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Xylotriose

Xylotriose O-XTR
Product code: O-XTR
€155.00

50 mg

Prices exclude VAT

Available for shipping

Content: 50 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 47592-59-6
Molecular Formula: C15H26O13
Molecular Weight: 414.4
Purity: > 95%
Substrate For (Enzyme): endo-1,4-β-Xylanase

High purity Xylotriose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Documents
Certificate of Analysis
Safety Data Sheet
FAQs Booklet
Publications
Megazyme publication
A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.

McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.

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.

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Publication
Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.

Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.

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.

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Systematic evaluation of the degraded products evolved from the hydrothermal pretreatment of sweet sorghum stems.

Sun, S., Wen, J., Sun, S. & Sun, R. C. (2015). Biotechnology for biofuels, 8(1), 37.

Background: Conversion of plant cell walls to bioethanol and bio-based chemicals requires pretreatment as a necessary step to reduce recalcitrance of cell walls to enzymatic and microbial deconstruction. In this study, the sweet sorghum stems were subjected to various hydrothermal pretreatment processes (110°C to 230°C, 0.5 to 2.0 h), and the focus of this work is to systematically evaluate the degraded products of polysaccharides and lignins in the liquor phase obtained during the pretreatment process. Results: The maximum yield of xylooligosaccharides (52.25%) with a relatively low level of xylose and other degraded products was achieved at a relatively high pretreatment temperature (170°C) for a short reaction time (0.5 h). Higher temperature (>170°C) and/or longer reaction time (>0.5 h at 170°C) resulted in a decreasing yield of xylooligosaccharides, but increased the concentration of arabinose and galactose. The xylooligosaccharides obtained are composed of xylopyranosyl residues, together with lower amounts of 4-O-Me-α-D-GlcpA units. Meanwhile, the concentrations of the degraded products (especially furfural) increased as a function of pretreatment temperature and time. Molecular weights of the water-soluble polysaccharides and lignins indicated that the degradation of the polysaccharides and lignins occurred during the conditions of harsh hydrothermal pretreatment. In addition, the water-soluble polysaccharides (rich in xylan) and water-soluble lignins (rich in β-O-4 linkages) were obtained at 170°C for 1.0 h. Conclusions: The present study demonstrated that the hydrothermal pretreatment condition had a remarkable impact on the compositions and the chemical structures of the degraded products. An extensive understanding of the degraded products from polysaccharides and lignins during the hydrothermal pretreatment will be beneficial to value-added applications of multiple chemicals in the biorefinery for bioethanol industry.

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Publication
Role of hemicellulases in production of fermentable sugars from corn stover.

Xin, D., Sun, Z., Viikari, L. & Zhang, J. (2015). Industrial Crops and Products, 74, 209-217.

In this work, the roles of hemicellulases, endoxylanase and β-xylosidase, in improving the hydrolysis of corn stover pretreated by aqueous ammonia (CS–AA) and dilute acid (CS–DA) were evaluated. Synergistic actions of endoxylanase and β-xylosidase were observed in the release of xylose in the hydrolysis of both isolated xylan and xylan in pretreated corn stover. Endoxylanase significantly reduced the negative effect of xylan on the action of cellulases, especially on cellobiohydrolase I. The addition of β-xylosidase from Selenomonas ruminantium increased the xylose yields from 9.6% and 13.0% to 31.7% and 47.6% in the hydrolysis of CS–AA by cellulases and xylanase at 40°C and 50°C, respectively. Furthermore, the addition of thermostable β-xylosidase from Entamoeba coli increased glucose yields from 40.3% and 20.7% to 44.0% and 26.6% in the hydrolysis of CS–AA and CS–DA by cellulases and xylanase at 50°C, respectively. β-xylosidase significantly reduced xylo-oligosaccharides inhibition on cellobiohydrolase I by converting most of xylo-oligosaccharides (93.6%) to the less inhibitory xylose, showing the importance and potential benefits of β-xylosidase in efficient and complete hydrolysis of lignocelluloses.

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Structural insights into the inhibition of cellobiohydrolase Cel7A by xylo‐oligosaccharides.

Momeni, M. H., Ubhayasekera, W., Sandgren, M., Ståhlberg, J. & Hansson, H. (2015). The FEBS journal, 282(11), 2167-2177.

The filamentous fungus Hypocrea jecorina (anamorph of Trichoderma reesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides (XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family 7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H. jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted ~ 2.4 Å towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition.

