Breakdown Of Hemicellulose Research Articles

The accessory domain changes the accessibility and molecular topography of the catalytic interface in monomeric GH39 β-xylosidases

β-Xylosidases (EC 3.2.1.37) are among the principal glycosyl hydrolases involved in the breakdown of hemicelluloses, catalyzing the reduction of xylooligosaccharides to free xylose. All GH39 β-xylosidases structurally characterized to date display a modular multi-domain organization that assembles a tetrameric quaternary structure. In this work, the crystal structure and the SAXS molecular envelope of a new GH39 β-xylosidase from Caulobacter crescentus (CcXynB2) have been determined. Interestingly, CcXynB2 is a monomer in solution and comparative structural analyses suggest that the shortened C-terminus prevents the formation of a stable tetramer. Moreover, CcXynB2 has a longer loop from the auxiliary domain (the long α-helix-containing loop) which makes a number of polar and hydrophobic contacts with the parental (α/β)(8)-barrel domain, modifying the accessibility and the molecular topography of the catalytic interface. These interactions also maintain the accessory domain tightly linked to the catalytic core, which may be important for enzyme function and stability.

A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover

BackgroundFungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study.ResultsA. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14 days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2 days and then maintained a steady state of 4% of the total biomass for the next 12 days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14 days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.ConclusionsOur results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1 day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.

One-Pot Conversion of Sugars and Lignin in Ionic Liquid and Recycling of Ionic Liquid

The mixture of ionic liquid (IL) and aqueous ethanol has been proven to be a promising biomass pretreatment solvent, facilitating the separation of cellulose and faster breakdown of hemicellulose through the disruption of lignin. However, lignin and sugar monomers from the hydrolysis of polysaccharides are soluble in IL and difficult to separate. This complicates the recycling of IL. To address this, CrCl3 and CrCl3·6H2O are used as catalysts for the one-pot conversion of lignin and sugars in the ionic liquid 1-butyl-3-methyl imidazolium chloride ([BMIM]Cl). Furfural resin or humin is produced and separated from IL after the reactions. When catalyzed by CrCl3.6H2O and at a high temperature (≥170 °C), almost all the lignin and sugars are transformed to humin, which leads to the high efficiency of IL recycling. And the mechanisms of the reactions are studied with guaiacylglycerol-beta-guaiacyl ether (GG) and glucose as model compounds.

Carbohydrate transporting membrane proteins of the rumen bacterium, Butyrivibrio proteoclasticus

The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation.

Particle geometry affects differentially substrate composition and enzyme profiles by Pleurotus ostreatus growing on sugar cane bagasse

The growth of Pleurotus ostreatus was analyzed on three particle sizes of sugar cane bagasse: 0.92 mm and 1.68 mm in diameter, in addition to heterogeneous fibers (average 2.9 mm in diameter). Specific growth rate on heterogeneous particles was lower (μ=0.043 h(-1)), although soluble protein production was maximal (809 μg/g dry wt). Higher μ values were reached on the other two particles sizes (0.049-0.05 h(-1)) with less soluble protein (500 μg/g dry wt). Xylanases and laccases were favored in heterogeneous particles; while the highest selectivity for xylanases over cellulases was observed in 1.68 mm particles, corresponding with the maximal hemicellulose breakdown. Lignin and cellulose were preferentially degraded in smallest particles. This study shows that the geometrical ratio, shape and size of sugar cane bagasse fibers strongly influence packing density for SSF substrate, with an impact in the production of extracellular enzymes, growth rates and composition changes in substrate.

An Effective Combination of Carbohydrases That Enables Reduction of Dietary Protein in Broilers: Importance of Hemicellulase

After observing the effects of purified cellulase (C), hemicellulase (H), pectinase (P), and their combinations on the in vitro digestibility of a corn-soybean meal broiler diet, we examined the associations between pectin breakdown and the digestibilities of CP and DM by using free galacturonic acid (GA) as an index of pectin breakdown. There was no significant effect of the single enzymes except for H. However, the enzyme combinations H + P, C + H, and C + H + P significantly increased CP and DM digestibilities, whereas the combination of C + P was not effective. Because H has activities of both H and P, these enzymes were considered to be important in stimulating digestion. Furthermore, when the enzymes increased CP and DM digestibilities, GA concentration was significantly higher, and clear correlations between CP and DM digestibilities and GA concentration were observed, whereas correlations between the digestibilities and concentration of glucose or xylose + mannose as indices of cellulose and hemicellulose breakdown, respectively, were not significant. From these observations, we hypothesized that a mixture of enzymes could increase the protein digestibility of broiler feed. Thus, in the in vivo experiment, low-protein (19% CP) diets made mainly of corn and soybean meal with or without mixed enzymes were prepared and given to broiler chicks. The birds given the diet containing mixed enzymes showed significantly higher BW gain, with higher CP and DM digestibilities than the birds given the diet without the mixed enzymes. Moreover, the growth rate was same as that of the birds given the normal (21% CP) diet. The results indicate that the mixed enzyme preparation can effectively degrade indigestible cell constituents and thus enable the protein of the broiler feed to become more digestible. Furthermore, the results indicate the importance of H as a rate-limiting factor of cell wall breakdown.

