Fructan biosynthesis in excised leaves of Lolium temulentum VII. Sucrose and fructan hydrolysis by a fructan-polymerizing enzyme preparation

Abstract

A partially purified enzyme preparation from leaves of Lolium temulentum L. was previously shown to catalyse the net synthesis of oligofructans and polyfructans from sucrose. Here the same preparation is shown to catalyse the hydrolysis of both sucrose and oligofructans. The magnitude and properties of these hydrolytic activities have been determined. The significance of these catabolic activities for studies of fructan polymerization both in vitro and in tissues in a physiologically anabolic state are discussed.

The preparation hydrolysed 1-kestose, 6-kestose, neokestose, inulin oligosaccharides of low degree of polymerization (DP 4 and 5) and endogenous oligofructans from L. temulentum, with the concomitant release of monosaccharide. The preparation also released reducing sugar at low rates from high molecular weight inulin but had no detectable activity against bacterial levan. Simultaneous incubation of sucrose and Neosugar (a commercially available mixture of predominantly β-2, 1 linked tri-, tetra- and penta-saccharides) showed that sucrose was preferentially hydrolysed by the preparation, with Neosugar fructans being protected from hydrolysis at sucrose concentrations >30 mol m⁻³. The kinetic properties for hydrolysis of both sucrose and Neosugar were determined. For sucrose and Neosugar respectively, Michaelis constants at 30°C and pH 6·0 were 7·7±0·5 and 14·1±1·1 mol m⁻³ (as terminal fructose) and maximum velocities were 6·5±0·1 and 2·7±0·1 mg g⁻¹ fr. wt h⁻¹ (equivalent to 10·0 and 4·2 nkat g⁻¹ as reducing sugar release). Maximal temperatures for activity were 45 and 44°C, and Arrhenius activation energies were 39·9 and 46·9 kJ mol⁻¹. Preincubations for 1 h at 49 and 48°C caused 50% loss of activity in subsequent assays at 30°C. The pHs for maximal activity for the two substrates were 5·2±0·1 and 5·5±0·1.

Using size exclusion chromatography (SEC), an activity catalysing the formation of fructan oligosaccharides and another catalysing sucrose hydrolysis, were not fully resolved, but exhibited distinct profiles of elution indicating M_r of 57 and 133 kD respectively. When assayed for the hydrolysis of Neosugar, the SEC eluate exhibited two peaks of activity indicative of M_r values of 57 and 133 kD.