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|Stability:||> 10 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-1,4-β-Xylanase, α-Arabinofuranosidase|
High purity 33-α-L-arabinofuranosyl-xylotetraose (XA3XX) for use in research, biochemical enzyme assays and in vitro diagnostic analysis. It can be used as an analytical standard or as a substrate to help characterise the activities of arabinoxylan degrading enzymes including endo-xylanase, β-xylosidase and α-L-arabinofuranosidase. This compound was prepared by the controlled enzymatic hydrolysis of wheat arabinoxylan.
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Cloning of an α-L-Arabinofuranosidase and Characterization of Its Action on Mono-and Di-Substituted Xylopyranosyl Units.
Wong, D. W. & Batt, S. (2022). Advances in Enzyme Research, 10(4), 75-82.
An α-L-arabinofuranosidase (ARF) gene of 1503 bp was synthesized, subcloned into pET26b vector, and expressed in Escherichia coli. The enzyme was purified in active form, and consisted of 500 amino acid residues, corresponding to 55 kD based on SDS-PAGE. The affinity-purified protein was characterized using arabinofuranosyl xylooligosaccharides (AXOS) as substrates. The pH effect was investigated showing an optimum at pH 5.5. XaARF catalyzed the cleavage of arabinose at C3 of the xylopyranosyl unit efficiently if the arabinofuranosyl substitution was at the terminal compared to internal xylose units. The enzyme was able to act on di-substituted xylopyranosyl units with the first cleavage at C3 followed by C2 linkages.Hide Abstract
Selfish uptake versus extracellular arabinoxylan degradation in the primary degrader Ruminiclostridium cellulolyticum, a new string to its bow.
Liu, N., Gagnot, S., Denis, Y., Byrne, D., Faulds, C., Fierobe, H. P. & Perret, S. (2022). Biotechnology for Biofuels and Bioproducts, 15(1), 1-16.
Background: Primary degraders of polysaccharides play a key role in anaerobic biotopes, where plant cell wall accumulates, providing extracellular enzymes to release fermentable carbohydrates to fuel themselves and other non-degrader species. Ruminiclostridium cellulolyticum is a model primary degrader growing amongst others on arabinoxylan. It produces large multi-enzymatic complexes called cellulosomes, which efficiently deconstruct arabinoxylan into fermentable monosaccharides. Results: Complete extracellular arabinoxylan degradation was long thought to be required to fuel the bacterium during this plant cell wall deconstruction stage. We discovered and characterized a second system of “arabinoxylan” degradation in R. cellulolyticum, which challenged this paradigm. This “selfish” system is composed of an ABC transporter dedicated to the import of large and possibly acetylated arabinoxylodextrins, and a set of four glycoside hydrolases and two esterases. These enzymes show complementary action modes on arabinoxylo-dextrins. Two α-L-arabinofuranosidases target the diverse arabinosyl side chains, and two exo-xylanases target the xylo-oligosaccharides backbone either at the reducing or the non-reducing end. Together, with the help of two different esterases removing acetyl decorations, they achieve the depolymerization of arabinoxylo-dextrins in arabinose, xylose and xylobiose. The in vivo study showed that this new system is strongly beneficial for the fitness of the bacterium when grown on arabinoxylan, leading to the conclusion that a part of arabinoxylan degradation is achieved in the cytosol, even if monosaccharides are efficiently provided by the cellulosomes in the extracellular space. These results shed new light on the strategies used by anaerobic primary degrader bacteria to metabolize highly decorated arabinoxylan in competitive environments. Conculsion: The primary degrader model Ruminiclostridium cellulolyticum has developed a “selfish” strategy consisting of importing into the bacterium, large arabinoxylan-dextrin fractions released from a partial extracellular deconstruction of arabinoxylan, thus complementing its efficient extracellular arabinoxylan degradation system. Genetic studies suggest that this system is important to support fitness and survival in a competitive biotope. These results provide a better understanding of arabinoxylan catabolism in the primary degrader, with biotechnological application for synthetic microbial community engineering for the production of commodity chemicals from lignocellulosic biomass.Hide Abstract
Rapid profiling strategy for oligosaccharides and polysaccharides by MALDI TOF mass spectrometry.
Wang, J., Zhao, J., Nie, S., Xie, M. & Li, S. (2021). Food Hydrocolloids, 124, 107237.
