Molecular architecture of softwood revealed by solid-state NMR

Economically important softwood from conifers is mainly composed of the polysaccharides cellulose, galactoglucomannan and xylan, and the phenolic polymer, lignin. The interactions between these polymers lead to wood mechanical strength and must be overcome in biorefining. Here, we use 13C multidimensional solid-state NMR to analyse the polymer interactions in never-dried cell walls of the softwood, spruce. In contrast to some earlier softwood cell wall models, most of the xylan binds to cellulose in the two-fold screw conformation. Moreover, galactoglucomannan alters its conformation by intimately binding to the surface of cellulose microfibrils in a semi-crystalline fashion. Some galactoglucomannan and xylan bind to the same cellulose microfibrils, and lignin is associated with both of these cellulose-bound polysaccharides. We propose a model of softwood molecular architecture which explains the origin of the different cellulose environments observed in the NMR experiments. Our model will assist strategies for improving wood usage in a sustainable bioeconomy

Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation

Background: Galactoglucomannan (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. Results: CjMan5A 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. Conclusions: Our 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.This work was funded by the Knut and Alice Wallenberg Foundation through the Wallenberg Wood Science Center (WWSC), which is gratefully acknowledged. FV and AMA also thank the Swedish Research Council (Project 621-2014-5295 to FV) for the financial support

Potent endogenous allelopathic compounds in Lepidium sativum seed exudate: effects on epidermal cell growth in Amaranthus caudatus seedlings

Many plants exude allelochemicals – compounds that affect the growth of neighbouring plants. This study reports further studies of the reported effect of cress (Lepidium sativum) seed(ling) exudates on seedling growth in Amaranthus caudatus and Lactuca sativa. In the presence of live cress seedlings, both species grew longer hypocotyls and shorter roots than cress-free controls. The effects of cress seedlings were allelopathic and not due to competition for resources. Amaranthus seedlings grown in the presence of cress allelochemical(s) had longer, thinner hypocotyls and shorter, thicker roots – effects previously attributed to lepidimoide. The active principle was more abundant in cress seed exudate than in seedling (root) exudates. It was present in non-imbibed seeds and releasable from heat-killed seeds. Release from live seeds was biphasic, starting rapidly but then continuing gradually for 24 h. The active principle was generated by aseptic cress tissue and was not a microbial digestion product or seed-treatment chemical. Crude seed exudate affected hypocotyl and root growth at ∼25 and ∼450 μg ml(−1) respectively. The exudate slightly (28%) increased epidermal cell number along the length of the Amaranthus hypocotyl but increased total hypocotyl elongation by 129%; it resulted in a 26% smaller hypocotyl circumference but a 55% greater epidermal cell number counted round the circumference. Therefore, the effect of the allelochemical(s) on organ morphology was imposed primarily by regulation of cell expansion, not cell division. It is concluded that cress seeds exude endogenous substances, probably including lepidimoide, that principally regulate cell expansion in receiver plants

Wood-derived dietary fibers promote beneficial human gut microbiota

Woody biomass is a sustainable and virtually unlimited source of hemicellulosic polysaccharides. The predominant hemicelluloses in softwood and hardwood are galactoglucomannan (GGM) and arabinoglucuronoxylan (AGX), respectively. Based on the structure similarity with common dietary fibers, GGM and AGX may be postulated to have prebiotic properties, conferring a health benefit on the host through specific modulation of the gut microbiota. In this study, we evaluated the prebiotic potential of acetylated GGM (AcGGM) and highly acetylated AGX (AcAGX) obtained from Norwegian lignocellulosic feedstocks in vitro. In pure culture, both substrates selectively promoted the growth of Bifidobacterium, Lactobacillus, and Bacteroides species in a manner consistent with the presence of genetic loci for the utilization of β-manno-oligosaccharides/β-mannans and xylo-oligosaccharides/xylans. The prebiotic potential of AcGGM and AcAGX was further assessed in a pH-controlled batch culture fermentation system inoculated with healthy adult human feces. Results were compared with those obtained with a commercial fructo-oligosaccharide (FOS) mixture. Similarly to FOS, both substrates significantly increased (P < 0.05) the Bifidobacterium population. Other bacterial groups enumerated were unaffected with the exception of an increase in the growth of members of the Bacteroides-Prevotella group, Faecalibacterium prausnitzii, and clostridial cluster IX (P < 0.05). Compared to the other substrates, AcGGM promoted butyrogenic fermentation whereas AcAGX was more propiogenic. Although further in vivo confirmation is necessary, these results demonstrate that both AcGGM and AcAGX from lignocellulosic feedstocks can be used to direct the promotion of beneficial bacteria, thus exhibiting a promising prebiotic ability to improve or restore gut health

