Abstract
BackgroundPlant cell wall polysaccharide composition varies substantially between species, organs and genotypes. Knowledge of the structure and composition of these polysaccharides, accompanied by a suite of well characterised glycosyl hydrolases will be important for the success of lignocellulosic biofuels. Current methods used to characterise enzymatically released plant oligosaccharides are relatively slow.ResultsA method and software was developed allowing the use of a DNA sequencer to profile oligosaccharides derived from plant cell wall polysaccharides (DNA sequencer-Assisted Saccharide analysis in High throughput, DASH). An ABI 3730xl, which can analyse 96 samples simultaneously by capillary electrophoresis, was used to separate fluorophore derivatised reducing mono- and oligo-saccharides from plant cell walls. Using electrophoresis mobility markers, oligosaccharide mobilities were standardised between experiments to enable reproducible oligosaccharide identification. These mobility markers can be flexibly designed to span the mobilities of oligosaccharides under investigation, and they have a fluorescence emission that is distinct from that of the saccharide labelling. Methods for relative and absolute quantitation of oligosaccharides are described. Analysis of a large number of samples is facilitated by the DASHboard software which was developed in parallel. Use of this method was exemplified by comparing xylan structure and content in Arabidopsis thaliana mutants affected in xylan synthesis. The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products.ConclusionsThe DASH method and DASHboard software can be used to carry out large-scale analyses of the compositional variation of plant cell walls and biomass, to compare plants with mutations in plant cell wall synthesis pathways, and to characterise novel carbohydrate active enzymes.
Highlights
Plant cell wall polysaccharide composition varies substantially between species, organs and genotypes
There is large variability in the structure of these polymers and in the composition of biomass from different plant organs, genotypes, species and between crops grown at different times [2], and it will be important to be able to study the composition of biomass and the sugars released by glycosyl hydrolase (GH) cocktails in relatively high throughput and with simple and robust equipment
The APTS labelled oligosaccharides were detected in the blue channel of the DNA sequencer
Summary
A method and software was developed allowing the use of a DNA sequencer to profile oligosaccharides derived from plant cell wall polysaccharides (DNA sequencer-Assisted Saccharide analysis in High throughput, DASH). Oligosaccharide mobilities were standardised between experiments to enable reproducible oligosaccharide identification These mobility markers can be flexibly designed to span the mobilities of oligosaccharides under investigation, and they have a fluorescence emission that is distinct from that of the saccharide labelling. Analysis of a large number of samples is facilitated by the DASHboard software which was developed in parallel. Use of this method was exemplified by comparing xylan structure and content in Arabidopsis thaliana mutants affected in xylan synthesis. The product profiles of specific xylanases were compared in order to identify enzymes with unusual oligosaccharide products
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