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Analysis of saccharification in Brachypodium distachyon stems under mild conditions of hydrolysis.

Gomez LD, Bristow JK, Statham ER, McQueen-Mason SJ - Biotechnol Biofuels (2008)

Bottom Line: The non-cellulosic monosaccharide composition of Brachypodium is closely related to grasses of agricultural importance and significantly different from the dicot model Arabidopsis thaliana.Diluted acid pretreatment of stem segments produced significant release of sugars and negatively affected the amount of sugars obtained by enzymatic hydrolysis.Scanning electron microscopy analysis of the treated materials showed progressive exposure of fibrils in the stem segments.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNAP, Department of Biology, University of York, Heslington, York YO10 5YW, UK. ldg3@york.ac.uk

ABSTRACT

Background: Brachypodium distachyon constitutes an excellent model species for grasses. It is a small, easily propagated, temperate grass with a rapid life cycle and a small genome. It is a self-fertile plant that can be transformed with high efficiency using Agrobacteria and callus derived from immature embryos. In addition, considerable genetic and genomic resources are becoming available for this species in the form of mapping populations, large expressed sequence tag collections, T-DNA insertion lines and, in the near future, the complete genome sequence. The development of Brachypodium as a model species is of particular value in the areas of cell wall and biomass research, where differences between dicots and grasses are greatest. Here we explore the effect of mild conditions of pretreatment and hydrolysis in Brachypodium stem segments as a contribution for the establishment of sensitive screening of the saccharification properties in different genetic materials.

Results: The non-cellulosic monosaccharide composition of Brachypodium is closely related to grasses of agricultural importance and significantly different from the dicot model Arabidopsis thaliana. Diluted acid pretreatment of stem segments produced significant release of sugars and negatively affected the amount of sugars obtained by enzymatic hydrolysis. Monosaccharide and oligosaccharide analysis showed that the hemicellulose fraction is the main target of the enzymatic activity under the modest hydrolytic conditions used in our assays. Scanning electron microscopy analysis of the treated materials showed progressive exposure of fibrils in the stem segments.

Conclusion: Results presented here indicate that under mild conditions cellulose and hemicellulose are hydrolysed to differing extents, with hemicellulose hydrolysis predominating. We anticipate that the sub-optimal conditions for hydrolysis identified here will provide a sensitive assay to detect variations in saccharification among Brachypodium plants, providing a useful analytical tool for identifying plants with alterations in this trait.

No MeSH data available.


Related in: MedlinePlus

Total sugars remaining in stem segments after pretreatment and hydrolysis. Segments were subjected to complete hydrolysis after pretreatment only (1% H2SO4, 20 min at 90°C) or pretreatment and enzymatic hydrolysis (4 FPU/g of material). Total sugars were determined using MBTH using glucose as standard. Results represent the mean (± SD) of three experiments.
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Figure 7: Total sugars remaining in stem segments after pretreatment and hydrolysis. Segments were subjected to complete hydrolysis after pretreatment only (1% H2SO4, 20 min at 90°C) or pretreatment and enzymatic hydrolysis (4 FPU/g of material). Total sugars were determined using MBTH using glucose as standard. Results represent the mean (± SD) of three experiments.

Mentions: One of the major aims of the work presented here is to establish conditions for use in high-throughput screens to enable the identification of alterations in saccharification potential in mutant populations. In contrast to industrial processing where maximum release of sugars is desirable, we wish to identify mild pretreatment conditions that allow the enzymes to release limited quantities of sugar from wild type stems and will enable changes in the ease of sugar mobilisation with enzymes in potential mutants to be detected. In a typical digestion we pretreat 4 mm whole stem segments with 1% H2SO4 at 90°C for 20 min. Subsequent digestion of the pretreated material is carried out for 1 h at 30°C, using an enzyme cocktail containing 4 FPU and 19 cellobiose units (CBU) (see Methods for details) per gram of material. Under these conditions, an average of 75% of the total sugars in the material remained undigested in the stems following pretreatment and enzyme hydrolysis, whilst 90% remained following enzyme hydrolysis without pretreatment (Figure 7). SEM imaging showed minor structural alterations of the Brachypodium stems following hydrolysis (Figure 4).


