FRUCTAN BIOSYNTHESIS IN EXCISED LEAVES OF LOLIUM-TEMULENTUM

A partially purified enzyme preparation from leaves of Lolium temulent um L. was previously shown to catalyse the net synthesis of oligofruct ans and polyfructans from sucrose. Here the same preparation is shown to catalyse the hydrolysis of both sucrose and oligofructans. The magn itude and properties of these hydrolytic activities have been determin ed. The significance of these catabolic activities for studies of fruc tan polymerization both in vitro and in tissues in a physiologically a nabolic state are discussed. The preparation hydrolysed 1-kestose, 6-k estose, neokestose, inulin oligosaccharides of low degree of polymeriz ation (DP 4 and 5) and endogenous oligofructans from L. temulentum, wi th the concomitant release of monosaccharide. The preparation also rel eased reducing sugar at low rates from high molecular weight inulin bu t had no detectable activity against bacterial levan. Simultaneous inc ubation of sucrose and Neosugar (a commercially available mixture of p redominantly beta-2, 1 linked tri-, tetra- and penta-saccharides) show ed that sucrose was preferentially hydrolysed by the preparation, with Neosugar fructans being protected from hydrolysis at sucrose concentr ations > 30 mol m(-3). The kinetic properties for hydrolysis of both s ucrose and Neosugar were determined. For sucrose and Neosugar respecti vely, Michaelis constants at 30 degrees C and pH 6.0 were 7.7+/-0.5 an d 14.1+/-1.1 mol m(-3) (as terminal fructose) and maximum velocities w ere 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 fo r activity were 45 and 44 degrees C, and Arrhenius activation energies were 39.9 and 16.9 kJ mol(-1). Preincubations for 1 h at 49 and 48 de grees C caused 50% loss of activity in subsequent assays at 30 degrees 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 cata lysing sucrose hydrolysis, were not fully resolved, but exhibited dist inct 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.