Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.
Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.
endo-1,4-β-Xylanase (EC 126.96.36.199) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.
Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-L-arabinofuranosidase and β-xylosidase.
McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Carbohydrate Research, 407, 79-96.
A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 32-α-L-Araf-(1-4)-β-D-xylobiose (A3X), 23-α-L-Araf-(1-4)-β-D-xylotriose (A2XX), 33-α-L-Araf-(1-4)-β-D-xylotriose (A3XX), 22-α-L-Araf-(1-4)-β-D-xylotriose (XA2X), 32-α-L-Araf (1-4)-β-D-xylotriose (XA3X), 23-α-L-Araf-(1-4)-β-D-xylotetraose (XA2XX), 33-α-L-Araf-(1-4)-β-D-xylotetraose (XA3XX), 23 ,33-di-α-L-Araf-(1-4)-β-D-xylotriose (A2+3XX), 23,33-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA2+3XX), 24,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA2+3XXX) and 33,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA3A3XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.
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.
Water extract of Triticum aestivum L. and its components demonstrate protective effect in a model of vascular dementia.
Han, H. S., Jang, J. H., Jang, J. H., Choi, J. S., Kim, Y. J., Lee, C., Lim, S. H., Lee, H. K. & Lee, J. (2010). Journal of Medicinal Food, 13(3), 572-578.
Although vascular dementia is the second leading cause of dementia and often underdiagnosed, there are no drugs yet approved for the treatment of vascular dementia. In this study, it is demonstrated that water extract of Triticum aestivum L. (TALE) and some of its components have protective effects against vascular dementia-induced damage by preserving the myelin sheath and inhibiting astrocytic activation. The memory test used a vascular dementia model utilizing bilateral ligation of the carotid arteries of rats. TALE, some of its components, such as starch, total dietary fiber (TDF), arabinoxylan, β-glucan, and degraded products of arabinoxylan, such as arabinose and xylose, were administered to the animals from day 8 to day 14, following the surgery. Twenty-one days after the surgery, the water maze test was performed for 5 days, and the time taken to find the platform during training trials (mean escape latency) was measured. The mean escape latency was decreased consistently in the TALE-, starch-, TDF-, arabinoxylan-, and arabinose-treated groups, compared with that in the vascular dementia group. To measure brain damage, Luxol fast blue staining and immunohistochemistry of myelin basic protein (MBP) were performed to observe myelin sheath in the white matter, and immunohistochemistry of glial fibrillary acidic protein (GFAP) was performed to observe the astrocytic reaction. Vascular dementia reduced the MBP level and increased the GFAP level. Arabinose effectively inhibited the MBP and GFAP change, whereas arabinoxylan inhibited the GFAP change only. These results suggest that TALE and some of its components can be used as a medicinal material for the development of neuroprotective agents against vascular dementia.
Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species.
Koutaniemi, S., Guillon, F., Tranquet, O., Bouchet, B., Tuomainen, P., Virkki, L., Petersen, H. L., Willats, W. G. T., Saulnier, L. & Tenkanen, M. (2012). Planta, 236(2), 739-751.
Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituents present in various xylans. We have produced a monoclonal antibody which specifically recognizes glucopyranosyl uronic acid (GlcA), or its 4-O-methyl ether (meGlcA), substituents in xylan and has no cross-reactivity with linear or arabinofuranosyl-substituted xylans. The UX1 antibody binds most strongly to (me)GlcA substitutions at the non-reducing ends of xylan chains, but has a low cross-reactivity with internal substitutions as well, at least on oligosaccharides. The antibody labeled plant cell walls from both mono- and dicotyledons, but in most tissues an alkaline pretreatment was needed for antibody binding. The treatment removed acetyl groups from xylan, indicating that the vicinity of glucuronic acid substituents is also acetylated. The novel labeling patterns observed in the xylem of tree species suggested that differences within the cell wall exist both in acetylation degree and in glucuronic acid content.
Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis.
Sakka, M., Tachino, S., Katsuzaki, H., van Dyk, J. S., Pletschke, B. I., Kimura, T. & Sakka, K. (2012). Enzyme and Microbial Technology, 51(4), 193-199.
The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. Subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-β-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-D-glucurono-D-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-L-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.
Understanding how noncatalytic carbohydrate binding modules can display specificity for xyloglucan.
Luís, A. S., Venditto, I., Temple, M. J., Rogowski, A., Baslé, A., Xue, J., Knox, J. P., Prates, J. A. M., Ferreira, L. M. A., Fontes, C. M. G. A., Najmudin, S. & Gilbert, H. J. (2013). Journal of Biological Chemistry, 288(7), 4799-4809.
Plant biomass is central to the carbon cycle and to environmentally sustainable industries exemplified by the biofuel sector. Plant cell wall degrading enzymes generally contain noncatalytic carbohydrate binding modules (CBMs) that fulfil a targeting function, which enhances catalysis. CBMs that bind β-glucan chains often display broad specificity recognizing β1,4-glucans (cellulose), β1,3-β1,4-mixed linked glucans and xyloglucan, a β1,4-glucan decorated with α-1,6-xylose residues, by targeting structures common to the three polysaccharides. Thus, CBMs that recognize xyloglucan target the β1,4-glucan backbone and only accommodate the xylose decorations. Here we show that two closely related CBMs, CBM65A and CBM65B, derived from EcCel5A, a Eubacterium cellulosolvens endoglucanase, bind to a range of β-glucans but, uniquely, display significant preference for xyloglucan. The structures of the two CBMs reveal a β-sandwich fold. The ligand binding site comprises the β-sheet that forms the concave surface of the proteins. Binding to the backbone chains of β-glucans is mediated primarily by five aromatic residues that also make hydrophobic interactions with the xylose side chains of xyloglucan, conferring the distinctive specificity of the CBMs for the decorated polysaccharide. Significantly, and in contrast to other CBMs that recognize β-glucans, CBM65A utilizes different polar residues to bind cellulose and mixed linked glucans. Thus, Gln106 is central to cellulose recognition, but is not required for binding to mixed linked glucans. This report reveals the mechanism by which β-glucan-specific CBMs can distinguish between linear and mixed linked glucans, and show how these CBMs can exploit an extensive hydrophobic platform to target the side chains of decorated β-glucans.
Peroxidase-mediated oxidative cross-linking and its potential to modify mechanical properties in water-soluble polysaccharide extracts and cereal grain residues.
Robertson, J. A., Faulds, C. B., Smith, A. C. & Waldron, K. W. (2008). Journal of Agricultural and Food Chemistry, 56(5), 1720-1726.
Analysis of wheat bran and spent grain shows that concentrations of ferulate and diferulates offer considerable scope to modify the cross-linking of feruloylated polysaccharides and hence the mechanical properties of these residues. In solution ferulic acid (FA) can be readily polymerized by horseradish peroxidase, but when esterified to a polysaccharide, the profile of diferulates becomes restricted. This situation also exists in muro and suggests structural constraints may limit the availability of FA for cross-linking. At relatively low polysaccharide concentration, (~3%), steric restrictions were apparent in gels prepared using isolated polysaccharides. Mechanical properties such as swelling also appear to be fixed at these relatively low polysaccharide concentrations. This limits the potential to modify mechanical properties in muro through oxidoreductase activity. To modify mechanical properties such treatments will need to focus on increasing the “flexibility” of the cell wall matrix and the accessibility of enzymes to the cell wall matrix.
A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14.
Zhou, J., Wu, Q., Zhang, R., Mo, M., Tang, X., Li, J., Xu, B., Ding, J., Lu, Q. & Huang, Z. (2014). Folia Microbiologica, 59(5), 423-431.