Analysis of saccharification in Brachypodium distachyon stems under mild conditions of hydrolysis.

Gomez LD, Bristow JK, Statham ER, McQueen-Mason SJ - Biotechnol Biofuels (2008)

Total sugars remaining in stem segments after pretreatment and hydrolysis. Segments were subjected to complete hydrolysis after pretreatment only (1% H2SO4, 20 min at 90°C) or pretreatment and enzymatic hydrolysis (4 FPU/g of material). Total sugars were determined using MBTH using glucose as standard. Results represent the mean (± SD) of three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577630&req=5

Figure 7: Total sugars remaining in stem segments after pretreatment and hydrolysis. Segments were subjected to complete hydrolysis after pretreatment only (1% H2SO4, 20 min at 90°C) or pretreatment and enzymatic hydrolysis (4 FPU/g of material). Total sugars were determined using MBTH using glucose as standard. Results represent the mean (± SD) of three experiments.
Mentions: One of the major aims of the work presented here is to establish conditions for use in high-throughput screens to enable the identification of alterations in saccharification potential in mutant populations. In contrast to industrial processing where maximum release of sugars is desirable, we wish to identify mild pretreatment conditions that allow the enzymes to release limited quantities of sugar from wild type stems and will enable changes in the ease of sugar mobilisation with enzymes in potential mutants to be detected. In a typical digestion we pretreat 4 mm whole stem segments with 1% H2SO4 at 90°C for 20 min. Subsequent digestion of the pretreated material is carried out for 1 h at 30°C, using an enzyme cocktail containing 4 FPU and 19 cellobiose units (CBU) (see Methods for details) per gram of material. Under these conditions, an average of 75% of the total sugars in the material remained undigested in the stems following pretreatment and enzyme hydrolysis, whilst 90% remained following enzyme hydrolysis without pretreatment (Figure 7). SEM imaging showed minor structural alterations of the Brachypodium stems following hydrolysis (Figure 4).

Bottom Line: The non-cellulosic monosaccharide composition of Brachypodium is closely related to grasses of agricultural importance and significantly different from the dicot model Arabidopsis thaliana.Diluted acid pretreatment of stem segments produced significant release of sugars and negatively affected the amount of sugars obtained by enzymatic hydrolysis.Scanning electron microscopy analysis of the treated materials showed progressive exposure of fibrils in the stem segments.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNAP, Department of Biology, University of York, Heslington, York YO10 5YW, UK. ldg3@york.ac.uk

ABSTRACT

Background: Brachypodium distachyon constitutes an excellent model species for grasses. It is a small, easily propagated, temperate grass with a rapid life cycle and a small genome. It is a self-fertile plant that can be transformed with high efficiency using Agrobacteria and callus derived from immature embryos. In addition, considerable genetic and genomic resources are becoming available for this species in the form of mapping populations, large expressed sequence tag collections, T-DNA insertion lines and, in the near future, the complete genome sequence. The development of Brachypodium as a model species is of particular value in the areas of cell wall and biomass research, where differences between dicots and grasses are greatest. Here we explore the effect of mild conditions of pretreatment and hydrolysis in Brachypodium stem segments as a contribution for the establishment of sensitive screening of the saccharification properties in different genetic materials.

Results: The non-cellulosic monosaccharide composition of Brachypodium is closely related to grasses of agricultural importance and significantly different from the dicot model Arabidopsis thaliana. Diluted acid pretreatment of stem segments produced significant release of sugars and negatively affected the amount of sugars obtained by enzymatic hydrolysis. Monosaccharide and oligosaccharide analysis showed that the hemicellulose fraction is the main target of the enzymatic activity under the modest hydrolytic conditions used in our assays. Scanning electron microscopy analysis of the treated materials showed progressive exposure of fibrils in the stem segments.

Conclusion: Results presented here indicate that under mild conditions cellulose and hemicellulose are hydrolysed to differing extents, with hemicellulose hydrolysis predominating. We anticipate that the sub-optimal conditions for hydrolysis identified here will provide a sensitive assay to detect variations in saccharification among Brachypodium plants, providing a useful analytical tool for identifying plants with alterations in this trait.

No MeSH data available.


Related in: MedlinePlus