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Separation of xylose oligomers from autohydrolyzed Miscanthus × giganteus using centrifugal partition chromatography.

Chen, M. H., Rajan, K., Carrier, D. J. & Singh, V. (2015). Food and Bioproducts Processing, 95, 125-132.

Autohydrolysis of cellulosic materials for saccharification generates xylose-oligosaccharides (XOS), due to the partial hydrolysis of xylan. Developing an efficient method for the separation and recovery of XOS from the prehydrolyzates would provide an excellent opportunity for the better utilization of the cellulosic material and for value-added co-product production. In this study, we investigated the use of centrifugal partition chromatography (CPC) for the fractionation of XOS from Miscanthus × giganteus (M × G). During autohydrolysis of miscanthus biomass at 180°C for 20 min, 63% of xylan was converted into XOS and xylose. The ensuing XOS concentrate contained up to 30% of XOS, which were distributed as 15.9% xylobiose (DP2), 5.9% xylotriose, (DP3), 5.6% xylotetraose (DP4), 0.8% xylopentaose (DP5) and 0.6% xylohexaose (DP6). The XOS concentrate was further fractionated by CPC with a solvent system composed of 4:1:4 (v/v/v) butanol:methanol:water. Using CPC techniques, 230 mg (80%) of DP2 to DP6 oligomers were fractionated from 1 g of XOS concentrate. The recoveries of individual XOS were 90.2% DP2, 64.5% DP3, 71.2% DP4, 61.9% DP5 and 68.9% DP6. The purities of DP2 to DP6 fractions were 61.9%, 63.2%, 44.5%, 31.5% and 51.3%, respectively. Presence of DP2 and DP3 in the CPC purified fractions was further validated by mass spectrometry analysis. The study provided information on fast recovery of individual XOS from crude biomass prehydrolyzate.

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Publication
Cloning, expression and characterization of β-xylosidase from Aspergillus niger ASKU28.

Choengpanya, K., Arthornthurasuk, S., Wattana-amorn, P., Huang, W. T., Plengmuankhae, W., Li, Y. K. & Kongsaeree, P. T. (2015). Protein expression and purification, 115, 132-140.

β-Xylosidases catalyze the breakdown of β-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 β-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5′ end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the β-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 β-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry.

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Publication
Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations.

Bowman, M. J., Dien, B. S., Vermillion, K. E. & Mertens, J. A. (2015). Carbohydrate research, 407, 42-50.

Switchgrass (Panicum virgatum, L.) is a potential renewable source of carbohydrates for use in microbial conversion to biofuels. Xylan comprises approximately 30% of the switchgrass cell wall. To understand the limitations of commercial enzyme mixtures, alkali-extracted, isolated switchgrass xylan was hydrolyzed by the action of two commercial enzyme cocktails, in the presence and absence of an additional α-arabinofuranosidase enzyme. The two most abundant enzymatic digestion products from each commercial enzyme treatment were separated and characterized by LC-MSn, linkage analysis, and NMR. The most abundant oligosaccharide from each commercial cocktail was susceptible to hydrolysis when supplemented with a GH62 α-arabinofuranosidase enzyme; further characterization confirmed the presence of (1 →3)-α-arabinose linkages. These results demonstrate the lack of the required selectivity for arabinose-containing substrates in the commercial enzyme preparations tested. One product from each condition remained intact and was found to contain (1 →2)-β-xylose-(1 →3)-α-arabinose side chains; this linkage acts as a source of oligosaccharide recalcitrance.

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Identification and characterization of plant cell wall degrading enzymes from three glycoside hydrolase families in the cerambycid beetle Apriona japonica.

Pauchet, Y., Kirsch, R., Giraud, S., Vogel, H. & Heckel, D. G. (2014). Insect Biochemistry and Molecular Biology, 49, 1-13.