A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52% amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5°C and pH 6.5 and retained more than 55% of the maximum activity when assayed at 40–75°C, 23% at 20°C, 16% at 85°C, and even 8% at 0°C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80°C, respectively. The enzyme exhibited more than 62% xylanase activity and stability at the concentration of 3–30% (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37°C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 µmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.
In vitro fermentation kinetics and end-products of cereal arabinoxylans and (1,3;1,4)-β-glucans by porcine faeces.
Williams, B. A., Mikkelsen, D., Le Paih, L. & Gidley, M. J. (2011). Journal of cereal science, 53(1), 53-58.
Purified and semi-purified polysaccharides characteristic of cereals were fermented in vitro with a pig faecal inoculum, using the cumulative gas production technique, to examine the kinetics and end-products of fermentation after 48 h. It was shown that arabinoxylan and mixed linkage (1,3;1,4) β-glucan were rapidly fermented if soluble, while less soluble substrates (insoluble arabinoxylan, maize and wheat starch granules, and bacterial cellulose) were more slowly fermented. Relevant monosaccharides were fermented at very similar rates to soluble polymeric arabinoxylan and β-glucan, showing that depolymerisation was not a limiting step, in contrast to some previous studies. Bacterial cellulose is shown to be a useful model substrate for fermentation of plant cellulose which is difficult to obtain without harsh chemical treatments. Fermentation end-products were related to kinetics, with slow carbohydrate fermentation resulting in increased protein fermentation. Ratios of short-chain fatty acid products were similar for all arabinoxylan and β-glucan substrates.
The use of β-xylanase for increasing the efficiency of biocatalytic conversion of crop residues to bioethanol.
Juodeikiene, G., Basinskiene, L., Vidmantiene, D., Makaravicius, T., Bartkiene, E. & Schols, H. (2011). Catalysis Today, 167(1), 113-121.
Proteinaceous inhibitors of xylanase naturally occur in cereals where they are involved in various roles in the plant defence metabolism. This study focused on the inhibitors of xylanase present in local rye cultivars, and their influence on the efficiency of the fermentation processes during bioethanol production from rye residues in comparison with common wheat. Different origin xylanases from Thermomyces lanuginosus and Trichoderma reesei were the objects of the investigations. Kinetic studies of these xylanases in the presence of proteins with inhibitory activity indicated that Th. lanuginosus was found more sensitive to proteinaceous xylanase inhibitors presented in rye than T. reesei. The highest yield of xylose and arabinose was achieved by adding T. reesei to cell wall substrates, while Th. lanuginosus converted to arabinoxylans only into xylooligosaccharides and monosaccharide were not released. The activity of xylanase in composition with α-amylase and glucoamylase was selected to achieve a higher ethanol yield in the distillate. It improved the quality of bioethanol by increasing the content of ethanol and decreasing the concentrations of propanol, isobutanol, isoamyl and amyl alcohols and the methanol concentration. No significant differences were found between the contents of ethanol from different type of bran.
Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome.
Sun, J., Wen, F., Si, T., Xu, J. H. & Zhao, H. (2012). Applied and Environmental Microbiology, 78(11), 3837-3845.
Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant Saccharomyces cerevisiae strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/CipA1) and up to three chimeric enzymes. The miniscaffoldin derived from Clostridium thermocellum contained one or three cohesin modules and was tethered to the cell surface through the S. cerevisiae a-agglutinin adhesion receptor. Up to three types of hemicellulases, an endoxylanase (XynII), an arabinofuranosidase (AbfB), and a β-xylosidase (XlnD), each bearing a C-terminal dockerin, were assembled onto the miniscaffoldin by high-affinity cohesin-dockerin interactions. Compared to uni- and bifunctional minihemicellulosomes, the resulting quaternary trifunctional complexes exhibited an enhanced rate of hydrolysis of arabinoxylan. Furthermore, with an integrated D-xylose-utilizing pathway, the recombinant yeast displaying the bifunctional minihemicellulosome CipA3-XynII-XlnD could simultaneously hydrolyze and ferment birchwood xylan to ethanol with a yield of 0.31 g per g of sugar consumed.
