Xylohexaose

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-β-D-xylopyranoside, with the highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50°C. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP₂) to six (DP₆), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with the crude cellulase cocktail of P. funiculosum with a degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig1⁸⁸, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig1⁸⁸ overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% or 7%, respectively, over the secretome of PfMig1⁸⁸, while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.

Xylans, a family of xylose-based polysaccharides, are dietary fibers resistant to digestion. They therefore reach the large intestine intact; there, they are utilized by members of the gut microbiota. They are initially broken down by primary degraders that utilize extracellular xylanases to cleave xylan into smaller oligomers. The resulting xylooligosaccharides (XOS) can either be further metabolized directly by primary degraders or cross-feed secondary consumers, including Bifidobacterium. While several Bifidobacterium species have metabolic systems for XOS, most grow poorly on longer-chain XOS and xylan substrates. In this study, we isolated strains of Bifidobacterium pseudocatenulatum and observed that some, including B. pseudocatenulatum ED02, displayed growth on XOS with a high degree of polymerization (DP) and straight-chain xylan, suggesting a primary degrader phenotype that is rare in Bifidobacterium. In silico analyses revealed that only the genomes of these xylan-fermenting (xylan⁺) strains contained an extracellular GH10 endo-β-1.4 xylanase, a key enzyme for primary degradation of xylan. The presence of an extracellular xylanase was confirmed by the appearance of xylan hydrolysis products in cell-free supernatants. Extracellular xylanolytic activity was only detected in xylan⁺ strains, as indicated by the production of XOS fragments with a DP of 2 to 6, identified by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Additionally, in vitro fecal fermentations revealed that strains with a xylan⁺ phenotype can persist with xylan supplementation. These results indicate that xylan⁺ B. pseudocatenulatum strains may have a competitive advantage in the complex environment of the gastrointestinal tract, due to their ability to act as primary degraders of xylan through extracellular enzymatic degradation.

Acidic xylooligosaccharides (XOS), also called aldouronics, are hetero-oligomers of xylose randomly branched with 4-O-methyl-D-glucuronic acid residues linked by α(1 → 2) bonds, which display bioactive properties. We have developed a simple and integrated method for the production of acidic XOS by enzymatic hydrolysis of a glucurono-xylan from beechwood. Among the enzymes screened, Depol 670L (a cellulolytic preparation from Trichoderma reesei) displayed the highest activity (70.3 U/mL, expressed in reducing xylose equivalents). High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis revealed the formation of a neutral fraction (corresponding to linear XOS, mainly xylose and xylobiose) and a group of more retained products (acidic XOS), which were separated using strong anion-exchange cartridges. The acidic fraction contained a major product, characterized by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and mono- and two-dimensional nuclear magnetic resonance spectroscopy (NMR) as 2′-O-α-(4-O-methyl-α-D-glucuronosyl)-xylobiose (X2_MeGlcA). Starting from 2 g of beechwood xylan, 1.5 g of total XOS were obtained, from which 225 mg (11% yield) corresponded to the aldouronic X2_MeGlcA. The acidic XOS exhibited higher antioxidant activity (measured by the ABTS^·+ discoloration assay) than xylan, whilst neutral XOS displayed no antioxidant activity. This work demonstrates that it is possible to obtain a safe and natural antioxidant by enzymatic biotransformation of hardwood hemicellulose.

BACKGROUND: Corn arabinoxylan (AX) is a complicated and multi-branched antinutritional factor, thereby proving the use of endo-xylanase (EX) to be marginally valid. This study focused on specific types of AX-degrading enzymes (ADE) to exert the synergy of debranching enzymes and track prebiotic potential of enzymatic hydrolysates. It aimed to investigate the effects of ADE on growth performance, intestinal histomorphology, absorption functions, changes of polysaccharide components, fermentation, and gut microbiota of broilers. To this purpose, 576 five-day-old Arbor Acres male broilers were randomly allocated into eight treatments with six replicates each. Corn basal diets supplemented with or without enzymes were fed for a 21-day period, specifically including EX, its compatible use with arabinofuranosidase (EXA) or ferulic acid esterase (EXF), and compound groups with the above three enzymes (XAF). RESULTS: Specific ADE stimulated the jejunal villus height and goblet cell number, and evidently decreased the crypt depth (P < 0.05), while the ratio of ileal villus height to crypt depth was significantly increased in EXF (P < 0.05). Maltase activities of ileal mucosa in XAF groups were extremely enhanced (P < 0.01), and EX boosted the activity of Na⁺ -K⁺ ATPase in the small intestine (P < 0.01). The insoluble AX concentrations comparatively lessened, thereby notably raising the sundry xylooligosaccharide (XOS) yield in the ileal chyme (P < 0.05), which was dominant in xylobiose and xylotriose. Improvements in the abundance and diversity of ileal microbial communities within EXA, EXF, XAF treatments were observed (P < 0.05). Positive correlations between microbiota and XOS were revealed, with xylobiose and xylotriose being critical for ten beneficial bacteria (P < 0.05). EXF escalated the BWG and FCR of broilers in this phase (P < 0.05), attributing to the thriving networks modified by Lactobacillus. The intracecal contents of acetic acid, butyric acid, and propionic acid were greatly enhanced in most ADE groups such as EXF (P < 0.05). CONCLUSIONS: Debranching enzymes appreciably targeted corn AX to release prebiotic XOS in the posterior ileum and facilitated intracaecal fermentation. It was beneficial for improving gut development, digestion and absorption, and modulating the microflora to promote early performance of broilers.

Microwave hydrothermal treatment (MHT) is considered a sustainable technology for the valorization of lignocellulosic materials, enabling the solubilization of hemicellulosic-derived compounds, especially in the form of oligosaccharides that may present potential in the chemical, pharmaceutical or nutraceutical industries. Hence, MHT at 200 and 230°C, at severity (S₀) among 2.92-4.66 were performed. S₀ = 3.98 permitted the recovery of about 80% of the initial xylan as xylooligosaccharides. In order to compare the effectiveness of MHT, conventional hydrothermal treatment (CHT) was performed at conditions leading to the maximum recovery of oligosaccharides (S₀ =3.98, non-isothermal regime at 203°C). Despite the structural features of oligomers in the three liquors were very similar, the spent solids presented different enzymatic digestibility, which implied a different effect of the treatments, reaching up to 80% of glucan to glucose conversion for the solid after MHT at 230°C for 0.5 min. Additionally, CHT consumed 2.1-2.8-fold greater energy than MHT, reflecting that microwave-assisted autohydrolysis is a sustainable and efficient technology to process PW.

Xylans are a diverse family of hemicellulosic polysaccharides found in abundance within the cell walls of nearly all flowering plants. Unfortunately, naturally occurring xylans are highly heterogeneous, limiting studies of their synthesis and structure− function relationships. Here, we demonstrate that xylan synthase 1 from the charophyte alga Klebsormidium flaccidum is a powerful biocatalytic tool for the bottom-up synthesis of pure β-1,4 xylan polymers that selfassemble into microparticles in vitro. Using uridine diphosphate-xylose (UDP-xylose) and defined saccharide primers as substrates, we demonstrate that the shape, composition, and properties of the selfassembling xylan microparticles could be readily controlled via the fine structure of the xylan oligosaccharide primer used to initiate polymer elongation. Furthermore, we highlight two approaches for bottom-up and surface functionalization of xylan microparticles with chemical probes and explore the susceptibility of xylan microparticles to enzymatic hydrolysis. Together, these results provide a useful platform for structural and functional studies of xylans to investigate cell wall biosynthesis and polymer−polymer interactions and suggest possible routes to new biobased materials with favorable properties for biomedical and renewable applications.

