Cloning and Expression of Genes Encoding Fructosyltransferases from Higher Plants in Food Technology

Abstract

Recently, we have investigated enzymic production of non-digestible oligosaccharides which have several functional activities as “tertiary functional ingredients” of foods. Fructo-oligosaccharides synthesized from sucrose by Eurotium repense fructosyltransferase had the effect of no elevation of blood glucose or insulin concentrations in rats. Fructosylxyloside formed from sucrose and xylose with fructosyltransferase action of Scopulariopsis brevicaulis cells suppressed serum glucose and insulin responses and / or promoted the absorption of calcium and magnesium ions in rats administered with sucrose. On the other hand, we studied purification and characterization of several fructosyltransferases; sucrose: sucrose 1-fructosyltransferase (1-SST), fructan: fructan 1-fructosyltransferase (1-FFT) and a new enzyme, fructan: fructan 6^G-fructosyltransferase (6G-FFT) from asparagus roots. Previously, we reported that the asparagus 1-FFT synthesized new functional oligosaccharides elongated with one or two additional fructose units by fructosyltransfer from 1-kestose to 4^G-β-D-galactosylsucrose and the saccharides selectively stimulated growth for Bifidobacteria. In this study, we have tried the isolation and expression of cDNAs encoding 6G-FFT, 1-FFT and 1-SST from asparagus for industrial use. The cDNAs encoding 6G-FFT, 1-FFT and 1-SST were isolated from a cDNA library of asparagus leaves or roots. The isolated cDNAs were named AoFT 1, AoFT 2 and AoFT 3, respectively. The deduced amino acid sequences of these cDNAs showed a high homology with those of plant fructosyltransferases. Expression of these cDNAs was done using Pichia pastoris. The recombinant protein from Pichia transformed with AoFT 1 produced 1^F,6^G-di-β-D-fructofuranosylsucrose, neokestose and sucrose from 1-kestose, while the transformant with an empty vector produced no saccharides. These results show that 6G-FFT is expressed in P. pastoris. In the same way, recombinant protein from Pichia transformed with AoFT 2 produced nystose from 1-kestose and AoFT 3 recombinant protein produced 1-kestose from sucrose. These results also show that 1-FFT and 1-SST are expressed in P. pastoris. Three recombinant proteins had enzymatic properties similar to those of 6G-FFT, 1-FFT and 1-SST from asparagus roots, respectively.