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Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes.

Li X, Jackson P, Rubtsov DV, Faria-Blanc N, Mortimer JC, Turner SR, Krogh KB, Johansen KS, Dupree P - Biotechnol Biofuels (2013)

Bottom Line: 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.The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Building O, Downing Site, University of Cambridge, Cambridge CB2 1QW, UK. p.dupree@bioc.cam.ac.uk.

ABSTRACT

Background: Plant 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.

Results: 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). 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.

Conclusions: The 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.

No MeSH data available.


Quantitative analysis of oligosaccharides released by wheat arabinoxylan hydrolysis with different xylanases. (A) Aligned electropherograms of duplicate hydrolysis with xylanase 1 to xylanase 6. QS3 and QS4 are annotated. (B) Hierarchical clustering of quantities of oligosaccharides released by enzymes 1 to 6 using standardized Euclidean distances and DASHboard. (C) Principal components analysis of quantities of oligosaccharides released by xylanases 1 to 6.
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Figure 7: Quantitative analysis of oligosaccharides released by wheat arabinoxylan hydrolysis with different xylanases. (A) Aligned electropherograms of duplicate hydrolysis with xylanase 1 to xylanase 6. QS3 and QS4 are annotated. (B) Hierarchical clustering of quantities of oligosaccharides released by enzymes 1 to 6 using standardized Euclidean distances and DASHboard. (C) Principal components analysis of quantities of oligosaccharides released by xylanases 1 to 6.

Mentions: Fingerprinting of polysaccharide hydrolase product profiles may reveal differences in enzyme activity. We investigated whether the technique could reveal product differences between related xylanase enzymes. Figure 7A shows aligned traces of duplicate digests of wheat flour arabinoxylan (WAX) with six different hydrolytic enzymes. The traces show that the enzymes released several arabinoxylan oligosaccharides. The oligosaccharides were quantified using two QS (DP6, DP7). The enzyme products were grouped by hierarchical clustering of these oligosaccharide abundances (Figure 7B). Pairs of duplicate digests clustered together. Based on the pattern of oligosaccharides released after digestion, the hydrolytic enzymes were clearly separated in two different groups. These groups correspond to enzymes from CAZy family GH10 (Enzymes 2, 3, 6) and GH11 (Enzymes 1, 4, 5). Principal component analysis (PCA) of the same data also resolved these two groups along PC1 (Figure 7C). However, PC2 additionally separated GH10 enzyme 3 from the other GH10 enzymes, indicating that the enzyme produced different oligosaccharide products.


Development and application of a high throughput carbohydrate profiling technique for analyzing plant cell wall polysaccharides and carbohydrate active enzymes.

Li X, Jackson P, Rubtsov DV, Faria-Blanc N, Mortimer JC, Turner SR, Krogh KB, Johansen KS, Dupree P - Biotechnol Biofuels (2013)

Quantitative analysis of oligosaccharides released by wheat arabinoxylan hydrolysis with different xylanases. (A) Aligned electropherograms of duplicate hydrolysis with xylanase 1 to xylanase 6. QS3 and QS4 are annotated. (B) Hierarchical clustering of quantities of oligosaccharides released by enzymes 1 to 6 using standardized Euclidean distances and DASHboard. (C) Principal components analysis of quantities of oligosaccharides released by xylanases 1 to 6.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3717103&req=5

Figure 7: Quantitative analysis of oligosaccharides released by wheat arabinoxylan hydrolysis with different xylanases. (A) Aligned electropherograms of duplicate hydrolysis with xylanase 1 to xylanase 6. QS3 and QS4 are annotated. (B) Hierarchical clustering of quantities of oligosaccharides released by enzymes 1 to 6 using standardized Euclidean distances and DASHboard. (C) Principal components analysis of quantities of oligosaccharides released by xylanases 1 to 6.
Mentions: Fingerprinting of polysaccharide hydrolase product profiles may reveal differences in enzyme activity. We investigated whether the technique could reveal product differences between related xylanase enzymes. Figure 7A shows aligned traces of duplicate digests of wheat flour arabinoxylan (WAX) with six different hydrolytic enzymes. The traces show that the enzymes released several arabinoxylan oligosaccharides. The oligosaccharides were quantified using two QS (DP6, DP7). The enzyme products were grouped by hierarchical clustering of these oligosaccharide abundances (Figure 7B). Pairs of duplicate digests clustered together. Based on the pattern of oligosaccharides released after digestion, the hydrolytic enzymes were clearly separated in two different groups. These groups correspond to enzymes from CAZy family GH10 (Enzymes 2, 3, 6) and GH11 (Enzymes 1, 4, 5). Principal component analysis (PCA) of the same data also resolved these two groups along PC1 (Figure 7C). However, PC2 additionally separated GH10 enzyme 3 from the other GH10 enzymes, indicating that the enzyme produced different oligosaccharide products.

Bottom Line: 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.The product profiles of specific xylanases were also compared in order to identify enzymes with unusual oligosaccharide products.The 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Building O, Downing Site, University of Cambridge, Cambridge CB2 1QW, UK. p.dupree@bioc.cam.ac.uk.

ABSTRACT

Background: Plant 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.

Results: 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). 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.

Conclusions: The 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.

No MeSH data available.