Characterization of the enzymatic polymerization of 2,6-linked fructan by leaf extracts from timothy grass (Phleum pratense)

Abstract

A fructan polymerase activity was partially purified and concentrated by sequential acid and salt precipitation from extracts of excised, illuminated leaves of timothy grass (Phleum pratense). The polymerase catalysed the de novo synthesis of oligo- and polyfructan from sucrose as sole substrate at near-physiological rates (0.5–0.9 mg g⁻¹ fresh mass h⁻¹; 0.9–1.5 nkat g⁻¹). Rates of in vitro polymerisation were high, at up to 4.1 mg cm⁻³ h⁻¹ (7.1 nkat cm⁻³) of total products of degree of polymerization greater than 2 (DP>2). The trisaccharides 1-kestose and 6-kestose together with oligosaccharides of up to DP[les ]c. 10 were synthesized in under 2 h at 30°C. In longer incubations, ethanol-precipitable polymers of DP=c. 10–35 (1.6–5.7 kDa) were detected by anion-exchange chromatography and pulsed amperometry. When this polymeric product was used as a primer and re-incubated with fresh enzyme and sucrose, abundant polymers of up to DP=50 (8.1 kDa) were formed. The structure of the polymeric enzyme product was compared with native fructan from timothy leaves and with standard inulin, using glycosyl-linkage analysis followed by identification of partially methylated alditol acetate derivatives by GC–MS. The deduced structure was a linear (unbranched) 2,6-linked fructose chain terminated with glucose and fructose. The linkage structures of the native and enzyme-generated polymers were identical, increasing confidence in the physiological relevance of the activity. After ultracentrifugation of tissue homogenates at 265 000 g_av, the polymerase remained in the supernatant, demonstrating no tight association with particulate components. The polymerizing reaction was dependent on enzyme concentration, requiring at least 3 g fresh mass equivalent cm⁻³ (c. 2.7 nkat cm⁻³) for the efficient in vitro generation of fructans of DP>3. In common with other trisaccharide-synthesizing and oligofructan-glycosylating enzymes from grasses, the polymerase reaction exhibited both a maximal velocity at pH 5.0–5.5 and a low affinity for sucrose. The polymerization reaction did not saturate fully even at 1.5 M sucrose, and the concentration causing half maximal velocity (apparent K_m) was c. 560 mM. The preparation contained substantial invertase activity (1.8 mg sucrose g⁻¹ fresh mass h⁻¹=1.5 nkat g⁻¹ fresh mass) with a K_m for sucrose hydrolysis of 5 mM. A single peak of polymerase activity with an M_r of 51 kDa was recovered from size-exclusion chromatography (SEC). Invertases of M_r 51 and 110 kDa were identified in the preparation. The 110-kDa invertase isoform exhibited no polymerase activity, but synthesized trisaccharide (mainly 1-kestose) from sucrose. The 51-kDa isoform co-eluted with the polymerase. The trisaccharide fraction produced by this isoform contained abundant 1- and 6-kestose. After SEC, the purification of the polymerase was 41-fold relative to the original tissue homogenate. The properties of enzymatic polymerization of fructan are discussed with respect to the physiology of accumulation in grass leaves and other systems.