Mapping the polysaccharide degradation potential of Aspergillus niger

<p>Abstract</p> <p>Background</p> <p>The degradation of plant materials by enzymes is an industry of increasing importance. For sustainable production of second generation biofuels and other products of industrial biotechnology, efficient degradation of non-edible plant polysaccharides such as hemicellulose is required. For each type of hemicellulose, a complex mixture of enzymes is required for complete conversion to fermentable monosaccharides. In plant-biomass degrading fungi, these enzymes are regulated and released by complex regulatory structures. In this study, we present a methodology for evaluating the potential of a given fungus for polysaccharide degradation.</p> <p>Results</p> <p>Through the compilation of information from 203 articles, we have systematized knowledge on the structure and degradation of 16 major types of plant polysaccharides to form a graphical overview. As a case example, we have combined this with a list of 188 genes coding for carbohydrate-active enzymes from <it>Aspergillus niger</it>, thus forming an analysis framework, which can be queried. Combination of this information network with gene expression analysis on mono- and polysaccharide substrates has allowed elucidation of concerted gene expression from this organism. One such example is the identification of a full set of extracellular polysaccharide-acting genes for the degradation of oat spelt xylan.</p> <p>Conclusions</p> <p>The mapping of plant polysaccharide structures along with the corresponding enzymatic activities is a powerful framework for expression analysis of carbohydrate-active enzymes. Applying this network-based approach, we provide the first genome-scale characterization of all genes coding for carbohydrate-active enzymes identified in <it>A. niger</it>.</p

Bioprospecting xylanase enzymes from environmental samples

Hemicelluloses are a group of heteropolysaccharides which accounts for 33% by dry weight of the total lignocellulosic biomass. Xylan is one of the most important and abundantly found hemicellulose which has diverse structures according to the source from which it is extracted. Due to its structural diversity, xylan is hydrolysed by a class of xylanase enzymes into its monomeric xylose subunits. These xylose residues when treated with yeasts get converted into ethanol which can be used as a supplement along with natural fuels. Ethanol production by biological means not only reduces the cost of production but also decreases the formation of inhibitors which are formed during chemical pretreatment of xylan that hampers the process of fermentation. Besides bioethanol production xylanases have also got many other productive uses in various industries such as paper pulp bleaching, oil extraction, food additives, bakeries, detergents, fodder industries, etc. Twelve strains were isolated from environmental samples like cow dung, leaf litter, waste water, soil, termites, etc. The isolated strains were confirmed for xylanase production on xylan agar plates and Congo red assay. Activity of endoxylanases (1-4 â-D xylanases) was determined by measuring the amount of reducing sugar from xylan which was estimated spectrophotometrically with 3,5 dinitrosalycilic acid using xylose as standard. The isolated strains produced maximum xylanase at 37ᵒC at a pH of 6.8 to 7.0 after 120 h of incubation. Xylanases extracted from different strains showed maximum activity at an optimal pH of 7.0 and optimal temperature 40ᵒC. The value of km and Vmax recorded for highest xylanase producing strain JS4(7) was recorded to be 0.25mg/ml and 83µg/ml/min. The morphology of the two highest xylanase producing strains JS3(4) and JS4(7) was found to be bacillus and coccus respectively under SEM imaging

Ultrastructural characterization (morphological and topochemical) of wood pulp fibres

Different electron microscopy techniques including SEM (scanning electron microscopy), FE-SEM (field emission-scanning electron microscopy), TEM (transmission electron microscopy) and Immuno-gold TEM (immuno-gold transmission electron microscopy) were applied in order to gain a better understanding of the influence of the native softwood fibre cell wall ultrastructure including morphology and topochemistry (i.e. lignin and glucomannan distribution) during mechanical pulping. In thermomechanical pulp (TMP) processing, wood fibres undergo structural changes (cell wall delamination and fibrillation) that are regulated by the native fibre micro- and ultrastructure. In addition, novel information was obtained on the fibre cell wall architecture. In contrast, the stoneground wood (SGW) process inflicted severe damage to the fibre structure resulting in transverse and longitudinal fibre breakage. However, juvenile wood SGW fibres showed improved properties (strength and light scattering) compared to mature wood. Ultrastructural aspects of fibre processing and development explained the differences in physical properties observed. During the SGW process, the native morphological fibre cell wall ultrastructure and microfibrillar organization governed the manner of juvenile wood fibre development similar to TMP fibres. Ultrastructural studies on Norway spruce and Scots pine TMPs revealed fundamental features that governed the different behaviour exhibited by the two wood species. Specific ultrastructural characteristics of pine TMP fibre cell walls were explored in relation to both morphology and topochemistry and that regulating the different pine fibre development mechanisms compared to spruce. The negative behaviour shown by Scots pine during TMP processing was most likely attributable to the observed fibre development mechanism. Histochemical techniques were applied to study wood resin associated problems during mechanical and kraft pulping. Studies provided information on the spatial micro-morphological distribution/redistribution of lipophilic extractives that were visualized on single fibre and cell wall fractions. Results from histochemical staining and chemical analysis performed on Norway spruce and Scots pine TMPs showed that there were morphological and chemical differences in the redistribution of extractives between the two species. This may further contribute to the effects of extractives on pulp- and paper properties and processing. Localization of lipophilic birch wood extractives involved in pitch problems was performed using histochemical techniques. Correlated information from gas chromatography-mass spectrometry and specific staining methods gave details on how extractives are removed during processing as well as information on the mechanisms of removal