Fructan: fructan fructosyltransferase (FFT) activity was purified about 300-fold from leaves of Lolium rigidum Gaudin by a combination of affinity chromatography, gel filtration, anion exchange and isoelectric focusing. The FFT activity was free of sucrose: sucrose fructosyltransferase and invertase activities. It had an apparent pI of 4·7 as determined by isoelectric focusing, and a molecular mass of about 50000 (gel filtration). The FFT activity utilized the trisaccharides 1-kestose and 6G-kestose as sole substrates, but was not able to use 6-kestose as sole substrate. The FFT activity was not saturated when assayed at concentrations of 1-kestose, 6G-kestose or (1,1)-kestotetraose of up to 400 mM. The rate of reaction of the FFT activity was most rapid when assayed with 1-kestose and was less rapid when assayed with 6G-kestose, (1,1)-kestotetraose or (1,1,1)-kestopentaose. The FFT activity when assayed at a relatively high concentration of enzyme activity (approximately equivalent to about half the activity in crude extracts per gram fresh mass) did not synthesize fructan of degree of polymerization > 6, even during extended assays of up to 10 h. When assayed with a combination of 1-kestose and uniformly labelled [14C]sucrose as substrates, the major reaction was the transfer of a fructosyl residue from 1-kestose to sucrose resulting in the re-synthesis of 1-kestose. Tetrasaccharide and 6G-kestose were also synthesized. When assayed with 6G-kestose and [14C]sucrose as substrates, the major reaction of the FFT activity was the synthesis of tetrasaccharide. However, some synthesis of 1-kestose and re-synthesis of 6G-kestose also occurred. When 6G-kestose was the sole substrate for the FFT activity, synthesis of tetrasaccharide was 2·7 to 3·4-fold slower than when 1-kestose was used as the sole substrate. Owing to differences in the fructan [ratio ] sucrose fructosyltransferase activity of the FFT with each of the trisaccharides, net synthesis of tetrasaccharide by the FFT was altered significantly in the presence of sucrose. The magnitude of this effect depended on the concentration of the trisaccharides. In the presence of sucrose, 6G-kestose could be a substrate of equivalent importance to 1-kestose for synthesis of tetrasaccharide.

The products formed by a fructan [ratio ] fructan fructosyltransferase (FFT) activity purified from Lolium rigidum Gaudin were identified after gas chromatography-mass spectrometry of partially methylated alditol acetates, electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography. The FFT activity synthesized oligofructans up to degree of polymerization (DP) 6, but did not synthesize fructans of DP > 6 even when assayed with (1,1,1)-kestopentaose for up to 10 h. The FFT activity when assayed with 1-kestose or 6G-kestose synthesized fructan with fructosyl residues almost exclusively linked by β-2,1-glycosidic linkages. When assayed with 1-kestose, the FFT activity synthesized tetrasaccharides and pentasaccharides with an internal glucosyl residue. The predominant tetrasaccharide was (1&6G)-kestotetraose and the predominant pentasaccharide was (1&6G,1)-kestopentaose. By comparison, tetrasaccharides and pentasaccharides extracted from L. rigidum also contained predominantly β-2,1-glycosidic linked fructans with an internal glucosyl residue. The only exception was that one of the pentasaccharides contained β-2,1- and β-2,6-glycosidic linked fructosyl residues. This pentasaccharide was not synthesized by the FFT activity. The role of this FFT activity in formation of oligofructans in L. rigidum is discussed.