Search

The products formed by a fructan : 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 tetrasaceharides 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. [ABSTRACT FROM AUTHOR]

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 6Gkestose 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 : sucrose fructosyltransferase acitity 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 the effect depended on the concentration of the trisaccharides. In the presence of sucrose, 6Gkestose could be a substrate of equvalent importance to 1-kestose for synthesis of tetrasaccharide. [ABSTRACT FROM AUTHOR]

This paper presents a method for rapid quantification of sucrose and fructans by thin-layer chromatography (TLC) and densitometry. In the optimized protocol, glass-backed TLC plates were developed in a solution of propan-1-ol:ethyl acetate:water (45:35:20, v/v/v) for 3.5 h at 30°C. In this system, sucrose, the fructan trisaccharides: 1-kestose, 6G-kestose and 6-kestose, fructan tetrasaccharide and fructan pentasaccharide were separated. An internal standard of stachyose was useful for most of the applications examined. The fructans were detected by spraying with urea-phosphoric acid and heating at 110 °C for 6 min. The blue colour developed was stable for 18 h provided that plates were kept in the dark. Quantification was achieved by reflectance densitometry and comparison with standards. The standard curves for the oligosaccharides were not linear, owing to the spreading of spots on the TLC plates, but were highly repeatable. The TLC quantification method provided a more rapid analysis of sucrose and fructan oligosaccharides than was possible by high-performance liquid chromatography analysis because time-consuming pre-treatment of samples was not needed. The method will be useful for both enzyme and physiological studies. [ABSTRACT FROM AUTHOR]

A partial separation of sucrose: sucrose fructosyltranferase (SST) activity from invertase activity extracted from leaves of Lolium rigidum Gaudin was achieved by gel filtration. The SST activity was purified 54-fold with a ratio of SST activity to invertase activity of 1:1:1. the partially purified SST activity eluted with an apparent molecular mass of about 68000, had an optimum pH of 5.5 and a Km of 0.12 M. When incubated with sucrose concentrations ranging from 10 to 600 mM the partially purified SST activity produced mainly 1-kestose and some 6-kestose (10-15% of trisaccharide produced). SST and invertase activities declined during storage at -25°C with invertase activity declining at a faster rate. After the loss of invertase activity in storage, the partially purified SST activity synthesized only 1-ketose. When incubated with 1-ketose the partially purified SST activity synthesized neokestose (1.9 nkat ml-1), nystose (2.2 nkat ml-1) and an unidentified tetrasaccharide (1.4 nkat ml-1) indicating that the fructosyltranferase preparation also had FFT-like activity. The partially purified invertase had significant sucrose-hydrolyzing activity (25 nkat ml-1) and contained no measurable SST activity when incubated with 10-400 mM sucrose, but produced trisaccharide (1-kestose and 6-kestose, ratio 1.5:1) when incubated with 600 mM sucrose. L. rigidum does not normally accumulate 6-ketose. The optimum pH of the sucrose-hydrolyzing activity was 4.5 and the optimum pH of the fructosyltranferase activity of the invertase preparation was 5.5. The partially purified invertase did not have significant FFT-like activity. The results support the hypothesis that SST activity synthesizes only 1-kestose, which is a key intermediate in the synthesis of fructan in L. rigidum. [ABSTRACT FROM AUTHOR]