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 (0n5-0n9 mg g −" fresh mass h −" ; 0n9-1n5 nkat g −" ). Rates of in vitro polymerisation were high, at up to 4n1 mg cm −$ h −" (7n1 nkat cm −$ ) 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 c. 10 were synthesized in under 2 h at 30mC. In longer incubations, ethanol-precipitable polymers of DP l c. 10-35 (1n6-5n7 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 l 50 (8n1 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 −$ (c. 2n7 nkat cm −$ ) 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 5n0-5n5 and a low affinity for sucrose. The polymerization reaction did not saturate fully even at 1n5 M sucrose, and the concentration causing half maximal velocity (apparent K m ) was c. 560 mM. The preparation contained substantial invertase activity (1n8 mg sucrose g −" fresh mass h −" l 1n5 nkat g −" 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

Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L.

“…An added complexity with invertases is the interpretation of published enzyme activity data due to the similarities between fructolsyltransferase and invertase activities when assayed in vitro Cairns et al, 1999). Without enzymic data and more detailed localization studies, it is dif®cult to comment with much certainty on the physiological signi®cance of the two invertases identi®ed in this study, other than to state that they have different modes of regulation implying different roles in sugar metabolism within the plant.…”

“…Fructans have been implicated in conferring tolerance to drought and cold stress (De Roover et al, 2000;Housley and Pollock, 1993;Pilon-Smits et al, 1995;Thomas and James, 1999;Volaire and Lelievre, 1997). Their synthesis occurs de novo in carbohydrate accumulating tissue, with sucrose as the major or sole precursor (Cairns and Pollock, 1988a;Cairns et al, 1999;Wagner et al, 1983). In L. temulentum, fructosyltransferase activity has been identi®ed and measured (Pollock, 1984).…”

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass

“…For example, timothy grass ( Phleum pratense ) and orchard grass ( Dactylis glomerata ) have linear β-2,6-linked fructans which are referred to as phleins or plant levans [23–25]. Mixed levans (graminans) which contain both, β-2,1 and β-2,6 linkages, are present in many Poales species (e.g.…”

Dactylis glomerata Growing Along a Light Gradient in the Central Appalachian Region of the Eastern USA: III. Nonstructural Carbohydrates and Nutritive Value

“…In general, polymers of fructose, or fructans, accumulate in stembases of cool-temperate origin grasses when photosynthate production exceeds carbohydrate translocation and utilization rates. Fructan can account for as much as 40% of plant dry mass and influence the pattern of relative carbon allocation among plant structures (Cairns et al 1999). Stored TNC is involved in environmental and competitive stress tolerance (Cheplick and Chui 2001), and also provides energy for regrowth after defoliation; however, defoliation frequency (brief interval between events) or intensity (minimal plant residual height after an event) can impair TNC replenishment.…”