Chemical pulping: Localization of xylan (native and added) during cooking

The aim of this study was to investigate how the xylan content is changed within a wood chip and within the fiber cell walls at different positions of the chip during cooking. It was hoped that such information would give an understanding regarding the critical factors for xylan deposition within the cell wall and possible ways of affecting strength delivery of the pulp. Two sets of cooking trials were performed; one Ref-pulp and one with beech xylan addition in the impregnation stage (10g/L). The cooks were interrupted at specific times and the chips taken out and analyzed for chemical composition and distribution of wood polymers within the chips as well as within the fiber cell walls. The pulps produced with beech-xylan addition had about 2 % higher xylan content at the end of cooking compared to the Ref-pulp. Results indicated that the chips already contained an increased amount of xylan after 10 minutes of impregnation when beech xylan was added to the cooking liquor. However, only chips at the end of the cook showed a substantial increase in xylan sorption. During cooking, a large gradient in lignin content was noted between the surface and the inside of the chips which always showed higher lignin content even at the end of the cook. A progressive increase in xylan content was also noted in the chips as cooking proceeded. From the studies made it was not possible however, to determine if the addition of xylan led to any increased xylan content within the fibre walls. In cooks with beech xylan added during early stages, an increase in xylan was noted in the fibre lumen areas. In later stages and with increased delignification, increased xylan content was also noted between fibres in degraded middle lamellae regions

Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

<p>Abstract</p> <p>Background</p> <p>Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology.</p> <p>Results</p> <p>Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections.</p> <p>Conclusion</p> <p>We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.</p

Chemical pulping: the influence of the molecular weight of added xylan on pulp properties

The aim of this study was to investigate if added beech xylan with low molecule weight (Mw) could penetrate into the fiber wall to a greater extent compared to xylan with high Mw. The high Mw (ca 11000) xylan was degraded using enzymes to obtain xylan with low Mw (ca 1800). The influence from the added xylan on the strength properties was evaluated. The cooks in this study were performed without mechanical damage introduced during the cooking process, thus any conclusions of the impact from adding low Mw xylan on the sensitivity towards mechanical damage was not possible. The different microscopy analyses performed could not show any evidence for a higher penetration of xylan into the fiber wall when producing the pulp with addition of low molecular weight (low Mw) xylan. The low Mw xylan did not contribute to any improved pulp properties, rather the opposite. Addition of low Mw-xylan did not result in straighter fibers compared to the Ref-pulp, which was the case for the high Mw-xylan-pulp. The tensile indexdevelopment (tensile vs PFI-beating and tensile vs density) for the low Mw-pulp was even worse compared to the Ref-pulp. The pulp produced with addition of the high Mw-xylan showed, as earlier seen, an improved tensile index development compared to the Ref-pul

CRUW chemical pulping sub-project 1: the influence of xylan on the sensitivity towards fiber damage

The aim of this study was to determine if the presence and position of xylan in the fiber wall are of importance for the degree of damage introduced into fibers during mechanical action in the cook. Kraft pulps from spruce with different amounts of xylan have been produced in the laboratory, either by adding birch xylan in different positions in the cook or by redistribution of spruce xylan. At the end of the cook, fiber damages were introduced by subjecting the fibers to shear and compression forces. The extra birch xylan had adsorbed on the fiber surfaces, the outer fiber walls (presumably S1/primary wall) as well as on the fiber cell lumen wall. Xylan penetration into the fiber wall was very low. A large variation in coverage of surface xylan within the fibers and between fibers was noted. No significant difference between pulps produced in the different ways or between the pulps produced with or without mechanical treatment could however be observed. The extra xylan added resulted as expected in an improved tensile strength development for these pulps. No direct indications were seen that the extra xylan added during the cook resulted in a lower amount of introduced damaged areas. But some positive tendencies could be noted for the pulps produced with extra xylan added including: a lower kink/mm and lower amount of cleavage/fiber measured by the HCL method; and the zero-span level and tear-tensile relationship were not inferior compared to the reference despite the higher xylan content. The removal and subsequent re-introduction of xylan into the cook seemed to negatively influence the strength properties, i.e. the tear-tensile relationship was inferior compared to the reference pulp. The redistribution procedure may have drained the fiber wall of xylan negatively influencing the strength propertie

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>A panel of synthetic xylan-binding CBMs, previously selected from a molecular library based on the scaffold of CBM4-2 from xylanase Xyn10A of <it>Rhodothermus marinus</it>, 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.</p> <p>Conclusion</p> <p>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.</p

Microdistribution of xyloglucan in differentiating poplar cells

Recent studies on the ultrastructure and composition of the gelatinous layer (g-layer) in poplar have reported findings of xyloglucan. Using correlated fluorescence, scanning- and transmission electron micros-copy, we found evidence for xyloglucan present in and surrounding the g-layer, using the fucosylated xyloglucan specific CCRC-M1 antibody and the carbohydrate binding module FXG-14b. However, labeling of isolated gelatinous layer remained negative

Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H2O2 in Brown Rot Decay of Wood▿

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (Km = 2.3 mM, kcat = 15.6 s−1). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H2O2, a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals