Wheat Arabinoxylan Research Articles

α-L-ARABINOFURANOSIDASE FROM AN EFFICIENT HEMICELLULOLYTIC FUNGUS Penicillium janthinellum CAPABLE OF HYDROLYZING WHEAT AND RYE ARABINOXYLAN TO ARABINOSE

This study reports Penicillium janthinellum strain, producing high levels of α-L-arabinofuranosidase (AFase) as well as other components of hemicelluloytic enzyme system (endoxylanase, β-xylosidase and acetyl xylan esterase) on rice straw and wheat bran containing solidified culture medium. Optimization of culture conditions was carried out using Box–Behnken design of experiment to study the influence of process variables (ammonium sulphate, pH and moisture level) on AFase production. Analysis of data showed R2 (0.9967) and adjusted R2 (0.9925) indicating model to be good fit and robust to predict culture conditions for AFase production. Under optimal culture conditions P. janthinellum produced high levels of AFase (212 units/g dw substrate) in addition to xylanase (1800 units/g dw substrate), β-xylosidase (31 units/g dw substrate), acetyl xylan esterase (231 units/g dw substrate) and feruloyl esterase (27 units/g dw substrate). AFase from P. janthinellum culture extract was purified to homogeneity and characterized to be a 64 kDa protein with a pI of 3.8. The peptide mass fingerprinting showed the AFase belonged to family GH54. The enzyme was optimally active at 50o C at pH 5.5 and its activity was positively modulated in presence of Fe3+ions. The enzyme preferentially catalyzed the hydrolysis of pNP- arabinofuranoside (pNPA) with Km and Vmax of 0.4mM and 260 unit mg-1 protein-1, respectively. Hydrolysis with purified AFase (3.0 units/g substrate) released arabinose from rye arabinoxylans (29.5 mg/g substrate) and wheat arabinoxylan (13.4 mg/g substrate), as the sole product indicating P. janthinellum as the important source of α-L-arabinofuranosidase for bioconversion of agro-residue to value added products.

Efficiency of xylanases from families 10 and 11 in production of xylo-oligosaccharides from wheat arabinoxylans

This study investigated the effect of xylanases from families 10 and 11 on the production of small chain arabinoxylo-oligosaccharides (AXOS) (X2–X4) from wheat arabinoxylans, and identified the impact that pH, temperature and time exposed to xylanase have on this process. Xylanase from family 10 had greater hydrolytic efficiency and resulted in heightened AXOS production compared with xylanase from family 11 or family 10 and 11 combined. The pH of the environment had a significant effect on AXOS production (P<0.001) and the greatest conversion of arabinoxylans into AXOS was observed at pH 2.5. The effects of temperature of the environment and amount of time the samples were exposed to the xylanase on AXOS production were inconsistent and were dictated by pH and xylanase family, as evidenced by interactions between temperature and pH (P=0.016) and xylanase family (P=0.032) and time and pH (P=0.007).

Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential

The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases and xylanolytic enzymes being the most abundant. A 2-DE-based secretome analysis of Aspergillus niger and the less-studied pathogenic fungus Fusarium poae grown on barley flour and wheat arabinoxylan resulted in identification of 82 A. niger and 31 F. poae proteins many of which were hydrolytic enzymes, including xylanases. Biological significanceThe microorganisms that inhabit the surface of cereal grains are specialized in production of enzymes such as xylanases, which depolymerize plant cell walls. Integration of gel-based proteomics approach with activity assays is a powerful tool for analysis and characterization of fungal secretomes and xylanolytic activities which can lead to identification of new enzymes with interesting properties, as well as provide insight into plant-fungal interactions, fungal pathogenicity and adaptation. Understanding the fungal response to host niche is of importance to uncover novel targets for potential symbionts, anti-fungal agents and biotechnical applications.

Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC

A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass.

Extraction and purification of an endo-1,4-β-xylanase from wheat malt

Wheat and wheat malt are known to be high quality raw materials in the beer brewing process. The size and structure of wheat arabinoxylans (AXs) and their enzymatic degradation products have a profound impact on beer properties, such as viscosity, turbidity, filtration rate and foaming properties. AXs are of great benefit to human health due to their anti-tumor properties and potential roles in the immune system. Endo-1,4-β-xylanase is a key enzyme for degradation of AXs and production of small molecule AXs, thereby altering the properties of AXs, such as solubility, viscosity, and turbidity. In this study, extraction and purification of an endo-1,4-β-xylanase from wheat malt was described. The results showed that, after 40%–60% ammonium sulfate precipitation, Q-Sepharose Fast Flow anion exchange chromatography, Phenyl-Sepharose 6 Fast Flow Hydrophobic chromatography, and twice sephacryl S-100 HR gel filtration, a final endo-1,4-β-xylanase was obtained. The purified wheat malt endo-1,4-β-xylanase showed a single band on SDS-PAGE with a molecular weight of 27.8 kDa, which had a purification fold of 12.08 and a specific activity of 4.47 μ/mg.

Arabinoxylan hydrolyzates as immunomodulators in Caco-2 and HT-29 colon cancer cell lines.

The use of plant derived polysaccharides as health promoters has gained immense interest in the past few years. Arabinoxylan (AX) is the predominant non-starch polysaccharide in cereals and grasses including wheat. The current research aimed to investigate the structure-function relationship of arabinoxylan hydrolyzates (AXH), obtained by the enzymatic hydrolysis of AX using xylanase and arabinofuranosidase as immunomodulators in two colon cancer cell lines: Caco-2 and HT-29. Fine structural details had a strong correlation with the immunological properties of the wheat AXH. As a general trend, as the presence of arabinose substitution increased in the AXH, the production of proinflammatory cytokines, IL-8 and TNF-α, decreased in both cell lines. Thus, AXH with a higher degree of arabinose substitution might be better adept in lowering inflammation in colon cancer cells.

Recombinant cellulolytic or xylanolytic complex comprising the full-length scaffolding protein RjCipA and cellulase RjCel5B or xylanase RjXyn10C of Ruminiclostridium josui.

Three cellulosomal subunits of Ruminiclostridium josui, the full-length scaffolding protein CipA (RjCipA), a cellulase Cel5B (RjCel5B) and a xylanase Xyn10C (RjXyn10C), were successfully produced by Escherichia coli recombinant clones. RjCel5B and RjXyn10C were characterized as an endoglucanase and an endoxylanase, respectively. RjCipA, RjCel5B and Xyn10C adsorbed to microcrystalline cellulose (Funacel) and rice straw powder. Interaction between RjCel5B and RjCipA, and RjXyn10C and RjCipA were confirmed by qualitative assays. When a fixed amount of RjCel5B was mixed with different amounts of RjCipA, i.e., at the molar ratio of 6:1 or 6:6, the 6:6 complex showed 6.6-fold higher activity toward Funacel and 11.5-fold higher activity toward rice straw powder than RjCel5B, whereas the 6:1 complex showed only 2.8- and 3.9-folds higher activities toward Funacel and rice straw powder, respectively, than RjCel5B. These results suggest that the family-3 carbohydrate binding module (CBM3) of RjCipA in the RjCel5B-RjCipA complex plays an important role for hydrolysis of cellulose and the substrate-targeting effect of the CBM is more significant than the proximity effect caused by the presence of plural catalytic subunits adjoining each other. In contrast, the 6:1 complex of RjXyn10C and RjCipA showed 45% and 28% of the activities of RjXyn10C toward insoluble wheat arabinoxylan and rice straw powder, respectively. These results suggest that both a negative proximity effect and substrate-isolating effect, but not substrate-targeting effect, are caused by the CBM3 with inappropriate polysaccharide specificity. Substrate-targeting, proximity and substrate-isolating effects are discussed.