Xylophagous insects have evolved to thrive in a highly challenging environment. For example, wood-boring beetles from the family Cerambycidae feed exclusively on woody tissues, and to efficiently access the nutrients present in this sub-optimal environment, they have to cope with the lignocellulose barrier. Whereas microbes of the insect's gut flora were hypothesized to be responsible for the degradation of lignin, the beetle itself depends heavily on the secretion of a range of enzymes, known as plant cell wall degrading enzymes (PCWDEs), to efficiently digest both hemicellulose and cellulose networks. Here we sequenced the larval gut transcriptome of the Mulberry longhorn beetle, Apriona japonica (Cerambycidae, Lamiinae), in order to investigate the arsenal of putative PCWDEs secreted by this species. We combined our transcriptome with all available sequencing data derived from other cerambycid beetles in order to analyze and get insight into the evolutionary history of the corresponding gene families. Finally, we heterologously expressed and functionally characterized the A. japonica PCWDEs we identified from the transcriptome. Together with a range of endo-β-1,4-glucanases, we describe here for the first time the presence in a species of Cerambycidae of (i) a xylanase member of the subfamily 2 of glycoside hydrolase family 5 (GH5 subfamily 2), as well as (ii) an exopolygalacturonase from family GH28. Our analyses greatly contribute to a better understanding of the digestion physiology of this important group of insects, many of which are major pests of forestry worldwide.

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Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering.

Liu, Z., Zhao, X. & Bai, F. (2013). Applied Microbiology and Biotechnology, 97(10), 4361-4368.

Xylanase is the enzyme complex that is responsible for the degradation of xylan; however, novel xylanase producers remain to be explored in marine environment. In this study, a Streptomyces strain M11 which exhibited xylanase activity was isolated from marine sediment. The 16S rDNA sequence of M11 showed the highest identity (99%) to that of Streptomyces viridochromogenes. The xylanase produced from M11 exhibited optimum activity at pH 6.0, and the optimum temperature was 70°C. M11 xylanase activity was stable in the pH range of 6.0–9.0 and at 60°C for 60 min. Xylanase activity was observed to be stable in the presence of up to 5 M NaCl. Antibiotic-resistant mutants of M11 were isolated, and among the various antibiotics tested, streptomycin showed the best effect on obtaining xylanase overproducer. Mutant M11-1(10) isolated from 10 µg/ml streptomycin-containing plate showed 14% higher xylanase activities than that of the wild-type strain. An analysis of gene rpsL (encoding ribosomal protein S12) showed that rpsL from M11-1(10) contains a K88R mutation. This is the first report to show that marine-derived S. viridochromogenes strain can be used as a xylanase producer, and utilization of ribosome engineering for the improvement of xylanase production in Streptomyces was also first successfully demonstrated.

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Publication
Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6.

Driss, D., Bhiri, F., Siela, M., Ghorbel, R. & Chaabouni, S. E. (2012). Applied Biochemistry and Biotechnology, 168(4), 851-863.

An extracellular, endo-β-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS–PAGE with an apparent molecular weight of 30 kDa. The xylanase activity was optimal at pH 3.0 and 65°C. The specific activity measured for oat spelt xylan was 2,368 U mg-1. The apparent Km and Vmax values were 8.33 mg ml-1 and 58.82 µmol min-1 ml-1, respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962 bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88 kDa.

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Modulation of cellulosome composition in Clostridium cellulolyticum: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate‐active enzyme analyses.

Blouzard, J. C., Coutinho, P. M., Fierobe, H. P., Henrissat, B., Lignon, S., Tardif, C., Pages, S. & de Philip, P. (2010). Proteomics, 10(3), 541-554.

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate-active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin-containing proteins bear catalytic modules from numerous carbohydrate-active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip-cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32-kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate-binding module 6 or carbohydrate-binding module 22 xylan-binding modules along dockerins. This newly identified xyl-doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip-cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.

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Structural analysis of a glycoside hydrolase family 43 arabinoxylan arabinofuranohydrolase in complex with xylotetraose reveals a different binding mechanism compared with other members of the same family.

Vandermarliere, E., Bourgois, T. M., Winn, M. D., Van Campenhout, S., Volckaert, G., Delcour, J. A., Strelkov, S. V., Rabijns, A. & Courtin, C. (2009). Biochem. J, 418, 39-47.

AXHs (arabinoxylan arabinofuranohydrolases) are α-L-arabinofuranosidases that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl backbone residues of arabinoxylan. Bacillus subtilis was recently shown to produce an AXH that cleaves arabinose units from O-2- or O-3-mono-substituted xylose residues: BsAXH-m2,3 (B. subtilis AXH-m2,3). Crystallographic analysis reveals a two-domain structure for this enzyme: a catalytic domain displaying a five-bladed β-propeller fold characteristic of GH (glycoside hydrolase) family 43 and a CBM (carbohydrate-binding module) with a β-sandwich fold belonging to CBM family 6. Binding of substrate to BsAXH-m2,3 is largely based on hydrophobic stacking interactions, which probably allow the positional flexibility needed to hydrolyse both arabinose substituents at the O-2 or O-3 position of the xylose unit. Superposition of the BsAXH-m2,3 structure with known structures of the GH family 43 exo-acting enzymes, β-xylosidase and α-L-arabinanase, each in complex with their substrate, reveals a different orientation of the sugar backbone.