The application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) in glycan was limited due to their poor ionization efficiency, compared with biomolecules such as proteins and peptides. Aiming to improve the ionization efficiency and simplify preparation procedure simultaneously during MALDI MS analysis, an on-target derivatization method using 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) as matrix was employed and it was conducted both in the positive and negative ion MALDI TOF MS. Results indicated that after on-target derivatization, the ions generated had substantially improved S/N ratios and sensitivity in the tandem mass spectra. The B/Y- type ions of 3-AQ-labeled glycans could be easily recognized, and cross-ring A- type ions provided additional information to reveal the linkage patterns. Specifically, positive ion mass spectra with protonated adduct as precursor ion produced a simple fragmentation pattern benefited for sequencing and observation of branches. Furthermore, this method was successfully applied in polysaccharides analysis, including arabinoxylan, xylan, arabinogalactan and dextran after enzymatic or acid degradation. This study demonstrated that it was feasible to analyze higher molecular weight polysaccharides by MALDI TOF MS using 3-AQ/CHCA matrix through appropriate hydrolysis, and it allowed much efficient structural interpretation with increased sensitivity and characteristic fragment ions.Hide Abstract
Lignocellulose degradation for the bioeconomy: the potential of enzyme synergies between xylanases, ferulic acid esterase and laccase for the production of arabinoxylo-oligosaccharides.
Schmitz, E., Leontakianakou, S., Norlander, S., Karlsson, E. N. & Adlercreutz, P. (2021). Bioresource Technology, 343, 126114.
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.Hide Abstract
Multiple transporters and glycoside hydrolases are involved in arabinoxylan-derived oligosaccharide utilization in Bifidobacterium pseudocatenulatum.
Saito, Y., Shigehisa, A., Watanabe, Y., Tsukuda, N., Moriyama-Ohara, K., Hara, T., Matsumoto, S., Tsuji, H. & Matsuki, T. (2020). Applied and Environmental Microbiology, 86(24).
Arabinoxylan hydrolysates (AXH) are the hydrolyzed products of the major components of the dietary fiber arabinoxylan. AXH include diverse oligosaccharides varying in xylose polymerization and side residue modifications with arabinose at the O-2 and/or O-3 position of the xylose unit. Previous studies have reported that AXH exhibit prebiotic properties on gut bifidobacteria; moreover, several adult-associated bifidobacterial species (e.g., Bifidobacterium adolescentis and Bifidobacterium longum subsp. longum) are known to utilize AXH. In this study, we tried to elucidate the molecular mechanisms of AXH utilization by Bifidobacterium pseudocatenulatum, which is a common bifidobacterial species found in adult feces. We performed transcriptomic analysis of B. pseudocatenulatum YIT 4072T, which identified three upregulated gene clusters during AXH utilization. The gene clusters encoded three sets of ATP-binding cassette (ABC) transporters and five enzymes belonging to glycoside hydrolase family 43 (GH43). By characterizing the recombinant proteins, we found that three solute-binding proteins of ABC transporters showed either broad or narrow specificity, two arabinofuranosidases hydrolyzed either single- or double-decorated arabinoxylooligosaccharides, and three xylosidases exhibited functionally identical activity. These data collectively suggest that the transporters and glycoside hydrolases, encoded in the three gene clusters, work together to utilize AXH of different sizes and with different side residue modifications. Thus, our study sheds light on the overall picture of how these proteins collaborate for the utilization of AXH in B. pseudocatenulatum and may explain the predominance of this symbiont species in the adult human gut.Hide Abstract
Simultaneous determination of cereal monosaccharides, xylo-and arabinoxylo-oligosaccharides and uronic acids using HPAEC-PAD.
Alyassin, M., Campbell, G. M., O'Neill, H. M. & Bedford, M. R. (2020). Food Chemistry, 315, 126221.
Xylo- and arabinoxylo-oligosaccharides (XOS and AXOS) are of interest for their prebiotic activity. The production of these oligomers might be accompanied with monosaccharides. The measurement of both oligosaccharides and monosaccharides usually requires two methods. The current work presents an HPAEC-PAD method based on gradient elution of aqueous solvents sodium hydroxide and sodium acetate, in contrast to conventional isocratic elution, for the simultaneous separation of 16 standards of monosaccharides, xylo-oligosaccharides, arabinoxylo-oligosaccharides and uronic acids using CarboPac PA 200 column. The presented method showed a stable baseline and high-resolution separation of the standards. The method showed acceptable accuracy and precision. Limits of Detection and Quantitation (LOD and LOQ) were estimated for all the standards. The method was applied to measure the activity of a commercial endoxylanase on wheat bran; a steady release of xylose monosaccharide was observed. Enzyme action on oligosaccharide standards showed a preference for the larger oligosaccharides.Hide Abstract
Strategy for structural elucidation of polysaccharides: elucidation of a maize mucilage that harbors diazotrophic bacteria.
Amicucci, M. J., Galermo, A. G., Guerrero, A., Treves, G., Nandita, E., Kailemia, M. J., Higdon, S. M., Pozzo, T., Labavitch, J. M., Bennett, A. B. & Lebrilla, C. B. (2019). Analytical Chemistry, 91(11), 7254-7265.