A thermotolerant Enterococcus faecium s6 strain exhibited homoferementative lactic acid (LA) production at high xylose concentration (≥50 g/L). In batch fermentation at 45°C and controlled pH of 6.5, strain E. faecium s6 produced up to 72.9 g/L of LA with a yield of 0.99 g/g_{-consumed xylose} and productivity of 1.74 g/L.h from 75 g/L xylose. An increased LA concentration and productivities with high yields were obtained in repeated batch fermentation that was conducted for 24 runs. In this mode, the strain could produce LA up 81.1 g/L within 5 h fermentation at a high productivity of 13.5 g/L.h and a yield of 1.02 g/g_{-consumed xylose}. The strain was unable to consume birchwood xylan as sole carbon source. However, it could efficiently utilize xylooligosaccharides of xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose. The intracellular xylosidase activity was induced by xylose. Using xylooligosaccharide (50 g/L)/xylose (5 g/L) mixtures, the strain was able to produce maximum LA at 48.2 g/L within 120 h at a yield of 1.0 g/g_{-consumed sugar} and productivity of 0.331 g/L.h. These results indicate that E. faecium s6 is capable of directly utilizing xylan-hydrolyzate and will enable the development of a feasible and economical approach to the production of LA from hemicellulosic hydrolysate.

Xylan is a major constituent of plant cell walls and is a potential source of biomaterials, and the derived oligosaccharides have been shown to have prebiotic effects. Xylans can be highly substituted with different sugar moieties, which pose steric hindrance to the xylanases that catalyse the hydrolysis of the xylan backbone. One such substituent is α-D-glucuronic acid, which is linked to the O2' position of the β-1,4 D-xylopyranoses composing the main chain of xylans. The xylan-specific α-glucuronidases from glycoside hydrolase family 115 (GH115) specifically catalyse the removal of α-D-glucuronic acid (GlcA) or methylated GlcA (MeGlcA). Here, the molecular basis by which the bacterial GH115 member wtsAgu115A interacts with the main chain of xylan and the indirect involvement of divalent ions in the formation of the Michaelis-Menten complex are described. A crystal structure at 2.65 Å resolution of wtsAgu115A originating from a metagenome from an anaerobic digester fed with wastewater treatment sludge was determined in complex with xylohexaose, and Asp303 was identified as the likely general acid. The residue acting as the general base could not be identified. However, a proton wire connecting the active site to the metal site was observed and hence a previous hypothesis suggesting a Grotthuss-like mechanism cannot be rejected. Only a single molecule was found in the asymmetric unit. However, wtsAgu115A forms a dimer with a symmetry-related molecule in the crystal lattice. The xylohexaose moieties of the xylohexaose are recognized by residues from both protomers, thus creating a xylohexaose recognition site at the dimer interface. The dimer was confirmed by analytical size-exclusion chromatography in solution. Kinetic analysis with aldouronic acids resulted in a Hill coefficient of greater than 2, suggesting cooperativity between the two binding sites. Three Ca²⁺ ions were identified in the wtsAgu115A structures. One Ca²⁺ ion is of particular interest as it is coordinated by the residues of the loops that also interact with the substrate. Activity studies showed that the presence of Mg²⁺ or Mn²⁺ resulted in a higher activity towards aldouronic acids, while the less restrictive coordination geometry of Ca²⁺ resulted in a decrease in activity.

The success of establishing bioeconomies replacing current economies based on fossil resources largely depends on our ability to degrade recalcitrant lignocellulosic biomass. This study explores the potential of employing various enzymes acting synergistically on previously pretreated agricultural side streams (corn bran, oat hull, soluble and insoluble oat bran). Degrees of synergy (oligosaccharide yield obtained with the enzyme combination divided by the sum of yields obtained with individual enzymes) of up to 88 were obtained. Combinations of a ferulic acid esterase and xylanases resulted in synergy on all substrates, while a laccase and xylanases only acted synergistically on the more recalcitrant substrates. Synergy between different xylanases (glycoside hydrolase (GH) families 5 and 11) was observed particularly on oat hulls, producing a yield of 57%. The synergistic ability of the enzymes was found to be partly due to the increased enzyme stability when in combination with the substrates.

