ORCID IDs: 0000-0003-0277-2269 (J.B.); 0000-0001-6732-2571 (J.W.); 0000-0003-3572-7798 (F.V.).Xylan is tightly associated with cellulose and lignin in secondary plant cell walls, contributing to its rigidity and structural integrity in vascular plants. However, the molecular features and the nanoscale forces that control the interactions among cellulose microfibrils, hemicelluloses, and lignin are still not well understood. Here, we combine comprehensive mass spectrometric glycan sequencing and molecular dynamics simulations to elucidate the substitution pattern in softwood xylans and to investigate the effect of distinct intramolecular motifs on xylan conformation and on the interaction with cellulose surfaces in Norway spruce (Picea abies). We confirm the presence of motifs with evenly spaced glycosyl decorations on the xylan backbone, together with minor motifs with consecutive glucuronation. These domains are differently enriched in xylan fractions extracted by alkali and subcritical water, which indicates their preferential positioning in the secondary plant cell wall ultrastructure. The flexibility of the 3-fold screw conformation of xylan in solution is enhanced by the presence of arabinofuranosyl decorations. Additionally, molecular dynamic simulations suggest that the glycosyl substitutions in xylan are not only sterically tolerated by the cellulose surfaces but that they increase the affinity for cellulose and favor the stabilization of the 2-fold screw conformation. This effect is more significant for the hydrophobic surface compared with the hydrophilic ones, which demonstrates the importance of nonpolar driving forces on the structural integrity of secondary plant cell walls. These novel molecular insights contribute to an improved understanding of the supramolecular architecture of plant secondary cell walls and have fundamental implications for overcoming lignocellulose recalcitrance and for the design of advanced wood-based materials.

Hemicelluloses, a family of heterogeneous polysaccharides with complex molecular structures, constitute a fundamental component of lignocellulosic biomass. However, the contribution of each hemicellulose type to the mechanical properties of secondary plant cell walls remains elusive. Here we homogeneously incorporate different combinations of extracted and purified hemicelluloses (xylans and glucomannans) from softwood and hardwood species into self-assembled networks during cellulose biosynthesis in a bacterial model, without altering the morphology and the crystallinity of the cellulose bundles. These composite hydrogels can be therefore envisioned as models of secondary plant cell walls prior to lignification. The incorporated hemicelluloses exhibit both a rigid phase having close interactions with cellulose, together with a flexible phase contributing to the multiscale architecture of the bacterial cellulose hydrogels. The wood hemicelluloses exhibit distinct biomechanical contributions, with glucomannans increasing the elastic modulus in compression, and xylans contributing to a dramatic increase of the elongation at break under tension. These diverging effects cannot be explained solely from the nature of their direct interactions with cellulose, but can be related to the distinct molecular structure of wood xylans and mannans, the multiphase architecture of the hydrogels and the aggregative effects amongst hemicellulose-coated fibrils. Our study contributes to understanding the specific roles of wood xylans and glucomannans in the biomechanical integrity of secondary cell walls in tension and compression and has significance for the development of lignocellulosic materials with controlled assembly and tailored mechanical properties.

SUMMARYThe macromolecular conformation of the constituent polysaccharides in lignocellulosic biomass influences their supramolecular interactions, and therefore their function in plants and their performance in technical products. The flexibility of glycosidic linkages from the backbone of hemicelluloses was studied by evaluating the conformational freedom of the φ and w dihedral angles using molecular dynamic simulations, additionally selected molecules were correlated with experimental data by nuclear magnetic resonance spectroscopy. Three types of b-(1?4) glycosidic linkages involving the monosaccharides (Glcp, Xylp and Manp) present in the backbone of hemicelluloses were defined. Different di-and tetrasaccharides with combinations of such sugar monomers from hemicelluloses were simulated, and free energy maps of the φ -w space and hydrogen-bonding patterns were obtained. The glycosidic linkage between Glc-Glc or Glc-Man (C-type) was the stiffest with mainly one probable conformation; the linkage from Man-Man or Man-Glc (M-type) was similar but with an increased probability for an alternative conformation making it more flexible, and the linkage between two Xyl-units (X-type) was the most flexible with two almost equally populated conformations. Glycosidic linkages of the same type showed essentially the same conformational space in both disaccharides and in the central region of tetrasaccharides. Different probabilities of glycosidic linkage conformations in the backbone of hemicelluloses can be directly estimated from the free energy maps, which to a large degree affect the overall macromolecular conformations of these polymers. The information gained contributes to an increased understanding of the function of hemicelluloses both in the cell wall and in technical products.