The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.Hide Abstract
Mechelke, M., Koeck, D. E., Broeker, J., Roessler, B., Krabichler, F., Schwarz, W. H., Zverlov, V. V. & Liebl, W. (2017). Journal of Biotechnology, 257, 122-130.
Herbinix hemicellulosilytica is a newly isolated, gram-positive, anaerobic bacterium with extensive hemicellulose-degrading capabilities obtained from a thermophilic biogas reactor. In order to exploit its potential as a source for new industrial arabinoxylan-degrading enzymes, six new thermophilic xylanases, four from glycoside hydrolase family 10 (GH10) and two from GH11, three arabinofuranosidases (1x GH43, 2x GH51) and one β-xylosidase (GH43) were selected. The recombinantly produced enzymes were purified and characterized. All enzymes were active on different xylan-based polysaccharides and most of them showed temperature-vs-activity profiles with maxima around 55–65°C. HPAEC-PAD analysis of the hydrolysates of wheat arabinoxylan and of various purified xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) was used to investigate their substrate and product specificities: among the GH10 xylanases, XynB showed a different product pattern when hydrolysing AXOS compared to XynA, XynC, and XynD. None of the GH11 xylanases was able to degrade any of the tested AXOS. All three arabinofuranosidases, ArfA, ArfB and ArfC, were classified as type AXH-m,d enzymes. None of the arabinofuranosidases was able to degrade the double-arabinosylated xylooligosaccharides XA2+3XX. β-Xylosidase XylA (GH43) was able to degrade unsubstituted XOS, but showed limited activity to degrade AXOS.Hide Abstract
Mechelke, M., Herlet, J., Benz, J. P., Schwarz, W. H., Zverlov, V. V., Liebl, W. & Kornberger, P. (2017). Analytical and Bioanalytical Chemistry, 1-13.
The rising importance of accurately detecting oligosaccharides in biomass hydrolyzates or as ingredients in food, such as in beverages and infant milk products, demands for the availability of tools to sensitively analyze the broad range of available oligosaccharides. Over the last decades, HPAEC-PAD has been developed into one of the major technologies for this task and represents a popular alternative to state-of-the-art LC-MS oligosaccharide analysis. This work presents the first comprehensive study which gives an overview of the separation of 38 analytes as well as enzymatic hydrolyzates of six different polysaccharides focusing on oligosaccharides. The high sensitivity of the PAD comes at cost of its stability due to recession of the gold electrode. By an in-depth analysis of the sensitivity drop over time for 35 analytes, including xylo- (XOS), arabinoxylo- (AXOS), laminari- (LOS), manno- (MOS), glucomanno- (GMOS), and cellooligosaccharides (COS), we developed an analyte-specific one-phase decay model for this effect over time. Using this model resulted in significantly improved data normalization when using an internal standard. Our results thereby allow a quantification approach which takes the inevitable and analyte-specific PAD response drop into account.Hide Abstract
Puchart, V & Biely, P. (2008). Journal of Biotechnology. 137(1-4), 34–43.
When grown on beech-wood glucuronoxylan, two strains of the thermophilic fungus Thermomyces lanuginosius, IMI 84400 and IMI 96213, secreted endo-β-1,4-xylanase of glycoside hydrolase family 11 and simultaneously accumulated an acidic pentasaccharide in the medium. The aldopentaouronic acid was purified and its structure was established by a combination of NMR spectroscopy and enzyme digestion with glycosidases as MeGlcA3Xyl4. Both strains showed limited growth on wheat arabinoxylan as a carbon source. An essential part of the polysaccharide was not utilized, and it was converted to a series of arabinoxylooligosaccharides differing in the degree of polymerization. The structure of the shorter arabinoxylooligosaccharides remaining in the wheat arabinoxylan-spent medium was established using mass spectrometry and digestion with glycosidases. Xylose and linear β-1,4-xylooligosaccharides generated extracellularly during growth on either hardwood or cereal xylan were efficiently taken up by the cells and metabolized intracellularly. The data suggest that due to a lack of extracellular β-xylosidase, α-glucuronidase, and α-L-arabinofuranosidase, the widely used T. lanuginosus strains might become efficient producers of branched xylooligosaccharides from both types of xylans.Hide Abstract
Pastell, H., Tuomainen, P., Virkki, L. & Tenkanen, M. (2008). Carbohydrate Research, 343(18), 3049-3057.