Empty fruit bunch (EFB), the largest amount of biomass from a palm oil mill, is a promising renewable source for many value-added products. This study aimed to utilize EFB for production of xylanase by Streptomyces thermocarboxydus ME742 and applied the enzyme to the production of xylooligosaccharides (XOS) from EFB derived xylan (EFB-xylan). The strain ME742 produced the maximum xylanase (473.21 ± 6.55 U/mL) in a medium containing 50 g/L EFB supplemented with 3.0 g/L peptone at 40°C for 4 days. Xylanase ME742 was purified 23.68 fold with specificity of 1229 U/mg by acetone precipitation and DEAE-Sepharose chromatography. It was stable in a wide range of pH (4.0-9.5) and in elevated temperatures up to 55°C. Xylanase ME742 effectively hydrolyzed EFB-xylan. HPLC analysis revealed that the hydrolysate was composed of xylose and XOS with degree of polymerization between 2 and 5 and xylobiose was the predominant product. The XOS yield ranging in 95.01-151.19 mg/g EFB-xylan was generated from enzymatic processing for 12-72 h. Interestingly, arabinosyl-xylooligosaccharides were also predicted to be produced from EFB-xylan. XOS mixture exhibited high antioxidant activity (71.36 ± 0.57%) against DPPH at 100 μg/mL concentration and moderate activity (45.18 ± 1.56%) against ABTS radical. These findings reveal the potential use of Streptomyces thermocarboxydus ME742 and xylanase produced in a sustainable bioprocess to add value of EFB waste.

At present, xylooligosaccharides (XOS) from corncob using acid-base conjugate system has not been reported. In this study, XOS production from corncob by two-step acetic acid/sodium acetate (AC/SA) conjugate system hydrolysis and xylanase hydrolysis was optimized, and monosaccharides were subsequently produced from corncob residues by cellulase hydrolysis. The XOS of 19.9 g/L was obtained from corncob (10%, w/v) using 0.15 M AC/SA hydrolysis at a molar ratio of 3.0 at 170 °C for 60 min, followed by xylanase hydrolysis. The second-step AC/SA hydrolysis of hydrolyzed corncob (10%, w/v) produced 3.1 g/L of XOS. Finally, the maximum XOS yield of 74.8% (based on xylan in corncob) was achieved, which is the highest yield among yields reported previously. The purity of XOS was high, whereas the contents of by-products were very low. This work presents a novel and promising strategy for co-production of XOS and monosaccharides from corncob without xylan isolation and purification.

Bacteroidetes are efficient degraders of complex carbohydrates, much thanks to their use of polysaccharide utilization loci (PULs). An integral part of PULs are highly specialized carbohydrate-active enzymes, sometimes composed of multiple linked domains with discrete functions-multicatalytic enzymes. We present the biochemical characterization of a multicatalytic enzyme from a large PUL encoded by the gut bacterium Bacteroides eggerthii. The enzyme, BeCE15A-Rex8A, has a rare and novel architecture, with an N-terminal carbohydrate esterase family 15 (CE15) domain and a C-terminal glycoside hydrolase family 8 (GH8) domain. The CE15 domain was identified as a glucuronoyl esterase (GE), though with relatively poor activity on GE model substrates, attributed to key amino acid substitutions in the active site compared to previously studied GEs. The GH8 domain was shown to be a reducing-end xylose-releasing exo-oligoxylanase (Rex), based on having activity on xylooligosaccharides but not on longer xylan chains. The full-length BeCE15A-Rex8A enzyme and the Rex domain were capable of boosting the activity of a commercially available GH11 xylanase on corn cob biomass. Our research adds to the understanding of multicatalytic enzyme architectures and showcases the potential of discovering novel and atypical carbohydrate-active enzymes from mining PULs.

The aim of this work was to investigate the use of isobutanol as organic solvent for the efficient delignification and fractionation of beechwood through the OxiOrganosolv process in the absence of any catalyst. The results demonstrate that cellulose-rich solid pulp produced after pretreatment is a source of fermentable sugars that can be easily hydrolyzed and serve as a carbon source in microbial fermentations for the production of omega-3 fatty acids and D-lactic acid. The C5 sugars are recovered in the aqueous liquid fractions and comprise a fraction rich in xylo-oligosaccharides with prebiotic potential. The maximum production of optically pure D-lactic from Lactobacillus delbrueckii sp. bulgaricus reached 51.6 g/L (0.57 g/g_biomass), following a simultaneous saccharification and fermentation strategy. Crypthecodenium cohnii accumulated up to 52.1 wt% lipids with a DHA content of 54.1 %, while up to 43.3 % hemicellulose recovery in form of oligosaccharides was achieved in the liquid fraction.