Water‐soluble organic compounds in biomass burning aerosols over Amazonia 1. Characterization by NMR and GC‐MS

As part of the European contribution to the Large‐Scale Atmosphere‐Biosphere Experiment in Amazonia (LBA‐EUSTACH), aerosols were sampled at representative pasture and primary rainforest sites in Rondônia, Brazil, during the 1999 “burning season” and dry‐to‐wet season transition (September‐October). Water‐soluble organic compounds (WSOCs) within the samples were characterized using a combination of1H Nuclear Magnetic Resonance (NMR) spectroscopy for chemical functional group analysis, and Gas Chromatography‐Mass Spectrometry (GC‐MS) for identification and quantification of individual low‐molecular‐weight compounds. The1H NMR analysis indicates that WSOCs are predominantly aliphatic or oxygenated aliphatic compounds (alcohols, carboxylic acids, etc.), with a minor content of aromatic rings carrying carboxylic and phenolic groups. Levoglucosan (1,6‐anhydro‐β‐D‐glucose), a well‐known cellulose combustion product, was the most abundant individual compound identified by GC‐MS (0.04–6.90 μg m−3), accounting for 1–6% of the total carbon (TC) and 2–8% of the water‐soluble organic carbon (WSOC). Other anhydrosugars, produced by hemicellulose breakdown, were detected in much smaller amounts, in addition to series of acids, hydroxyacids, oxoacids, and polyalcohols (altogether 2–5% of TC, 3–6% of WSOC). Most correlated well with organic carbon, black carbon, and potassium, indicating biomass burning to be the major source. A series of sugar alcohols (mannitol, arabitol, erythritol) and sugars (glucose, fructose, mannose, galactose, sucrose, trehalose) were identified as part of the natural background aerosol and are probably derived from airborne microbes and other biogenic material. The bulk of the WSOCs (86–91% WSOC) eluded analysis by GC‐MS and may be predominantly high‐molecular weight in nature.

Formation and mobilization pathways of dissolved organic matter: evidence from chemical structural studies of organic matter fractions in acid forest floor solutions

Dissolved organic matter (DOM) is well recognized to influence the geochemistry of ecosystems. This study was conducted to determine the pathways of DOC mobilization in the forest floor of coniferous forests. DOM from the forest floor of two acid forest soils in the Fichtelgebirge (Germany) was fractionated into hydrophobic acids and neutrals, and hydrophilic acids and neutrals. Carbon distribution at both sites was similar: 53 and 52% hydrophobic acids, 23 and 22% hydrophilic acids, 10 and 11% hydrophobic neutrals, and 7 and 8% hydrophilic neutrals, respectively. Structural composition of the DOM fractions was determined using chemical degradation, FT-IR and 13C NMR spectroscopy, as well as pyrolysis-field ionization mass spectrometry. Results indicate different chemical composition of the DOM fractions within each site. Hydrophobic acids show high concentrations of carboxyl and hydroxyl groups, and a high ratio of vanillic acid to vanillin ( ca 1.0), both indicating a high degree of biodegradation of plant-derived compounds. Carbohydrates are covalently bound to apolar moieties and from carbohydrate composition a lignocellulose nature of the hydrophobic acid fraction is suggested. Hydrophilic acids can be differentiated from the hydrophobic acids by their higher degree of oxidative biodegradation. The hydrophilic acid fraction also exhibits a higher ratio of microbially released polysaccharides versus plant-derived polysaccharides. Hydrophobic neutrals show the closest relationships to the refractory soil humin, with less degraded dimeric ‘condensed’ lignin subunits and relatively high contents of non-carbohydrate aliphatics. Hydrophilic neutrals are enriched in carbohydrates mobilized by enzymatic cellulose and hemicellulose breakdown, as well as from microbial origin. We conclude that DOM release into forest floor solution is related to microbial activity by oxidative degradation of plant-derived organic matter (e.g. water-soluble lignin and lignocellulose fragments) and by production of microbial metabolites (e.g. polysaccharides). Overall evidence suggests that hydrophobic acids (i) represent intermediates in organic matter decomposition, which can be further degraded to hydrophilic acids and CO 2, and (ii) are precursors of humic substances in illuvial horizons after precipitation/adsorption.

Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.

Xylanase and β‐xylosidase production by Aspergillus ochraceus during growth on lignocelluloses

Xylan is a polysaccharide located in the cell wall of higher plants which is one of the major components of lignocellulosic materials. In some higher plants and agricultural wastes the xylan is 20-40% of the dry material.' It consists of p-1, 4-linked D-xylose chain. The breakdown of hemicellulose is accomplished by the synergistic action of xylanase (1,4+-~-xylan xylanohydrolase, EC 3.2.1.8) and P-xylosidase (P-D-xyloside xylohydrolase, EC 3.2.1.37). Such enzymes are useful in the degradation of biomaterials containing hemicellulose for the production of monosaccharides from which single-cell protein, single-cell oil, or ethanol could be produced. '-I1 The production and properties of enzymes capable of breaking down cellulose have been extensively st~died. '~-~' Although there are reports on the production of xylanase,'8-26 the behavior of xylanases and its involvement in the utilization of complex lignocellulosic material is still incomplete. Therefore, the present communication deals with the utilization of some lignocellulosic materials by A . ochraceus for xylanase and pxylosidase production. The use of purified xylan enhances the cost of enzyme production and is a major limitation to the economic feasibility of bioconversion and utilization of lignocellulosic materials. Therefore, in this work, easily available substrates like wheat straw, wheat bran, and sugarcane bagasse were used in the experiment to obtain optimum levels of xylanases by the fungal strain A . ochruceus.

Hindrance of Hemicellulose and Cellulose Hydrolysis by Pectic Substances

Treating the tissue of the grapefruit segment membrane with pectinase and cellulase decreased the content of xylose and glucose significantly more than the sum of the separate effects. The synergistic effect obtained by the combination of pectinase and cellulase showed that the pectic substance sterically masked the hemicellulose and cellulose. Breaking down the barrier of the pectic substance with pectinase allowed a significant increase in the hydrolysis of the hemicellulose and cellulose. Preparation of alchol‐insoluble solids from fresh tissue modified the structure of the pectin in the cell wall and prevented its steric hindrance to breakdown of hemicellulose and cellulase. Extraction of pectic substances from the tissue by NaOH greatly increased cellulose hydrolysis by cellulase.

Recycle of the cellulase— enzyme complex after hydrolysis of steam-exploded wood

Cellulase can be recovered in high yields by contacting fresh substrate with hydrolysis filtrate and by extraction of spent hydrolysis residue with pH 7 buffer. Recycled enzymes give hydrolysis rates about equal to those with fresh enzymes. Steam-exploded wood (SEW) is washed with water to remove sugars and byproducts from breakdown of hemicellulose, and recycle of enzymes proceeds better if lignin is also removed prior to hydrolysis. Oven drying of SEW interferes with recycle, and the recovery of enzymes is only one-half of that with SEW that is kept moist. Effectiveness of enzyme recovery depends on the completeness of hydrolysis, as determined by contact time and enzyme concentration. For cost-effective operation, enzyme should not be recovered until appreciable filter paper activity and carboxylmethylcellulase activity appear in the hydrolysate. 6 references.

Conservation of herbage of varying dry matter content in air‐tight metal containers with reference to the carbohydrate fraction

AbstractHerbage at four different dry matter levels, 19·8, 28·0, 37·5 and 45·0%, was ensiled in air‐tight metal containers. There was no loss as inedible waste and this together with the low losses of constituents demonstrated the efficiency of this system of conservation. The mean dry matter loss from the silos was 6%. There was no discernible trend in the dry matter losses with increasing dryness of the ensiled material.The presence of butyric acid and the low levels of volatile nitrogen indicated a saccharolytic type of clostridial activity.Arabinose, galactose and xylose arising from the breakdown of hemicellulose were detected in the silages but the loss of the hemicellulose fraction was low.The presence of mannitol, which was most prevalent in the wilted‐grass silages, indicated a substantial degree of a heterolactic fermentation of fructose. In the material of low dry matter concentration a homolactic fermentation was predominant.