Shearzyme (GH10 endo-1,4-β-D-xylanase) and two different α-L-arabinofuranosidases (AXH-m and AXH-d3) were used stepwise to manufacture arabinoxylo-oligosaccharides (AXOS) with α-L-Araf (1→2)-monosubstituted β-D-Xylp residues or α-L-Araf (1→2)- and (1→3) doubly substituted β-D-Xylp residues from wheat arabinoxylan (AX) in a rather straightforward way. Four major AXOS (d-I, d-II, m-I and m-II) were formed in two separate hydrolyses. The AXOS were purified and the structures were confirmed using TLC, HPAEC-PAD, MALDI-TOF-MS and 1D and 2D NMR spectroscopy. The samples were identified as d-I: α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-D-Xylp, d-II: α-L-Araf-(1→2)-[α-L-Araf-(1→3)]-β-D-Xylp-(1→4)-D-Xylp, m-I: α-L-Araf-(1→2)-β-D-Xylp-(1→4)-β-D-Xylp-(1→4)-D-Xylp and m-II: α-L-Araf-(1→2)-β-D-Xylp-(1→4)-D-Xylp. To our knowledge, this is the first report on structural 1H and 13C NMR analysis of xylobiose-derived AXOS d-II and m-II. The latter compound has not been reported previously. The doubly substituted AXOS were produced for the first time in good yields, as d-I and d-II corresponded to 11.8 and 5.6 wt% of AX, respectively. Singly α-L-Araf (1→2)-substituted AXOS could also be prepared in similar yields by treating the doubly substituted AXOS further with AXH-d3.Hide Abstract
Van Laere, K. M. J., Hartemink, R., Bosveld, M., Schols, H. A. & Voragen, A. G. J. (2000). Journal of Agricultural and Food Chemistry, 48(5), 1644–1652.
New types of nondigestible oligosaccharides were produced from plant cell wall polysaccharides, and the fermentation of these oligosaccharides and their parental polysaccharides by relevant individual intestinal species of bacteria was studied. Oligosaccharides were produced from soy arabinogalactan, sugar beet arabinan, wheat flour arabinoxylan, polygalacturonan, and rhamnogalacturonan fraction from apple. All of the tested substrates were fermented to some extent by one or more of the individual species of bacteria tested. Bacteroides spp. are able to utilize plant cell wall derived oligosaccharides besides their reported activity toward plant polysaccharides. Bifidobacterium spp. are also able to utilize the rather complex plant cell wall derived oligosaccharides in addition to the bifidogenic fructooligosaccharides. Clostridium spp., Klebsiella spp., and Escherichia coli fermented some of the selected substrates in vitro. These studies do not allow prediction of the fermentation in vivo but give valuable information on the fermentative capability of the tested intestinal strains.Hide Abstract
Kormelink, F. J. M., Hoffmann, R. A., Gruppen, H., Voragen, A. G. J., Kamerling, J. P. & Vliegenthart, J. F. G. (1993). Carbohydrate Research, 249(2), 369-382.
Alkali-extractable wheat-flour arabinoxylan, treated with endo-(1→4)-β-D-xylanase III from Aspergillus awamori CMI 142717, was fractionated by Bio-Gel P-2 size exclusion chromatography at 60°C. Column fractions, corresponding to oligosaccharides with degrees of polymerisation from 5 to 10, were collected, and subfractionated by high performance anion-exchange chromatography on CarboPac PA-1. The structures of the oligosaccharides thus obtained were elucidated by 1H NMR spectroscopy, showing chains of (1→4)-linked β-D-xylopyranosyl residues differently substituted at O-3 and / or O-2,3 with α-L-arabinofuranosyl groups. The structures were different from those obtained with endo-(1→4)-β-D-xylanase I of the same xylanolytic enzyme system.Hide Abstract
Hoffmann, R. A., Leeflang, B. R., de Barse, M. M., Kamerling, J. P. & Vliegenthart, J. F. (1991). Carbohydrate Research, 221, 63-81.
Characterisation by 1H-n.m.r. spectroscopy of oligosaccharides, derived from arabinoxylans of white endosperm of wheat, that contain the elements ----4)[alpha-L-Araf-(1----3)]-beta-D-Xylp-(1---- or ----4)[alpha- L-Araf-(1----2)][alpha-L-Araf-(1----3)]-beta-D-Xylp-(1----. The structure of penta- to hepta-saccharides, generated by digestion of purified wheat-endosperm arabinoxylan with endo-(1----4)-beta-D-xylanase and isolated by gel-permeation chromatography on Bio-Gel P-6 followed by high-performance anion-exchange chromatography with pulsed amperometric detection, was established using monosaccharide and methylation analysis, f.a.b.-m.s., and 1H-n.m.r. spectroscopy. The oligosaccharides had a core of (1----4)-linked beta-D-xylopyranosyl residues 3- or 2,3-substituted with single alpha-L-arabinofuranosyl groups, and gave 1H-n.m.r. spectra typical for each type.Hide Abstract