Xylan Chain Research Articles

The X‐Ray Crystallographic Structure of Endoxylanase Xyn10C from Xanthomonas axonopodis at 1.6 Angstrom Resolution

The recent biochemical characterization of the xylanases of glycoside hydrolase family 10 (GH 10) has identified a distinctive endolytic mode of action, hydrolyzing the β-1,4 xylan chain at a specific site directed by the position of an α-1,2-linked glucuronate moiety. Xylanase C (XynC), the GH10 xylanase from Xanthomonas axonopodis, has been cloned, overexpressed and crystallized. Previous attempts to crystallize the protein using commercially available screens were unsuccessful, but analysis of the crystallization condition of a homologous protein allowed us to construct a novel grid screen and obtain small (< 0.5 mm) crystals of the protein in 24 hours via hanging drop vapor diffusion. Crystals grew in 100 mM sodium phosphate citrate, 200 mM NaCl, 25% (w/v) PEG 8000, 5% (v/v) glycerol, pH 5.5. The crystals belonged to the orthorhombic space group C2221 and had unit cell parameters of 71 Å, 80 Å and 161 Å and α=β=γ=90°. Diffraction data were collected to 1.6 Å resolution and an initial phasing model was created using molecular replacement techniques. Multiple rounds of model building and refinement were performed using Coot and Phenix, respectively. The protein was found to possess the typical (β/α)8 fold observed in all members of GH10. A large loop on the aglycone side of the active site was found to adopt a unique conformation. This loop was in a position that would prevent it from binding substrates larger than three xylose units in size and would be expected to produce a xylobiose and a free xylose upon cleavage of such a trimer. This work will help to identify the structural determinants that grant the exceptional specificity of this enzyme and the role it plays in the biological depolymerization and processing of glucuronoxylan.

Construction of porous chitosan–xylan–TiO2 hybrid with highly efficient sorption capability on heavy metals

A facile and environmentally benign method was developed to incorporate TiO2 into a chitosan–xylan polysaccharide network. The chitosan–xylan–TiO2 hybrid (CXTH) with highly porous structure was constructed via Schiff base linkages between the aldehyde groups on xylan chain and the amino groups on chitosan. The CXTH was characterized by FTIR, TGA, XRD, SEM and TEM. Moreover, the porous structure of CXTH could be controlled by employing nanoTiO2 particles as nanofillers. The results showed that the nanoTiO2 powders can significantly improve the porous structure of hybrid. The water absorption of CXTH reached 1507wt% in purified water and 821wt% in 0.9wt% NaCl solution, respectively. The adsorption capabilities for heavy metals on CXTH was investigated as the function of pH, temperature, contact time and the initial concentration of metal ions. The maximum adsorption capacities of CXTH for Cu2+, Cr6+, Ni2+, Cd2+ and Hg2+ were 158.7, 97.1, 96.2, 78.1 and 76.3mgg−1, respectively. Langmuir isotherm equation and the pseudo-second-order kinetic model illustrated that the adsorption was spontaneous and endothermic chemical adsorption. The regeneration experiments showed CXTH can be used effectively for numbers of adsorption–desorption cycles.

Production of xylooligosaccharides by immobilized His-tagged recombinant xylanase from Penicillium occitanis on nickel-chelate Eupergit C.

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49%. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH2.0-4.0) than that of the free PoXyn2 (pH3.0). The free PoXyn2 had an optimum temperature of 50°C, whereas that of the immobilized enzyme was shifted to 65°C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2% (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.