Differential growth of bowel commensal Bacteroides species on plant xylans of differing structural complexity

Alterations to the composition of the bowel microbiota (dysbioses) are associated with particular diseases and conditions of humans. There is a need to discover new, indigestible polysaccharides which are selective growth substrates for commensal bowel bacteria. These substrates (prebiotics) could be added to food in intervention studies to correct bowel dysbiosis. A collection of commensal bacteria was screened for growth in culture using a highly-branched xylan produced by New Zealand flax. Two, Bacteroides ovatus ATCC 8483 and Bacteroides xylanisolvens DSM 18836 grew well on this substrate. The utilisation of the xylan was studied chromatographically and by constituent sugar analysis. The two closely related species utilised the xylan in different ways, and differently from their use of wheat arabinoxylan. The growth of Bacteroides species on other plant xylans having differing chemical structures was also investigated. Novel xylans expand the choice of potential prebiotics that could be used to correct bowel dysbioses.

An unusual feruloyl esterase from Aspergillus oryzae: two tryptophan residues play a crucial role for the activity

The genome of Aspergillus oryzae encodes a hypothetical protein (annotated as AO090701000884), designated AoFaeD, which exhibits sequence identity to feruloyl esterases from Aspergillus clavatus, Coprinopsis cinerea, Neurospora crassa, and Pseudomonas fluorescens subsp. cellulosa. In this study, we cloned the AofaeD coding sequence into Pichia pastoris and demonstrated heterologous expression and secretion of active recombinant enzyme (rAoFaeD) as a 30-kDa protein in the culture medium. Purified rAoFaeD exhibited optimal activity at pH 7.0 and at 45°C, but was stable at a pH range of 7.0–10.0 and a temperature of 40°C. While wild-type rAoFaeD failed to cleave the methyl esters of ferulic acid (MFA), p-coumaric acid (MpCA), caffeic acid (MCA), and sinapic acid (MSA) and ethyl ester of ferulic acid (EFA), enzyme exhibited esterase activity when wheat arabinoxylan was used as a substrate, and this reaction was enhanced synergistically by the addition of xylanase. Notably, rAoFaeD variants in which one of the tryptophan residues (Trp142 or Trp144) located adjacent to the putative catalytic serine residue at position 143 (Ser143) was replaced with phenylalanine and tyrosine (W142F and W144Y), respectively, exhibited hydrolytic activity toward MFA, MpCA, MCA, MSA, and EFA. Moreover, these variants exhibited enhanced production of ferulic acid from wheat arabinoxylan compared to the wild-type protein, and the activity of these proteins was enhanced by the addition of xylanase.

Replacement of carbohydrate binding modules improves acetyl xylan esterase activity and its synergistic hydrolysis of different substrates with xylanase.

BackgroundAcetylation of the xylan backbone was a major obstacle to enzymatic decomposition. Removal of acetyl groups by acetyl xylan esterases (AXEs) is essential for completely enzymatic hydrolysis of xylan. Appended carbohydrate binding modules (CBMs) can promote the enzymatic deconstruction of plant cell walls by targeting and proximity effects. Fungal acetyl xylan esterases are strictly appended to cellulose-specific CBM1. It is still unclear whether xylan-specific CBMs have a greater advantage than CBM1 in potentiating the activity of fungal deacetylating enzymes and its synergistic hydrolysis of different substrates with xylanase.ResultsThree recombinant AXE1s fused with different xylan-specific CBMs, together with wild-type AXE1 with CBM1 and CBM1-deleted mutant AXE1dC, were constructed in this study. The optimal temperature and pH of recombinant AXE1s was 50 °C and 8.0 (except AXE1dC-CBM6), respectively. Cellulose-specific CBM1 in AXE1 obviously contributed to its catalytic action against substrates compared with AXE1dC. However, replacement of CBM1 with xylan-specific CBM4-2 significantly enhanced AXE1 thermostability and catalytic activity against soluble substrate 4-methylumbelliferyl acetate. Whereas replacements with xylan-specific CBM6 and CBM22-2 were more effective in enzymatic release of acetic acid from destarched wheat bran, NaClO2-treated wheat straw, and water-insoluble wheat arabinoxylan compared to AXE1. Moreover, replacement with CBM6 and CBM22-2 also resulted in higher degree releases of reducing sugar and acetic acid from different substrates when simultaneous hydrolysis with xylanase. A good linear relationship exists between the acetic acid and reducing sugar release.ConclusionsOur findings suggested that the replacement with CBM6 and CBM22-2 not only significantly improved the catalysis efficiency of AXE1, but also increased its synergistic hydrolysis of different substrates with xylanase, indicating the significance of targeting effect in AXE1 catalysis mediated by xylan-specific CBMs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0305-6) contains supplementary material, which is available to authorized users.