Xylooligosaccharides (XOS) are usually produced by xylan isolation from lignocellulose by alkaline followed by enzymatic hydrolysis, but the process is complicated. Recently acid hydrolysis for XOS preparation has become popular as it is faster and easier. This study investigated a novel strategy for producing XOS from corncob using propionic acid (PA) hydrolysis, then producing monosaccharides from solid residues by cellulase hydrolysis. The effect of alkaline post-treatment on enzymatic hydrolysis was studied. The maximum XOS yield of 68.5% was achieved using 5% PA at 170°C for 50 min. About 84% of lignin in PA-hydrolyzed corncob was removed using alkaline post-treatment. The yields of glucose and xylose reached 89.8% and 80.1%, respectively, using 5 FPU cellulase/g dry matter. The results indicated that alkaline post-treatment reduced 50% cellulase loading and improved the saccharification of PA-hydrolyzed corncob. This study presents an innovative option for efficient production of XOS and monosaccharides from corncob.

In the present study, ozone assisted autohydrolysis (OAAH) was evaluated for enhanced generation of xylooligosaccharide (XOS) from wheat bran. The total XOS yield with optimum ozone dose of 3% (OAAH-3) was found to be 8.9% (w/w biomass) at 110°C in comparison to 7.96% at 170°C by autohydrolysis (AH) alone. Although, there was no significant difference in oligomeric composition (DP 2-6), significant decrease in degradation products namely furfural (2.78-fold), HMF (3.15-fold), acrylamide (nil) and acetic acid (1.06-fold), was observed with OAAH-3 as a pretreatment option. There was 1-fold higher xylan to XOS conversion and OAAH-hydrolysate had higher DPPH radical scavenging activity than AH. PCA plots indicated clear enhancement in XOS production and lower generation of inhibitors with decrease in treatment temperature. Results of the study therefore suggest OAAH can be an effective pretreatment option that can further be integrated with downstream processing for concentration and purification of XOS.

Autohydrolysis is used for producing xylan-derived oligosaccharides from lignocellulosic biomass. Although numerous studies report optimized autohydrolysis conditions for various plants, few of these studies correlate process parameters with the resulting structural properties to their impact on intestinal bacterial communities. Thus, to further clarify these relationships, beechwood xylan (BWX)-derived substrates, processed under five conditions, were fermented in vitro by human gut microbiota. Autohydrolysis reduced the mean molecular size and substitutions of BWX. Distinct fermentation kinetics were observed with differing processing of BWX substrates, which correlated with impacts on community species evenness. The relative abundances of Bacteroides, Fusicatenibacter, Bifidobacterium, and Megasphaera within the fermentations varied with processing conditions. While the total short-chain fatty acid concentrations were the same among the treatments, processing conditions varied the extent of propionate and butyrate generation. Autolysis parameters may be an important tool for optimizing beneficial effects of xylan-derived fibers on human gut microbiota structure and function.

The heteropolysaccharide xylan is a valuable source of sustainable chemicals and materials from renewable biomass sources. A complete hydrolysis of this major hemicellulose component requires a diverse set of enzymes including endo-β-1,4-xylanases, β-xylosidases, acetylxylan esterases, α-l-arabinofuranosidases, and α-glucuronidases. Notably, the most studied xylanases from glycoside hydrolase family 11 (GH11) have exclusively been endo-β-1,4- and β-1,3-xylanases. However, a recent analysis of a metatranscriptome library from a microbial lignocellulose community revealed GH11 enzymes capable of releasing solely xylobiose from xylan. Although initial biochemical studies clearly indicated their xylobiohydrolase mode of action, the structural features that drive this new activity still remained unclear. It was also not clear whether the enzymes acted on the reducing or nonreducing end of the substrate. Here, we solved the crystal structure of MetXyn11 in the apo and xylobiose-bound forms. The structure of MetXyn11 revealed the molecular features that explain the observed pattern on xylooligosaccharides released by this nonreducing end xylobiohydrolase.

Sugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121°C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121°C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0-0.1 M NaOH) in the time and temperature range 10-30 min and 50-150°C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121°C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with β-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions.