, and a cDNA encoding 1-FFT was isolated from the plant. Two 1-FFTs named 1-FFTa and 1-FFTb were purified from extract of edible burdock by ammonium sulfate precipitation, followed by chromatographies on DEAE-Sepharose CL-6B, Toyopearl HW-55S and Sephadex G-100 columns. Inulin-type fructan such as nystose and fructooligosaccharides with higher DP were produced from 1-kestose by purified 1-FFTs. The general properties of both purified enzymes were very similar to each other. The purified 1-FFTa and 1-FFTb showed a single band by native PAGE and two bands, relative molecular masses of about 46,000 and 17,500 for 1-FFTa and 46,000 and 17,000 for 1-FFTb, by SDS-PAGE, respectively. The N-terminal sequences of the 46,000 peptides of both enzymes were the same, and those of the 17,500 and the 17,000 peptides were also identical. Based on the sequences, the 1-FFT cDNA named alft1 was cloned. The alft1 encoded a polypeptide of 617 amino acids. The relative molecular mass and pI of the mature protein region of deduced polypeptide were calculated to be 60,213 and 4.89, respectively. The characteristics of recombinant protein produced by Pichia pastoris closely resembled those of the native 1-FFTs purified from edible burdock. In this study, we demonstrated that alft1 encoding edible burdock 1-FFT was involved in the elongation of fructosyl chains of fructooligosaccharides in edible burdock.