Adsorption of arabinoxylan on cellulosic surfaces: influence of degree of substitution and substitution pattern on adsorption characteristics.
Köhnke, T., Östlund, Å. & Brelid, H. (2011). Biomacromolecules, 12(7), 2633-2641.
This study presents results that show that the fine structure of arabinoxylan affects its interaction with cellulosic surfaces, an important understanding when designing and evaluating properties of xylan–cellulose-based materials. Arabinoxylan samples, with well-defined structures, were prepared from a wheat flour arabinoxylan with targeted enzymatic hydrolysis. Turbidity measurements and analyses using NMR diffusometry showed that the solubility and the hydrodynamic properties of arabinoxylan are determined not only by the degree of substitution but also by the substitution pattern. On the basis of results obtained from adsorption experiments on microcrystalline cellulose particles and on cellulosic model surfaces investigated with quartz crystal microbalance with dissipation monitoring, it was also found that arabinoxylan adsorbs irreversibly on cellulosic surfaces and that the adsorption characteristics, as well as the properties of the adsorbed layer, are controlled by the fine structure of the xylan molecule.
Binding selectivity of dietary polyphenols to different plant cell wall components: quantification and mechanism.
Phan, A. D. T., Flanagan, B. M., D'Arcy, B. R. & Gidley, M. J. (2017). Food Chemistry, 233, 216-227.
Selected polyphenols exhibited binding selectivity to different cellulose-based composites and apple cell walls. For catechin, cellulose is the dominant binding component, whereas hemicelluloses (xyloglucan and arabinoxylan) apparently did not contribute to polyphenol adsorption in the presence of cellulose. In contrast, ferulic acid and cyanidin-3-glucoside bound to cellulose-based composites and apple cell walls with different affinities, showing that both electrostatic interactions and plant cell wall microstructure were important. Negatively-charged pectin-containing cell walls exhibited the most extensive binding of positively-charged cyanidin-3-glucoside, and bound negatively-charged ferulic acid least effectively. Langmuir binding isotherms predicted the maximum amount of adsorbed polyphenols to be in the range of 30–150% plant cell wall mass. NMR and CLSM analysis support the interactions between polyphenols and plant cell walls and show that although polyphenols are associated with plant cell walls under hydrated conditions, they are not immobilised on polymer surfaces.
Effect of Water-Extractable Arabinoxylans from Wheat Aleurone and Bran on Lipid Peroxidation and Factors Influencing their Antioxidant Capacity.
Malunga, L. N., Izydorczyk, M. & Beta, T. (2017). Bioactive Carbohydrates and Dietary Fibre, In Press.
Dietary lipid hydroperoxides (LOOH) are implicated in the rise of colon cancers. We investigated the effect of water-extractable arabinoxylans (WEAX) on lipid peroxidation under simulated gastric conditions. Water-extractable fractions containing mostly arabinoxylans were isolated from wheat aleurone and wheat bran and fractionated by stepwise precipitation with (NH4)2SO4 at 50 and 75% saturation. LOOH concentration (121.2 ± 3.8 µM/g) of grilled chicken breast muscle increased by 30%; however, it remained unaltered or decreased (≤35%) depending on type and concentration of WEAX fractions. Antioxidant capacity of WEAX fractions based on DPPH, ABTS and ORAC was 28.2 – 147.9, 91.2 – 355.3, and 185.9 – 527.5 µM TE/g, respectively. The content of ferulic acid residues in WEAX fractions (R = 0.99) and relative proportions of monosubstituted xylose residues (R = 0.80) influenced the antioxidant capacity. Consumption of diets rich in feruloylated WEAX may offer protection against oxidative damage in the gastrointestinal tract.
Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.
van Munster, J. M., Thomas, B., Riese, M., Davis, A. L., Gray, C. J., Archer, D. B. & Flitsch, S. L. (2017). Scientific Reports, 7.
Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.