Structure elucidation of an immunostimulatory arabinoxylan-type polysaccharide prepared from young barley leaves (Hordeum vulgare L.)

We recently isolated an immunostimulating polysaccharide (BLE-P-I), which possesses a large proportion of arabinoxylan, from young barley leaves. In the present study, to elucidate the structural details of BLE-P-I, it was fractionated into enzyme-resistant fraction (BLE-P-I-X1) and arabinoxylan-rich oligosaccharide fraction (BLE-P-I-X2) after endo-xylanase hydrolysis. Commercial wheat arabinoxylan was also fractionated into WAX-XI and WAX-X2 after the same treatment. BLE-P-I-X2 consisted of arabinose and xylose, including 11 types of neutral glycosidic linkages, whereas WAX-X2 had less, with 6 types of linkages. Mass spectrometric results indicated that WAX-X2 was composed of a xylan backbone bearing only arabinose. In contrast, BLE-P-I-X2 consisted of a xylan backbone with acetate, arabinose, galactose, glucuronic acid, and 4-O-methylglucuronic acid, resembling structural characteristics as glucuronoarabinoxylan. Macrophage-stimulatory activity showed that BLE-P-I-X2 has a role in the expression of the activity. These results indicate that the structural complexity of arabinoxylan seems to be responsible for the immunomodulatory activity in barley leaf.

Synergistic degradation of arabinoxylan by free and immobilized xylanases and arabinofuranosidase

Effective degradation of hemicellulose is of utmost importance in a wide variety of applications in bioindustry. Five endoxylanases from different glycoside hydrolase families and microorganisms were tested with an arabinofuranosidase, Araf51A, for the hydrolysis of insoluble wheat arabinoxylan, which is a structural component of hemicellulose. The optimized combination was XynZ/Xyn11A/Araf51A with a loading ratio of 2:2:1, and the value of degree of synergy increased with the increase of Araf51A proportion in the enzyme mixture. Afterwards, selected enzymes were immobilized on commercial magnetic nanoparticles through covalent bonding. Both free and immobilized enzymes showed a similar conversion to reducing sugars after hydrolysis for 48h. After 10 cycles, approximately 20% of the initial enzymatic activity of both the individual or mixture of immobilized enzymes was retained. A 5.5-fold increase in the production of sugars was obtained with a mixture of enzymes immobilized after 10 cycles in total compared with free enzymes. Importantly, a sustainable synergism between immobilized arabinofuranosidase and immobilized endoxylanases in the hydrolysis of arabinoxylan was demonstrated.

Addition of arabinoxylan and mixed linkage glucans in porcine diets affects the large intestinal bacterial populations.

