A novel glycoside hydrolase 43-like enzyme from Clostridium boliviensis is an endo-xylanase and a candidate for xylooligosaccharide production from different xylan substrates.

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE: The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.

Xylanases and high-degree wet milling improve soluble dietary fibre content in liquid oat base.

The growth of plant-based food and drink substitutes has led to increased interest in oat-based milk substitute as a dairy milk alternative. Conventional liquid oat base (LOB) production results in a fibre-rich insoluble by-product and loss of valuable macronutrients. This study investigates the use of xylanase enzymes to release insoluble arabinoxylan (AX) fibre and employs different degrees of milling in the LOB manufacturing process, with the aim to reduce insoluble waste and simultaneously increase soluble dietary fibre in oat-based milk substitutes. The combination of decreased mill gap space from 1 to 0.05 mm and addition of GH10 xylanase, resulted in a homogenous LOB product and solubilization of all available AX. Potential prebiotic arabinoxylooligosaccharides of DP3-7 from GH10 hydrolysis were identified using HPAEC-PAD and MS analysis. These findings demonstrate the value of utilizing xylanases and fine-milling in LOB manufacturing, offering a sustainable approach to maximize health benefits of oat-based beverages.

Novel Function of CtXyn5A from Acetivibrio thermocellus: Dual Arabinoxylanase and Feruloyl Esterase Activity in the Same Active Site.

Enzymes' uncharacterised side activities can have significant effects on reaction products and yields. Hence, their identification and characterisation are crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus, besides its well-known arabinoxylanase activity, for the first time. Activity analysis of enzyme variants mutated in the catalytic nucleophile, Glu279, confirmed removal of all activity for E279A and E279L, and increased esterase activity while removing xylanase activity for E279S, thus allowing the proposal that both reaction types are catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than that of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity.

Novel thermostable GH5_34 arabinoxylanase with an atypical CBM6 displays activity on oat fiber xylan for prebiotic production.

Carbohydrate active enzymes are valuable tools in cereal processing to valorize underutilized side streams. By solubilizing hemicellulose and modifying the fiber structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica, HhXyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibers: arabinoxylo-oligosaccharides. The purified two-domain HhXyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature Tm of 61°C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan. HhXyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, whereas the sole catalytic domain of HhXyn5A did not demonstrate detectable activity. HhXyn5A demonstrated no side activity on oat ß-glucan. In contrast to the commercially available homolog CtXyn5A, HhXyn5A gave a more specific HPAEC-PAD oligosaccharide product profile when using wheat arabinoxylan and alkali extracted oat bran fibers as the substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modeling of HhXyn5A and docking simulations with ligands XXXA3, XXXA3XX and X5 concluded that the active site of HhXyl5A catalytic domain is highly conserved and can accommodate both shorter and longer ligands. However, significant structural dissimilarities between HhXyn5A and CtXyn5A in the binding cleft of CBM6, due to the lack of important ligand-interacting residues, is suggested to cause the observed differences in substrate specificity and product formation.

Lignocellulose degradation for the bioeconomy: The potential of enzyme synergies between xylanases, ferulic acid esterase and laccase for the production of arabinoxylo-oligosaccharides.

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.

Non-aqueous reversed phase liquid chromatography with charged aerosol detection for quantitative lipid analysis with improved accuracy.

There is a great need for efficient analysis of the composition of vegetable oils and fats, since it affects the physical and technical properties. However, due to the complex nature of these kind of samples, it is often difficult and costly. In the present study, we developed a Non-Aqueous Reversed-Phase HPLC method that can be used to separate and quantify different free fatty acids, fatty acid esters, monoacylglycerides, diacylglycerides and triacylglycerides, including regioisomers such as SOS/SSO and 1,2- and 1,3-diolein. Two 25 cm Nucleodur C18 Isis columns in series, sub-ambient column temperature and a mobile phase gradient composed of acetonitrile, acetic acid, isopropanol and heptane were used for the separation. The lipids were detected and quantified using a charged aerosol detector and it was found that the peak shape highly affected the detector response as well as the response uniformity, even when inverse gradient compensation was employed. Thus, calibration and determination of response factors were necessary for reliable quantification. A correlation between response factors and peak width at half peak height was found and used for quantification of non-calibrated components. A quantification approach was suggested including an appropriate selection of calibrated components, depending on sample composition and the accuracy required. It was shown in a complex oil sample that the reduced calibration approach, using only 6 instead of 33 calibrated components, resulted in virtually the same composition, but yielded a more accurate result compared to using relative area that neglects response factors. The method validation showed good reproducibility and accuracy, making it an excellent tool for extensive analysis of complex lipid mixtures.

Chemical and biochemical bleaching of oat hulls: The effect of hydrogen peroxide, laccase, xylanase and sonication on optical properties and chemical composition.

Oat hulls are an excellent dietary fibre source for food supplements due to their rich lignocellulose composition as well as their great abundance as low-value agricultural side stream. For the production of white fibre supplements, a mild, but effective bleaching of the hulls is required. Chemical bleaching with hydrogen peroxide and sodium hydroxide was here found to be a suitable method increasing the CIE L* value (corresponds to a lightness value) above 85. The developed method is mild, retaining the hull's chemical composition. Only a minor decrease in coniferaldehyde structures upon bleaching was detected. Colour and chemical variabilities of oat hulls from different growth seasons did not influence the required bleaching conditions to achieve the desired optical properties. The inclusion of biochemical bleaching steps utilizing the xylanase Pentopan Mono BG, the laccase NS51003 and sonication was industrially not feasible as they could not reduce the required amount of subsequently applied bleaching chemicals significantly.

Novel xylan-degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205.

Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-ß-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43_1 in the same PUL, is an intracellular ß-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43_12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.

Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)-catalyzed transglycosylation.

Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation of a long-carbohydrate-chain alkyl glycoside and cyclodextrin glucanotransferase (CGTase)-catalyzed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl ß- d-maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase-catalyzed coupling/disproportionation reactions with α-cyclodextrin and dodecyl ß- d-maltoside diuronic acid (DDM-2COOH) or octyl ß- d-glucuronic acid (OG-COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non-ionic substrates (dodecyl ß- d-maltoside and octyl ß- d-glucoside), Toruzyme exhibited high catalytic activity at pH 5-6, but for the acidic substrates (DDM-2COOH and OG-COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM-2COOH and OG-COOH, which exist at lower pH (pKa about 3).

Xylooligosaccharides Increase Bifidobacteria and Lachnospiraceae in Mice on a High-Fat Diet, with a Concomitant Increase in Short-Chain Fatty Acids, Especially Butyric Acid.

Effects of xylooligosaccharides (XOSs) as well as a mixture of XOS, inulin, oligofructose, and partially hydrolyzed guar gum (MIX) in mice fed a high-fat diet (HFD) were studied. Control groups were fed an HFD or a low-fat diet. Special attention was paid to the cecal composition of the gut microbiota and formation of short-chain fatty acids, but metabolic parameters were also documented. The XOS group had significantly higher cecum levels of acetic, propionic, and butyric acids than the HFD group, and the butyric acid content was higher in the XOS than in the MIX group. The cecum microbiota of the XOS group contained more Bifidobacteria, Lachnospiraceae, and S24-7 bacteria than the HFD group. A tendency of lower body weight gain was observed on comparing the XOS and HFD groups. In conclusion, the XOS was shown to be a promising prebiotic candidate. The fiber diversity in the MIX diet did not provide any advantages compared to the XOS diet.

Engineering CGTase to improve synthesis of alkyl glycosides.

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.

Efficient laccase/TEMPO oxidation of alkyl glycosides: Effects of carbohydrate group and alkyl chain length.

Alkyl glycosides with long hydrophobic chains have attractive surfactant properties, but their wider application is hampered by their low solubility in water. Here, a route to increased solubility by introduction of carboxyl groups via laccase/TEMPO oxidation is presented. The oxidation pathways for dodecyl ß-maltoside and hexadecyl ß-maltoside were studied in detail. Close to full conversion was achieved for both substrates and conditions were found under which the diacid products dominated, with only minor amounts of under-oxidized and over-oxidized products. Dodecyl ß-maltoside oxidation was improved to give a yield of 85 % of the diacid derivative. Interestingly, in spite of low substrate solubility the oxidation of hexadecyl ß-maltoside was very efficient in aqueous medium, due to the higher solubility of the products. Addition of organic cosolvents did not provide additional advantages. The method is promising for producing soluble anionic derivatives of alkyl glycosides in an environmentally friendly and efficient way.

Chemoenzymatic synthesis of the pH responsive surfactant octyl ß-D-glucopyranoside uronic acid.

Methodology was developed to expand the range of benign alkyl glycoside surfactants to include also anionic types. This was demonstrated possible through conversion of the glycoside to its carboxyl derivative. Specifically, octyl ß-D-glucopyranoside (OG) was oxidised to the corresponding uronic acid (octyl ß-D-glucopyranoside uronic acid, OG-COOH) using the catalyst system T. versicolor laccase/2,2,6,6-tetramethylpiperidinyloxy (TEMPO) and oxygen from air as oxidant. The effects of oxygen supply methodology, concentrations of laccase, TEMPO and OG as well as reaction temperature were evaluated. At 10 mM substrate concentration, the substrate was almost quantitatively converted into product, and even at a substrate concentration of 60 mM, 85% conversion was reached within 24 h. The surfactant properties of OG-COOH were markedly dependent on pH. Foaming was only observed at low pH, while no foam was formed at pH values above 5.0. Thus, OG-COOH can be an attractive low-foaming surfactant, for example for cleaning applications and emulsification, in a wide pH range (pH 1.5-10.0).

Endo-xylanases as tools for production of substituted xylooligosaccharides with prebiotic properties.

Xylan has a main chain consisting of ß-1,4-linked xylose residues with diverse substituents. Endoxylanases cleave the xylan chain at cleavage sites determined by the substitution pattern and thus give different oligosaccharide product patterns. Most known endoxylanases belong to glycoside hydrolase (GH) families 10 and 11. These enzymes work well on unsubstituted xylan but accept substituents in certain subsites. The GH11 enzymes are more restricted by substituents, but on the other hand, they are normally more active than the GH10 enzymes on insoluble substrates, because of their smaller size. GH5 endoxylanases accept arabinose substituents in several subsites and require it in the - 1 subsite. This specificity makes the GH5 endoxylanases very useful for degradation of highly arabinose-substituted xylans and for the selective production of arabinoxylooligosaccharides, without formation of unsubstituted xylooligosaccharides. The GH30 endoxylanases have a related type of specificity in that they require a uronic acid substituent in the - 2 subsite, which makes them very useful for the production of uronic acid substituted oligosaccharides. The ability of dietary xylooligosaccharides to function as prebiotics in humans is governed by their substitution patterns. Endoxylanases are thus excellent tools to tailor prebiotic oligosaccharides to stimulate various types of intestinal bacteria and to cause fermentation in different parts of the gastrointestinal tract. Continuously increasing knowledge on the function of the gut microbiota and discoveries of novel endoxylanases increase the possibilities to achieve health-promoting effects.

Hydrophilization of bixin by lipase-catalyzed transesterification with sorbitol.

Bixin is one of the most used yellow-orange food colorants in the food industry. The polyene chain of bixin makes it highly hydrophobic and less suitable for water-based food formulations. Lipase-catalyzed reactions of bixin with sorbitol were studied to synthesize a new derivative of bixin with potential hydrophilic properties. Interestingly, we show that the lipase-catalyzed reaction of bixin leads to a transesterification reaction and formation of a transesterified product, sorbitol ester of norbixin (SEN). The reaction efficiency was optimized with various immobilized lipases at different water activity levels in the organic solvent, 2-methyl-2-butanol. Among the examined lipases, immobilized Candida antarctica lipase B (Novozyme 435) provided the highest reaction yield at a water activity close to zero. Tetrahydrofuran (THF) was used as co-solvent to improve bixin solubility. The optimization of the reaction conditions with 20% THF lead to a total reaction yield of 50% of SEN.

Screening of organic solvents for bioprocesses using aqueous-organic two-phase systems.

The application of conventional organic solvents has been essential in several steps of bioprocesses in order to achieve sufficient economic efficiency. The use of organic solvents is frequently used either to partly or fully replace water in the reaction medium or as a process aid for downstream separation. Nowadays, manufacturers are increasingly requested to avoid and substitute solvents with hazardous potential. Therefore, the solvent selection must account for potential environmental hazards, health and safety problems, in addition to fulfilling the ideal characteristics for application in a process. For the first time, criteria including Environment, Health and Safety (EHS), as well as the technical requirements for reaction and separation have been reviewed, collected and integrated in a single organic solvent screening strategy to be used as a guideline for narrowing down the list of solvents to test experimentally. Additionally, we have also included a solvent selection guide based on the methodology developed in the Innovative Medicines Initiative CHEM21 (IMI CHEM21) project and applied specifically to water-immiscible solvents commonly used in bioprocesses.

ß-Mannanase-catalyzed synthesis of alkyl mannooligosides.

ß-Mannanases catalyze the conversion and modification of ß-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), ß-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable ß-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 ß-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate ß-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three ß-mannanases using methanol or 1-hexanol as acceptor. Among the three ß-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1-hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted ß-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using ß-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable ß-mannans.

Xylo- and arabinoxylooligosaccharides from wheat bran by endoxylanases, utilisation by probiotic bacteria, and structural studies of the enzymes.

Xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) were produced from the insoluble arabinoxylan fraction of pretreated wheat bran by endoxylanases. The glycoside hydrolase (GH) family 10 xylanases GsXyn10A from Geobacillus stearothermophilus and RmXyn10A-CM from Rhodothermus marinus produced the AXOS A3X, A2XX and A2 + 3XX in addition to XOS. RmXyn10A-CM also produced XA2 + 3XX due to its non-conserved aglycone region accommodating additional arabinose substitutions in subsite +2. The GH11 enzymes, Pentopan from Thermomyces lanuginosus and NpXyn11A from Neocallimastix patriciarum had minor structural differences affecting hydrogen bonds in subsites -3 and +3, with similar hydrolysis profiles producing XA3XX as major AXOS and minor amounts of XA2XX but different ratios of X3/X2. In vitro analysis of the prebiotic properties of (A)XOS produced by Pentopan revealed nearly complete uptake of X2 and X3 by the probiotic bacteria Lactobacillus brevis and Bifidobacterium adolescentis. In contrast to previous reports, the GH43 arabinofuranosidase BaAXHd-3 from B. adolescentis cleaved α-1,3-linked arabinose on some single substituted AXOS.

Valorization of Brewer's spent grain to prebiotic oligosaccharide: Production, xylanase catalyzed hydrolysis, in-vitro evaluation with probiotic strains and in a batch human fecal fermentation model.

Brewer's spent grain (BSG) accounts for around 85% of the solid by-products from beer production. BSG was first extracted to obtain water-soluble arabinoxylan (AX). Using subsequent alkali extraction (0.5 M KOH) it was possible to dissolve additional AX. In total, about 57% of the AX in BSG was extracted with the purity of 45-55%. After comparison of nine xylanases, Pentopan mono BG, a GH11 enzyme, was selected for hydrolysis of the extracts to oligosaccharides with minimal formation of monosaccharides. Growth of Bifidobacterium adolescentis (ATCC 15703) was promoted by the enzymatic hydrolysis to arabinoxylooligosaccharides, while Lactobacillus brevis (DSMZ 1264) utilized only unsubstituted xylooligosaccharides. Furthermore, utilization of the hydrolysates by human gut microbiota was also assessed in a batch human fecal fermentation model. Results revealed that the rates of fermentation of the BSG hydrolysates by human gut microbiota were similar to that of commercial prebiotic fructooligosaccharides, while inulin was fermented at a slower rate. In summary, a sustainable process to valorize BSG to functional food ingredients has been proposed.

Arabinoxylanase from glycoside hydrolase family 5 is a selective enzyme for production of specific arabinoxylooligosaccharides.

An arabinose specific xylanase from glycoside hydrolase family 5 (GH5) was used to hydrolyse wheat and rye arabinoxylan, and the product profile showed that it produced arabinose substituted oligosaccharides (AXOS) having 2-10 xylose residues in the main chain but no unsubstituted xylooligosaccharides (XOS). Molecular modelling showed that the active site has an open structure and that the hydroxyl groups of all xylose residues in the active site are solvent exposed, indicating that arabinose substituents can be accommodated in the glycone as well as the aglycone subsites. The arabinoxylan hydrolysates obtained with the GH5 enzyme stimulated growth of Bifidobacterium adolescentis but not of Lactobacillus brevis. This arabinoxylanase is thus a good tool for the production of selective prebiotics.