Functional roles of rice glycosyltransferase family GT43 in xylan biosynthesis

Xylan is the major hemicellulose present in both primary and secondary cell walls of rice vegetative tissues. Since xylan is one of the factors contributing to biomass recalcitrance, understanding how xylan is synthesized in rice will potentially provide tools to modify grass biomass composition better suited for biofuel production. Studies of xylan biosynthesis in Arabidopsis have revealed that family GT43 glycosyltransferases, which form 2 functionally nonredundant groups, IRX9/IRX9 homolog and IRX14/IRX14 homolog, are required for xylan backbone elongation. The rice genome harbors 10 genes encoding family GT43 members and it is currently unknown whether they are all involved in xylan biosynthesis. In this report, we performed biochemical analysis of xylan xylosyltransferase activity in rice stem microsomes and investigated the roles of 4 representative rice GT43 members, OsGT43A (LOC_Os05 g03174), OsGT43E (LOC_Os05 g48600), OsGT43H (LOC_Os04 g01280), and OsGT43J (LOC_Os06 g47340), in xylan biosynthesis. OsGT43 proteins were shown to be localized in the Golgi, where xylan biosynthesis occurs. Complementation analysis by expression of OsGT43s in Arabidopsis irx9 and irx14 mutants demonstrated that OsGT43A and OsGT43E but not OsGT43H and OsGT43J were able to rescue the mutant phenotypes conferred by the irx9 mutation, including defective stem mechanical strength, vessel morphology, xylan content, GlcA side chains, xylan chain length, and xylosyltransferase activity. On the other hand, OsGT43J but not OsGT43A, OsGT43E, and OsGT43H restored the defective xylan phenotype in the irx14 mutant. These results indicate that the rice GT43 family evolved to retain the involvement of 2 functionally nonredundant groups, OsGT43A and OsGT43E (IRX9 homologs) vs. OsGT43J (an IRX14 homolog), in xylan backbone biosynthesis.

Facile fabrication of chitosan active film with xylan via direct immersion

Chitosan film was immersed in NaOH solution with xylan to simply prepare active chitosan/xylan film. FT-IR, XRD, FE-SEM, AFM and XPS were used to evaluate the effects of xylan on the structure and morphology of chitosan film, and a wide variety of material characteristics of the chitosan/xylan composite films were investigated. The results showed that the xylan chains entered into the gap of chitosan film and became nodules, leading to strong hydrogen bonds and electrostatic interactions between chitosan and xylan. Moreover, the introduction of xylan not only resulted in stronger crystallinity and a more compact structure of chitosan film, but also had an important effect on the properties of chitosan film. The tensile strength, breaking elongation and anti-ultraviolet performance of the chitosan/xylan films were improved greatly with the increasing concentration of xylan; the water vapor transmission rate, water absorption rate and oxygen barrier property of chitosan/xylan composite films were higher than those of chitosan film; chitosan/xylan composite films still showed hydrophobicity when the xylan concentration was more than 1 %. The chitosan/xylan composite film has more potential to be used as food packaging than pure chitosan film.

Carbohydrate composition of compost during composting and mycelium growth of Agaricus bisporus

Changes of plant cell wall carbohydrate structures occurring during the process to make suitable compost for growth of Agaricus bisporus are unknown. In this paper, composition and carbohydrate structures in compost samples collected during composting and mycelium growth were analyzed. Furthermore, different extracts of compost samples were prepared with water, 1M and 4M alkali and analyzed. At the beginning of composting, 34% and after 16 days of mycelium growth 27% of dry matter was carbohydrates. Carbohydrate composition analysis showed that mainly cellulose and poorly substituted xylan chains with similar amounts and ratios of xylan building blocks were present in all phases studied. Nevertheless, xylan solubility increased 20% over the period of mycelium growth indicating partial degradation of xylan backbone. Apparently, degradation of carbohydrates occurred over the process studied by both bacteria and fungi, mainly having an effect on xylan-chain length and solubility.

The Arabidopsis DUF231 Domain-Containing Protein ESK1 Mediates 2-O- and 3-O-Acetylation of Xylosyl Residues in Xylan

Xylan, a major polysaccharide in plant lignocellulosic biomass, is acetylated at O-2 and/or O-3 and its acetylation impedes the use of biomass for biofuel production. Currently, it is not known what genes encode acetyltransferases that are responsible for xylan O-acetylation. In this report, we demonstrate an essential role for the Arabidopsis gene ESKIMO1 (ESK1) in xylan O-acetylation during secondary wall biosynthesis. ESK1 expression was found to be regulated by the secondary wall master regulator SND1 (secondary wall-associated NAC domain protein1) and specifically associated with secondary wall biosynthesis. Its encoded protein was localized in the Golgi, the site of xylan biosynthesis. The esk1 mutation caused reductions in secondary wall thickening and stem mechanical strength. Chemical analyses of cell walls revealed that although the esk1 mutation did not cause apparent alterations in the xylan chain length and the abundance of the reducing end sequence, it resulted in a significant reduction in the degree of xylan acetylation. The reduced acetylation of esk1 xylan rendered it more accessible and digestible by endoxylanase, leading to generation of shorter xylooligomers compared with the wild type. Further structural analysis of xylan showed that the esk1 mutation caused a specific reduction in 2-O- and 3-O-monoacetylation of xylosyl residues but not in 2,3-di-O-acetylation or 3-O-acetylation of xylosyl residues substituted at O-2 with glucuronic acid. Consistent with ESK1's involvement in xylan O-acetylation, an activity assay revealed that the esk1 mutation led to a significant decrease in xylan acetyltransferase activity. Together, these results demonstrate that ESK1 is a putative xylan acetyltransferase required for 2-O- and 3-O-monoacetylation of xylosyl residues and indicate the complexity of the biochemical mechanism underlying xylan O-acetylation.

Long-Chain Anhydride Modification: A New Strategy for Preparing Xylan Films

Xylan, which is a widely abundant plant polymer, has been considered as an alternative for film preparation. Up to now, however, xylan films have suffered from brittleness, low mechanical strength, and humidity sensitivity. This paper describes a new and effective strategy to prepare xylan films with high mechanical strength and less moisture-sensitive properties by introducing long carbon chains into the xylan backbone. Furthermore, this work revealed some important details on the relationships between structure (molecular structure, aggregation behaviors, and surface morphology) and properties (film-forming performance, flexibility, tensile strength, and hydrophilicity) of xylan film. It was found that the hydrophobic carbon chains (2-octenylsuccinic anhydride half-ester groups) in the xylan backbone acted as steric hindrance and could effectively prevent xylan chains from aggregation. 2-Octenylsuccinic anhydride (2-OSA) modified xylan (2-OSA-X) demonstrated amorphous structure and had better film-forming performance than the unmodified xylan. 2-OSA-X films were smooth, flexible, and less moisture-sensitive and showed significantly increasing tensile strength at a low degree of substitution.

Designing Cell Walls for Improved Bioenergy Production

Dwindling supply and unpredictable price fluctuations of fossil fuels have necessitated a renewed worldwide search for alternative fuels. Plant cell walls, rich in cellulose and hemicellulose, are actively pursued for their use in production of second-generation biofuels. It has been envisioned that degradation of these carbohydrate polymers to their sugar monomer subunits (saccharification) will provide necessary substrates for their yeast-mediated fermentation to ethanol. But the major component of plant cell walls, cellulose, is not easily amenable to this type of deconstruction and is tightly interlinked with hemicelluloses and lignin, making cell walls highly recalcitrant to saccharification.

Behavior of xylans from Eucalyptus species. Part 1. The influence of structural features of eucalyptus xylans on their retention in kraft pulp

Abstract 4-O-methylglucuronoxylans were isolated from the wood and pulp of different Eucalyptus species (E. dunni, E. grandis, E. nitens, E. urograndis and E. urophylla) and characterized by 1H NMR spectroscopy and size-exclusion chromatography (SEC). The content of 4-O-methylglucuronic acid (MeGlcA) varied between the eucalyptus species from 1.90 to 2.80 expressed as MeGlcA/10 xyloses, whereas in the pulp, the total uronic acid content was approximately 1.02, from which the half was hexenuronic acid (HexA). From the MeGlcA linked to the xylan chain 9–26% are substituted in position O-2 by hexose units. The Mw of the xylans extracted from wood ranged between 26.1 and 28.4 kDa. The retention of 4-O-methylglucuronoxylans (51.3–58.7%) during kraft pulping was high, and the retention is related to the content of MeGlcA group, the content of MeGlcA group substituted at O-2, and the average molecular weight of the xylans. The parameters observed affect the overall pulp yield and pulp properties.

Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species

Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituents present in various xylans. We have produced a monoclonal antibody which specifically recognizes glucopyranosyl uronic acid (GlcA), or its 4-O-methyl ether (meGlcA), substituents in xylan and has no cross-reactivity with linear or arabinofuranosyl-substituted xylans. The UX1 antibody binds most strongly to (me)GlcA substitutions at the non-reducing ends of xylan chains, but has a low cross-reactivity with internal substitutions as well, at least on oligosaccharides. The antibody labeled plant cell walls from both mono- and dicotyledons, but in most tissues an alkaline pretreatment was needed for antibody binding. The treatment removed acetyl groups from xylan, indicating that the vicinity of glucuronic acid substituents is also acetylated. The novel labeling patterns observed in the xylem of tree species suggested that differences within the cell wall exist both in acetylation degree and in glucuronic acid content.

Structure and organization within films of arabinoxylans extracted from wheat flour as revealed by various NMR spectroscopic methods

Arabinoxylans (AXs) extracted from wheat flour were characterized by using three different techniques of NMR spectroscopy. Liquid-state (1)H NMR and solid-state (13)C NMR allowed the investigation of the fine structure of the three specific fractions of AXs representative of the structural heterogeneity of AX in wheat tissues. Three pure AX fractions exhibiting an arabinose to xylose ratio of 0.33, 0.53, and 0.73 were compared relative to their substitution feature and also to their assembly into thin films. Measurements of M(2), i.e. the second moment of proton dipolar interactions between the polysaccharide chains, were achieved using time-domain (TD) (1)H NMR at different water contents and temperatures. Transitions of the M(2) values were observed at a certain temperature close to the glass transition temperature T(g) values of AXs in films. Comparison of the different AX films containing various water contents pointed out stronger dipolar interactions for lowly substituted AX. This indicated that, in films, contiguous unsubstituted xylan chains can interact together through hydrogen bonding resulting in a compact structure with small nanopores because of the lower chain motions and the shorter average distances between the lowly substituted AX chains.

Production of xylooligosaccharides using immobilized endo-xylanase of Bacillus halodurans

Endo-xylanase secreted by the alkaliphilic Bacillus halodurans was immobilized onto an anionic exchange resin via the ionic linkage and the highest immobilized amount was achieved at pH 8. Approximately 0.4mg of enzyme could be coupled onto 1g of anionic exchanger. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains as the substrate and to produce more xylobiose and xylotriose initially. On the contrary, when free enzyme was employed, products at initial stage of the reaction exhibited higher level of oligomers with degree of polymerization greater than 4, suggesting that free xylanase worked on both longer (insoluble) and shorter (soluble) xylan chains. At the end of 24h reaction, XOS mixture contained a total of 25.2% and 32.5% (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The conversions for converting substrate xylan to soluble XOS with immobilized xylanase was determined to be 80.9%, which was lower than the use of free xylanase (99.8%). The combination of free and immobilized xylanase can be employed to further improve the conversion of XOS.

Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase

Xylanase A from the phytopathogenic bacterium Erwinia chrysanthemi is classified as a glycoside hydrolase family 30 enzyme (previously in family 5) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid β-D-xylopyranosyl-(1→4)-[4-O-methyl-α-D-glucuronosyl-(1→2)]-β-D-xylopyranosyl-(1→4)-D-xylose as a ligand. The crystal structure of the enzyme-ligand complex was solved at 1.39 Å resolution. The ligand xylotriose moiety occupies subsites -1, -2 and -3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guanidinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear β-1,4-xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate. Structural and experimental data are available in the Protein Data Bank database under accession number 2y24 [45].

Selective chemical oxidation and depolymerization of switchgrass (Panicum virgatum L.) xylan with oligosaccharide product analysis by mass spectrometry

Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose; consequently, pretreatment conditions are (in part) optimized for maximal xylan depolymerization or displacement. Xylan consists of a long chain of β-1,4-linked xylose units substituted with arabinose (typically α-1,3-linked in grasses) and glucuronic acid (α-1,2-linked). Xylan has been proposed to have a structural function in plants and therefore may play a role in determining biomass reactivity to pretreatment. It has been proposed that substitutions along xylan chains are not random and, based upon studies of pericarp xylan, are organized in domains that have specific structural functions. Analysis of intact xylan is problematic because of its chain length (> degree of polymerization (d.p.) 100) and heterogeneous side groups. Traditionally, enzymatic end-point products have been characterized due to the limited products generated. Analysis of resultant arabino-xylo-oligosaccharides by mass spectrometry is complicated by the isobaric pentose sugars that primarily compose xylan. In this report, the variation in pentose ring structures was exploited for selective oxidation of the arabinofuranose primary alcohols followed by acid depolymerization to provide oligosaccharides with modified arabinose branches intact. Switchgrass samples were analyzed by hydrophilic interaction chromatography (HILIC)-liquid chromatography (LC)-mass spectrometry/mass spectrometry (MSMS) and off-line nanospray MS to demonstrate the utility of this chemistry for determination of primary hydroxyl groups on oligosaccharide structures, with potential applications for determining the sequence of arabino-xylo-oligosaccharides present in plant cell wall material. Published in 2011 by John Wiley & Sons, Ltd.

Selective chemical oxidation and depolymerization of switchgrass (Panicum virgatum L.) xylan with oligosaccharide product analysis by mass spectrometry

Xylan is a barrier to enzymatic hydrolysis of plant cell walls. It is well accepted that the xylan layer needs to be removed to efficiently hydrolyze cellulose; consequently, pretreatment conditions are (in part) optimized for maximal xylan depolymerization or displacement. Xylan consists of a long chain of β-1,4-linked xylose units substituted with arabinose (typically α-1,3-linked in grasses) and glucuronic acid (α-1,2-linked). Xylan has been proposed to have a structural function in plants and therefore may play a role in determining biomass reactivity to pretreatment. It has been proposed that substitutions along xylan chains are not random and, based upon studies of pericarp xylan, are organized in domains that have specific structural functions. Analysis of intact xylan is problematic because of its chain length (> degree of polymerization (d.p.) 100) and heterogeneous side groups. Traditionally, enzymatic end-point products have been characterized due to the limited products generated. Analysis of resultant arabino-xylo-oligosaccharides by mass spectrometry is complicated by the isobaric pentose sugars that primarily compose xylan. In this report, the variation in pentose ring structures was exploited for selective oxidation of the arabinofuranose primary alcohols followed by acid depolymerization to provide oligosaccharides with modified arabinose branches intact. Switchgrass samples were analyzed by hydrophilic interaction chromatography (HILIC)-liquid chromatography (LC)-mass spectrometry/mass spectrometry (MSMS) and off-line nanospray MS to demonstrate the utility of this chemistry for determination of primary hydroxyl groups on oligosaccharide structures, with potential applications for determining the sequence of arabino-xylo-oligosaccharides present in plant cell wall material.

Cloning, expression and characterization of glycoside hydrolase family 11 endoxylanase from Bacillus pumilus ARA

An endoxylanase gene, xynA, was cloned from Bacillus pumilus ARA and expressed in Escherichia coli. The open reading frame of the xynA gene was 687bp encoding a signal peptide and a mature xylanase with a molecular mass of 23kDa. The enzyme was categorized as a glycosyl hydrolase family 11 member based on the sequence analysis of the putative catalytic domain. The recombinant XynA (Bpu XynA) was purified to homogeneity by Ni-NTA and ion exchange chromatography on DEAE-Sepharose FF. The enzyme exhibited highest activity at pH 6.6 and 50°C. The purified Bpu XynA was stable for at least 2h at 45°C, and retained over 50% residual activity after being incubated at 60°C for 1h. The activity of the xylanase was not significantly affected by metal ions and EDTA. The K ( m ) and K ( cat ) /K ( m ) of Bpu XynA for oat-spelt xylan were 5.53mg/ml and 10.14ml/mgs at 50°C and pH 6.6. The main product of hydrolysis by Bpu XynA was xylooligosaccharide. The results revealed that the consumption of grass xylan by B. pumilus ARA depended on the synergistic reactions of Bpu XynA and Bpu arabinosidase, and that a typical GH11 xylanase e.g. Tla XynA had capability to remove the side chain of xylan. The properties Bpu XynA make it promising for application in the production of Bifidobacterium growth-promoting factors and in feed industry.

Ligand Bound Structures of a Glycosyl Hydrolase Family 30 Glucuronoxylan Xylanohydrolase