To investigate the effects of two cereal soluble dietary fibres (SDF), wheat arabinoxylan (AX) and oat-mixed linkage glucans (MLG), on fermentative end-products and bacterial community profiles of the porcine caecum (Cae) and distal colon (DC). We hypothesised that feeding pigs these SDF would stimulate Cae and DC carbohydrate fermentation, resulting in a modification of the resident bacterial communities. Five groups of six pigs were each fed one diet based on wheat starch (WS) only, or treatment diets in which some WS was replaced by 10% AX, or 10% MLG, a combination of 5% AX:5% MLG (AXMLG), or completely replaced with ground whole wheat. Post-euthanasia, Cae and DC digesta were collected for analysis of fermentative end-products, and bacterial community profiles were determined by 16S rRNA gene amplicon 454 pyrosequencing. Across all the SDF-containing diets, predominantly in the proximal region of the large intestine, Prevotella, Lactobacillus, Mitsuokella and Streptococcus were most significantly influenced (P<0.05), while notable changes were observed for the Ruminococcaceae and Lachnospiraceae families in the Cae and DC. The addition of MLG or AXMLG had the greatest effect of influencing bacterial profiles, reducing sequence proportions assigned to the genus Clostridium, considered detrimental to gut health, with associated increases in short-chain fatty acid and reduced ammonia concentrations. This study demonstrated how the cereal SDF AX and MLG altered the large intestinal bacterial community composition, particularly proximally, further giving insights into how diets rich in specific complex carbohydrates shift the bacterial population, by increasing abundance and promoting greater diversity of those bacteria considered beneficial to gut health.

Optimization of hemicellulose saccharification by recombinant hemicellulase cocktail in the water soluble wheat arabinoxylan and pretreated barley straw

Optimization of hemicellulose saccharification by recombinant hemicellulase cocktail in the water soluble wheat arabinoxylan and pretreated barley straw

A commercial GH 11 xylanase mediates xylan solubilisation and degradation in wheat, rye and barley as demonstrated by microscopy techniques and wet chemistry methods

Xylanases in animal feed are primarily utilized to alleviate the antinutritional effects of arabinoxylan, a predominant polymer in cereal cell walls consisting of a β-1,4-xylose polymer, substituted with various sugars. Although xylanases efficiently hydrolyze unsubstituted and substituted arabinoxylans, currently available enzymes are not always able to access highly decorated forms of the polysaccharide, such as arabinoxylans (AX) that contain arabinofuranose substitutions. The highly substituted regions are enriched in both 2- and 3-disubstituted xylose residues. Wheat AX contains very small amounts of 2-monosubstituted xylose residues, whereas barley AXs are characterised by a larger amount of 2-monosubstitution. The structure of rye AX is similar to that of wheat AX, but differences in the abundances of the 2- monosubstituted and 2-and 3- disubstituted xylose residues have been reported. In the current study the efficiency of a monocomponent xylanase was demonstrated on structurally different arabinoxylans present in wheat, rye and barley kernels using microscopic visualization, viscosity and arabinoxylan solubilisation measured as the sum of arabinose and xylose in an analysis of the non-starch polysaccharide (NSP) constituents, as well as xylose alone. Degradation of arabinoxylan components upon exposure to the enzyme was visualized by scanning electron microscopy, autofluorescence and with fluorescence immunocytochemistry and confocal microscopy using commercial antibodies recognizing arabinoxylan in the cereal cell walls. Viscosity data clearly demonstrated effective depolymerisation of arabinoxylans in water extracts from wheat and rye and thereby hydrolytic capacity of the monocomponent enzyme on structurally diverse arabinoxylans. The viscosity reducing effect observed in barley was limited, most likely due to the presence of highly viscous mixed linked ß-d-glucans. Wet chemistry analyses of the complete solubilisation of arabinoxylan (oligomers and polysaccharides) using a standardized xylose assay as well as NSP analyses (measuring polysaccharides, DP>10) on wheat, rye and barley, provided additional and complementary information to the microscopy conducted on the same cereal samples.The study has shown that microscopy techniques visualizing cell wall solubilising effects of xylanase can be useful for understanding enzyme mode of action and efficacy on different feed ingredients.

Modification of the Secondary Binding Site of Xylanases Illustrates the Impact of Substrate Selectivity on Bread Making.