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Thermostability and xylan-hydrolyzing property of endoxylanase expressed in yeast Saccharomyces cerevisiae.

Lee, J. H., Heo, S. Y., Lee, J. W., Yoon, K. H., Kim, Y. H. & Nam, S. W. (2009). Biotechnology and Bioprocess Engineering, 14(5), 639-644.

The endoxylanase gene (xynB, GeneBank access code U51675), including its signal sequence, from Bacillus spp. was amplified and connected in frame downstream of yeast ADH1 promoter and then the resulting plasmid, pAEDX-1, was introduced into Saccharomyces cerevisiae. When the yeast transformants were grown on YPD medium, the majority of endoxylanase activity was detected in the extracellular culture medium, indicating that the signal peptide of Bacillus endoxylanase functioned well in yeast. In the batch cultivation of yeast transformants, the total expression level of endoxylanase and secretion efficiency were measured to be about 9.8 U/mL and 66.2%, respectively. The extracellular endoxylanase expressed in yeast showed an enhanced thermal stability due to the N-linked glycosylation. Through the hydrolysis of birchwood xylan with the endoxylanase, it was found that xylobiose and xylotriose were produced as major products with equimolar ratio.

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Mode of action of glycoside hydrolase family 5 glucuronoxylan xylanohydrolase from Erwinia chrysanthemi.

Vršanská, M., Kolenová, K., Puchart, V. & Biely, P. (2007). FEBS Journal, 274(7), 1666-1677.

The mode of action of xylanase A from a phytopathogenic bacterium, Erwinia chrysanthemi, classified in glycoside hydrolase family 5, was investigated on xylooligosaccharides and polysaccharides using TLC, MALDI-TOF MS and enzyme treatment with exoglycosidases. The hydrolytic action of xylanase A was found to be absolutely dependent on the presence of 4-O-methyl-D-glucuronosyl (MeGlcA) side residues in both oligosaccharides and polysaccharides. Neutral linear β-1,4-xylooligosaccharides and esterified aldouronic acids were resistant towards enzymatic action. Aldouronic acids of the structure MeGlcA3Xyl3 (aldotetraouronic acid), MeGlcA3Xyl4 (aldopentaouronic acid) and MeGlcA3Xyl5 (aldohexaouronic acid) were cleaved with the enzyme to give xylose from the reducing end and products shorter by one xylopyranosyl residue: MeGlcA2Xyl2, MeGlcA2Xyl3 and MeGlcA2Xyl4. As a rule, the enzyme attacked the second glycosidic linkage following the MeGlcA branch towards the reducing end. Depending on the distribution of MeGlcA residues on the glucuronoxylan main chain, the enzyme generated series of shorter and longer aldouronic acids of backbone polymerization degree 3–14, in which the MeGlcA is linked exclusively to the second xylopyranosyl residue from the reducing end. Upon incubation with β-xylosidase, all acidic hydrolysis products of acidic oligosaccharides and hardwood glucuronoxylans were converted to aldotriouronic acid, MeGlcA2Xyl2. In agreement with this mode of action, xylose and unsubstituted oligosaccharides were essentially absent in the hydrolysates. The E. chrysanthemi xylanase A thus appears to be an excellent biocatalyst for the production of large acidic oligosaccharides from glucuronoxylans as well as an invaluable tool for determination of the distribution of MeGlcA residues along the main chain of this major plant hemicellulose.

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Crystallization and preliminary X-ray analysis of an arabinoxylan arabinofuranohydrolase from Bacillus subtilis.

Vandermarliere, E., Bourgois, T. M., Van Campenhout, S., Strelkov, S. V., Volckaert, G., Delcour, J. A., Courtin, C. M. & Rabijns, A. (2007). Acta Crystallographica Section F: Structural Biology and Crystallization Communications, 63(8), 692-694.

Arabinoxylan arabinofuranohydrolases (AXH) are α-L-arabinofuranosidases (EC 3.2.1.55) that specifically hydrolyse the glycosidic bond between arabinofuranosyl substituents and xylopyranosyl residues from arabinoxylan, hence their name. In this study, the crystallization and preliminary X-ray analysis of the AXH from Bacillus subtilis, a glycoside hydrolase belonging to family 43, is described. Purified recombinant AXH crystallized in the orthorhombic space group P212121, with unit-cell parameters a= 68.7, b= 73.7, c= 106.5 Å. X-ray diffraction data were collected to a resolution of 1.55 Å.

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A thermostable alkaline active endo-β-1-4-xylanase from Bacillus halodurans S7: Purification and characterization.

Mamo, G., Hatti-Kaul, R. & Mattiasson, B. (2006). Enzyme and Microbial Technology, 39(7), 1492-1498.

A thermostable, alkaline active xylanase was purified to homogeneity from the culture supernatant of an alkaliphilic Bacillus halodurans S7, which was isolated from a soda lake in the Ethiopian Rift Valley. The molecular weight and the pI of this enzyme were estimated to be around 43 kDa and 4.5, respectively. When assayed at 70°C, it was optimally active at pH 9.0–9.5. The optimum temperature for the activity was 75°C at pH 9 and 70°C at pH 10. The enzyme was stable over a broad pH range and showed good thermal stability when incubated at 65°C in pH 9 buffer. The enzyme activity was strongly inhibited by Mn2+. Partial inhibition was also observed in the presence of 5 mM Cu2+, Co2+ and EDTA. Inhibition by Hg2+ and dithiothreitol was insignificant. The enzyme was free from cellulase activity and degraded xylan in an endo-fashion.

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Structural insight into the ligand specificity of a thermostable family 51 arabinofuranosidase, Araf51, from Clostridium thermocellum.

Taylor, E. J., Smith, N. L., Turkenburg, J. P., D'souza, S., Gilbert, H. J. & Davies, G. J. (2006). Biochem. J, 395, 31-37.

The digestion of the plant cell wall requires the concerted action of a diverse repertoire of enzyme activities. An important component of these hydrolase consortia are arabinofuranosidases, which release L-arabinofuranose moieties from a range of plant structural polysaccharides. The anaerobic bacterium Clostridium thermocellum, a highly efficient plant cell wall degrader, possesses a single α-L-arabinofuranosidase (EC 3.2.1.55), CtAraf51A, located in GH51 (glycoside hydrolase family 51). The crystal structure of the enzyme has been solved in native form and in ‘Michaelis’ complexes with both α-1,5-linked arabinotriose and α-1,3 arabinoxylobiose, both forming a hexamer in the asymmetric unit. Kinetic studies reveal that CtAraf51A, in contrast with well-characterized GH51 enzymes including the Cellvibrio japonicus enzyme [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607–614], catalyses the hydrolysis of α-1,5-linked arabino-oligosaccharides and the α-1,3 arabinosyl side chain decorations of xylan with equal efficiency. The paucity of direct hydrogen bonds with the aglycone moiety and the flexible conformation adopted by Trp178, which stacks against the sugar at the +1 subsite, provide a structural explanation for the plasticity in substrate specificity displayed by the clostridial arabinofuranosidase.

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Determination of reducing end sugar residues in oligo- and polysaccharides by gas–liquid chromatography.

Courtin, C. M., Van den Broeck, H. & Delcour, J. A. (2000). Journal of Chromatography A, 866(1), 97-104.

Reducing end sugar residues in maltodextrins and arabinoxylans are determined as alditol acetates by gas-liquid chromatography following reduction, acid hydrolysis and acetylation of the samples. After this conversion to alditol acetates, the reducing end sugars are thus separated from their acetylated aldose counterparts. The method allows to identify individual reducing end sugars quantitatively and is a good alternative for colorimetric reducing sugar assays and 1H-NMR analysis. To demonstrate the advantages of the method, an application in a study of enzymic solubilisation and degradation of water unextractable arabinoxylan from a flour squeegee fraction is described.

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Evidence for the presence of arabinoxylan hydrolysing enzymes in European wheat flours.

Cleemput, G., Bleukx, W., Van Oort, M., Hessing, M. & Delcour, J. A. (1995). Journal of Cereal Science, 22(2), 139-145.

Water extracts of flour samples prepared from six sound European wheat varieties hydrolyse p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabino-furanoside and, in addition, release soluble, dyed fragments from azurine crosslinked xylan. Incubation of water soluble wheat arabinoxylan with water extracts of flour results in the release of low molecular weight fractions consisting mainly of arabinose and xylose and small proportions of oligosaccharides as detected by high performance anion exchange chromatography. Gel permeation profiles of the incubation mixtures show a clear breakdown of the arabinoxylan.

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