Xylanases of glycosyl hydrolase family 30 (GH30) have been shown to cleave β-1,4 linkages of 4-O-methylglucuronoxylan (MeGXn) as directed by the position along the xylan chain of an α-1,2-linked 4-O-methylglucuronate (MeGA) moiety. Complete hydrolysis of MeGXn by these enzymes results in singly substituted aldouronates having a 4-O-methylglucuronate moiety linked to a xylose penultimate from the reducing terminal xylose and some number of xylose residues toward the nonreducing terminus. This novel mode of action distinguishes GH30 xylanases from the more common xylanase families that cleave MeGXn in accessible regions. To help understand this unique biochemical function, we have determined the structure of XynC in its native and ligand-bound forms. XynC structure models derived from diffraction data of XynC crystal soaks with the simple sugar glucuronate (GA) and the tetrameric sugar 4-O-methyl-aldotetrauronate resulted in models containing GA and 4-O-methyl-aldotriuronate, respectively. Each is observed in two locations within XynC surface openings. Ligand coordination occurs within the XynC catalytic substrate binding cleft and on the structurally fused side β-domain, demonstrating a substrate targeting role for this putative carbohydrate binding module. Structural data reveal that GA acts as a primary functional appendage for recognition and hydrolysis of the MeGXn polymer by the protein. This work compares the structure of XynC with a previously reported homologous enzyme, XynA, from Erwinia chrysanthemi and analyzes the ligand binding sites. Our results identify the molecular interactions that define the unique function of XynC and homologous GH30 enzymes.

The role of acetyl xylan esterase in the solubilization of xylan and enzymatic hydrolysis of wheat straw and giant reed

BackgroundDue to the complexity of lignocellulosic materials, a complete enzymatic hydrolysis into fermentable sugars requires a variety of cellulolytic and xylanolytic enzymes. Addition of xylanases has been shown to significantly improve the performance of cellulases and to increase cellulose hydrolysis by solubilizing xylans in lignocellulosic materials. The goal of this work was to investigate the effect of acetyl xylan esterase (AXE) originating from Trichoderma reesei on xylan solubilization and enzymatic hydrolysis of cellulose.ResultsThe solubilization of xylan in pretreated wheat straw and giant reed (Arundo donax) by xylanolytic enzymes and the impact of the sequential or simultaneous solubilization of xylan on the hydrolysis of cellulose by purified enzymes were investigated. The results showed that the removal of acetyl groups in xylan by AXE increased the accessibility of xylan to xylanase and improved the hydrolysis of xylan in pretreated wheat straw and giant reed. Solubilization of xylan led to an increased accessibility of cellulose to cellulases and thereby increased the hydrolysis extent of cellulose. A clear synergistic effect between cellulases and xylanolytic enzymes was observed. The highest hydrolysis yield of cellulose was obtained with a simultaneous use of cellulases, xylanase and AXE, indicating the presence of acetylated xylan within the cellulose matrix. Acetylated xylobiose and acetylated xylotriose were produced from xylan without AXE, as confirmed by atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry.ConclusionsThe results in this paper demonstrate that supplementation of xylanase with AXE enhances the solubilization of xylan to some extent and, consequently, increases the subsequent hydrolysis of cellulose. The highest hydrolysis yield was, however, obtained by simultaneous hydrolysis of xylan and cellulose, indicating a layered structure of cellulose and xylan chains in the cell wall substrate. AXE has an important role in the hydrolysis of lignocellulosic materials containing acetylated xylan.

Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid.

The plant-pathogenic bacterium Dickeya dadantii (formerly Erwinia chrysanthemi) produces a large array of plant cell wall-degrading enzymes. Using an in situ detection test, we showed that it produces two feruloyl esterases, FaeD and FaeT. These enzymes cleave the ester link between ferulate and the pectic or xylan chains. FaeD and FaeT belong to the carbohydrate esterase family CE10, and they are the first two feruloyl esterases to be identified in this family. Cleavage of synthetic substrates revealed strong activation of FaeD and FaeT by ferulic acid. The gene faeT appeared to be weakly expressed, and its product, FaeT, is a cytoplasmic protein. In contrast, the gene faeD is strongly induced in the presence of ferulic acid, and FaeD is an extracellular protein secreted by the Out system, responsible for pectinase secretion. The product of the adjacent gene faeR is involved in the positive control of faeD in response to ferulic acid. Moreover, ferulic acid acts in synergy with polygalacturonate to induce pectate lyases, the main virulence determinant of soft rot disease. Feruloyl esterases dissociate internal cross-links in the polysaccharide network of the plant cell wall, suppress the polysaccharide esterifications, and liberate ferulic acid, which contributes to the induction of pectate lyases. Together, these effects of feruloyl esterases could facilitate soft rot disease caused by pectinolytic bacteria.