To investigate the importance of substrate selectivity for xylanase functionality in bread making, the secondary binding site (SBS) of xylanases from Bacillus subtilis (XBS) and Pseudoalteromonas haloplanktis was modified. This resulted in two xylanases with increased relative activity toward water-unextractable wheat arabinoxylan (WU-AX) compared to water-extractable wheat arabinoxylan, i.e., an increased substrate selectivity, without changing other biochemical properties. Addition of both modified xylanases in bread making resulted in increased loaf volumes compared to the wild types when using weak flour. Moreover, maximal volume increase was reached at a lower dosage of the mutant compared to wild-type XBS. The modified xylanases were able to solubilize more WU-AX and decreased the average degree of polymerization of soluble arabinoxylan in dough more during fermentation. This possibly allowed for additional water release, which might be responsible for increased loaf volumes. Altered SBS functionality and, as a result, enhanced substrate selectivity most probably caused these differences.

Combination of Xylanase and Debranching Enzymes Specific to Wheat Arabinoxylan Improve the Growth Performance and Gut Health of Broilers.

Arabinoxylan (AX) is the major antinutritional factor of wheat. This study evaluated the synergistic effects of xylanase and debranching enzymes (arabinofuranosidase [ABF] and feruloyl esterase [FAE]) on AX. During in vitro tests, the addition of ABF or FAE accelerated the hydrolysis of water-soluble AX (WE-AX) and water-insoluble AX (WU-AX) and produced more xylan oligosaccharides (XOS) than xylanase alone. XOS obtained from WE-AX stimulated greater proliferation of Lactobacillus brevis and Bacillus subtilis than did fructo-oligosaccharides (FOS) and glucose. During in vivo trials, xylanase increased the average daily growth (ADG), decreased the feed-conversion ratio (FCR), and reduced the digesta viscosity of jejunum and intestinal lesions of broilers fed a wheat-based diet on day 36. ABF or FAE additions further improved these effects. Broilers fed a combination of xylanase, ABF, and FAE exhibited the best growth. In conclusion, the synergistic effects among xylanase, ABF, and FAE increased AX degradation, which improve the growth performance and gut health of broilers.

Characterization and biotechnological application of recombinant xylanases from Aspergillus nidulans

Two xylanases from Aspergillus nidulans, XlnB and XlnC, were expressed in Pichia pastoris, purified and characterized. XlnB and XlnC achieved maximal activities at 60°C and pH 7.5 and at 50°C and pH 6.0, respectively. XlnB showed to be very thermostable by maintaining 50% of its original activity after 49h incubated at 50°C. XlnB had its highest activity against wheat arabinoxylan while XlnC had the best activity against beechwood xylan. Both enzymes were completely inhibited by SDS and HgCl2. Xylotriose at 1mg/ml also totally inibited XlnB activity. TLC analysis showed that the main product of beechwood xylan hydrolysis by XlnB and XlnC was xylotetraose. An additive effect was shown between XlnB and XlnC and the xylanases of two tested commercial cocktails. Sugarcane bagasse saccharification results showed that these two commercial enzymatic cocktails were able to release more glucose and xylose after supplementation with XlnB and XlnC.

Metagenomic mining of glycoside hydrolases from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) and the characterization of a multimodular β-1,4-xylanase (GH11).

In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high-performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high-value bio-based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α-fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular β-1,4-xylanase, consisting of a catalytic domain and two carbohydrate-binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50°C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.

Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

BackgroundPlant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1AT.ResultsTranscriptomic analyses of B. xylanisolvens XB1AT grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70.ConclusionThis study shows that xylan degradation by B. xylanisolvens XB1AT is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens XB1AT has specialized in the degradation of xylans of low complexity. This functional feature may provide a niche to all xylanolytic bacteria harboring similar PULs. Further functional and ecological studies on fibrolytic Bacteroides species are needed to better understand their role in dietary fiber degradation and their impact on intestinal health.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2680-8) contains supplementary material, which is available to authorized users.