Galactoglucomannan (GGM) from spruce was studied with respect to the degradation behavior in alkaline solution. Three reference systems including galactomannan from locust bean gum, glucomannan from konjac and the linear water-soluble carboxymethyl cellulose were studied with focus on molecular weight, sugar composition, degradation products, as well as formed oligomers, to identify relative structural changes in GGM. Initially all mannan polysaccharides showed a fast decrease in the molecular weight, which became stable in the later stage. The degradation of the mannan polysaccharides could be described by a function corresponding to the sum of two first order reactions; one slow that was ascribed to peeling, and one fast that was connected with hydrolysis. The galactose side group was stable under conditions used in this study (150 min, 90°C, 0.5 M NaOH). This could suggest that, apart from the covalent connection to C6 in mannose, the galactose substitutions also interact non-covalently with the backbone to stabilize the structure against degradation. Additionally, the combination of different backbone sugars seems to affect the stability of the polysaccharides. For carboxymethyl cellulose the degradation was linear over time which further suggests that the structure and sugar composition play an important role for the alkaline degradation. Molecular dynamics simulations gave details about the conformational behavior of GGM oligomers in water solution, as well as interaction between the oligomers and hydroxide ions.

Plant β-mannans are complex heteropolysaccharides that represent an abundant resource from lignocellulosic biomass. The influence of the molecular motifs of plant mannans on the backbone flexibility, solubility, and the interaction with cellulose was investigated by computational and experimental approaches. The regioselectivity of the acetyl substitutions at C2 and C3 distinctively influenced backbone flexibility in aqueous media, as revealed by molecular dynamic simulations. The molecular weight and degree of acetylation were tailored for two model seed mannans (galactomannan and glucomannan) and compared to spruce acetylated galactoglucomannan. The thermal stability was enhanced with increasing acetyl substitutions, independently of the type of mannan. Dynamic light scattering and atomic force microscopy revealed that the occurrence of galactosylation and a low degree of acetylation (similar to that of native acetylated galactoglucomannans) enhanced solubility/dispersibility of mannans, whereas the solubility/dispersibility decreased for higher degrees of acetylation. Mannan solubility influenced their interactions with cellulose at water–cellulose interfaces in terms of adsorbed mass and viscoelastic properties of the adsorbed mannan layers. Our results reveal that modulating the molecular motifs of plant β-mannans influences their macromolecular conformation and physicochemical properties, with fundamental implications for their role in the plant cell wall and the design of wood-based materials.

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BackgroundGalactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. GGM can be hydrolyzed into fermentable sugars, or used as a polymer in films, gels, and food additives. Endo-β-mannanases, which can be found in the glycoside hydrolase families 5 and 26, specifically cleave the mannan backbone of GGM into shorter oligosaccharides. Information on the activity and specificity of different mannanases on complex and acetylated substrates is still lacking. The aim of this work was to evaluate and compare the modes of action of two mannanases from Cellvibrio japonicus (CjMan5A and CjMan26A) on a variety of mannan substrates, naturally and chemically acetylated to varying degrees, including naturally acetylated spruce GGM. Both enzymes were evaluated in terms of cleavage patterns and their ability to accommodate acetyl substitutions.ResultsCjMan5A and CjMan26A demonstrated different substrate preferences on mannan substrates with distinct backbone and decoration structures. CjMan5A action resulted in higher amounts of mannotriose and mannotetraose than that of CjMan26A, which mainly generated mannose and mannobiose as end products. Mass spectrometric analysis of products from the enzymatic hydrolysis of spruce GGM revealed that an acetylated hexotriose was the shortest acetylated oligosaccharide produced by CjMan5A, whereas CjMan26A generated acetylated hexobiose as well as diacetylated oligosaccharides. A low degree of native acetylation did not significantly inhibit the enzymatic action. However, a high degree of chemical acetylation resulted in decreased hydrolyzability of mannan substrates, where reduced substrate solubility seemed to reduce enzyme activity.ConclusionsOur findings demonstrate that the two mannanases from C. japonicus have different cleavage patterns on linear and decorated mannan polysaccharides, including the abundant and industrially important resource spruce GGM. CjMan26A released higher amounts of fermentable sugars suitable for biofuel production, while CjMan5A, producing higher amounts of oligosaccharides, could be a good candidate for the production of oligomeric platform chemicals and food additives. Furthermore, chemical acetylation of mannan polymers was found to be a potential strategy for limiting the biodegradation of mannan-containing materials.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1115-y) contains supplementary material, which is available to authorized users.

The impact of various degrees of acetylation on improving the thermal stability of xylan isolated from different botanical source has been studied; methylglucuronoxylan from birch and eucalyptus, arabinoglucuronoxylan from spruce and glucuronoarabinoxylan from sugarcane bagasse and straw. The lower molecular weight of non-acetylated methylglucuronoxylan (17.7-23.7 kDa) and arabinoglucuronoxylan (16.8 kDa) meant that they were more soluble in water than glucuronoarabinoxylan (43.0-47.0 kDa). The temperature at the onset of degradation increased by 17-61°C and by 70-145°C for 2 low and high acetylated xylans respectively, as a result of acetylation. A glass transition temperature in the range of 121-132°C was observed for the samples non-acetylated and acetylated at low degree of acetylation (0.0-0.6). The acetylation to higher degrees (1.4-1.8) increased the glass transition temperature of the samples to 188-206°C. Acetylation proved to be an efficient method for functionalization of the xylan to increase the thermal stability.