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Synthetic xylan-binding modules for mapping of pulp fibres and wood sections.

Filonova L, Gunnarsson LC, Daniel G, Ohlin M - BMC Plant Biol. (2007)

Bottom Line: However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties.Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules.Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: WURC, Department of Wood Science, Swedish University of Agricultural Sciences, PO Box 7008, SE-750 07 Uppsala, Sweden. lada.filonova@trv.slu.se

ABSTRACT

Background: The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.

Results: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

Conclusion: In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.

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Related in: MedlinePlus

Sequence differences between the five different modules. Each protein is 165 amino acid residues long not counting the hexa-histidine tag, used in this investigation. Amino acids are represented by the standard one-letter code. Identities with the wt CBM4-2 sequence are shown by dots. Residue numbering is in accordance with Simpson et al. [25].
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Figure 1: Sequence differences between the five different modules. Each protein is 165 amino acid residues long not counting the hexa-histidine tag, used in this investigation. Amino acids are represented by the standard one-letter code. Identities with the wt CBM4-2 sequence are shown by dots. Residue numbering is in accordance with Simpson et al. [25].

Mentions: The wild type (wt) CBM4-2 from the xylanase Xyn 10A of Rhodothermis marinus is a type B CBM [20] displaying a groove-shaped binding site that fits polysaccharide chains well [25]. This CBM binds not only to xylan but also other non-crystalline plant carbohydrates [26,27]. In a previous study, we had constructed a combinatorial library of CBM4-2 [28] and used the phage-display system to select for CBM-variants specific for different ligands. The binding properties of three CBMs, X-2, X-6 and X-13, all selected using the insoluble part of birch wood xylan as a selection target were further investigated in this work with the aim of defining the potential to use such modules in analysis of xylans in plant tissues. The sequences of these proteins and a control module unable to bind plant carbohydrates are all very similar and they differ from the wt protein (of 167 residues) by only 4–9 amino acid substitutions (Figure 1). Despite the fact that they originate from the same selection, these CBMs have different affinities and specificities for a number of tested carbohydrates. Affinity electrophoresis demonstrated that the X-6 variant, like wt CBM4-2, binds to a range of carbohydrates (Figure 2). In contrast, both X-2 and X-13 are substantially more specific for xylans and do not recognise xyloglucan or glucan-containing carbohydrates (Figure 2). In addition, isothermal titration calorimetry studies further confirmed that X-2 was much more prone to bind xylans than any other carbohydrate targets investigated further demonstrating its modified specificity [27]. Relative binding affinities, determined by affinity electrophoresis, revealed a decrease in affinity for xylan of X-2 and X-13 and a somewhat higher affinity for xylan of X-6 compared to the wt CBM4-2 (Table 1). Altogether we have demonstrated our access to a range of very sequence-similar CBMs with diverse binding properties that can be used as a utility in analysis of the carbohydrate composition in lignocellulose materials.


Synthetic xylan-binding modules for mapping of pulp fibres and wood sections.

Filonova L, Gunnarsson LC, Daniel G, Ohlin M - BMC Plant Biol. (2007)

Sequence differences between the five different modules. Each protein is 165 amino acid residues long not counting the hexa-histidine tag, used in this investigation. Amino acids are represented by the standard one-letter code. Identities with the wt CBM4-2 sequence are shown by dots. Residue numbering is in accordance with Simpson et al. [25].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2099432&req=5

Figure 1: Sequence differences between the five different modules. Each protein is 165 amino acid residues long not counting the hexa-histidine tag, used in this investigation. Amino acids are represented by the standard one-letter code. Identities with the wt CBM4-2 sequence are shown by dots. Residue numbering is in accordance with Simpson et al. [25].
Mentions: The wild type (wt) CBM4-2 from the xylanase Xyn 10A of Rhodothermis marinus is a type B CBM [20] displaying a groove-shaped binding site that fits polysaccharide chains well [25]. This CBM binds not only to xylan but also other non-crystalline plant carbohydrates [26,27]. In a previous study, we had constructed a combinatorial library of CBM4-2 [28] and used the phage-display system to select for CBM-variants specific for different ligands. The binding properties of three CBMs, X-2, X-6 and X-13, all selected using the insoluble part of birch wood xylan as a selection target were further investigated in this work with the aim of defining the potential to use such modules in analysis of xylans in plant tissues. The sequences of these proteins and a control module unable to bind plant carbohydrates are all very similar and they differ from the wt protein (of 167 residues) by only 4–9 amino acid substitutions (Figure 1). Despite the fact that they originate from the same selection, these CBMs have different affinities and specificities for a number of tested carbohydrates. Affinity electrophoresis demonstrated that the X-6 variant, like wt CBM4-2, binds to a range of carbohydrates (Figure 2). In contrast, both X-2 and X-13 are substantially more specific for xylans and do not recognise xyloglucan or glucan-containing carbohydrates (Figure 2). In addition, isothermal titration calorimetry studies further confirmed that X-2 was much more prone to bind xylans than any other carbohydrate targets investigated further demonstrating its modified specificity [27]. Relative binding affinities, determined by affinity electrophoresis, revealed a decrease in affinity for xylan of X-2 and X-13 and a somewhat higher affinity for xylan of X-6 compared to the wt CBM4-2 (Table 1). Altogether we have demonstrated our access to a range of very sequence-similar CBMs with diverse binding properties that can be used as a utility in analysis of the carbohydrate composition in lignocellulose materials.

Bottom Line: However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties.Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules.Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: WURC, Department of Wood Science, Swedish University of Agricultural Sciences, PO Box 7008, SE-750 07 Uppsala, Sweden. lada.filonova@trv.slu.se

ABSTRACT

Background: The complex carbohydrate composition of natural and refined plant material is not known in detail but a matter that is of both basic and applied importance. Qualitative assessment of complex samples like plant and wood tissues requires the availability of a range of specific probes. Monoclonal antibodies and naturally existing carbohydrate binding modules (CBMs) have been used in the past to assess the presence of certain carbohydrates in plant tissues. However, the number of natural CBMs is limited and development of carbohydrate-specific antibodies is not always straightforward. We envisage the use of sets of very similar proteins specific for defined targets, like those developed by molecular evolution of a single CBM scaffold, as a suitable strategy to assess carbohydrate composition. An advantage of using synthetic CBMs lies in the possibility to study fine details of carbohydrate composition within non-uniform substrates like plant cell walls as made possible through minor differences in CBM specificity of the variety of binders that can be developed by genetic engineering.

Results: A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of Rhodothermus marinus, was used in this study. The wild type CBM4-2 and evolved modules both showed binding to wood sections. However, differences were observed in the staining patterns suggesting that these modules have different xylan-binding properties. Also the staining stability varied between the CBMs, the most stable staining being obtained with one (X-2) of the synthetic modules. Treatment of wood materials resulted in altered signal intensities, thereby also demonstrating the potential application of engineered CBMs as analytical tools for quality assessment of diverse plant material processes.

Conclusion: In this study we have demonstrated the usefulness of synthetic xylan-binding modules as specific probes in analysis of hemicelluloses (xylan) in wood and fibre materials.

Show MeSH
Related in: MedlinePlus