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.
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.
Chromatographic determination of 1,4-β-xylooligosaccharides of different chain lengths to follow xylan deconstruction in biomass conversion.
Li, H., Qing, Q., Kumar, R. & Wyman, C. E. (2013). Journal of Industrial Microbiology & Biotechnology, 40(6), 551-559.
Xylooligosaccharides released in hydrothermal pretreatment of lignocellulosic biomass can be purified for high-value products or further hydrolyzed into sugars for fermentation or chemical conversion. In addition, characterization of xylooligosaccharides is vital to understand hemicellulose structure and removal mechanisms in pretreatment of cellulosic biomass. In this study, gel permeation chromatography was applied to fractionate xylooligosaccharides produced from birchwood xylan according to their specific degree of polymerization (DP). Then, each fraction was identified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF–MS); and their concentrations were determined by a downscaled post-hydrolysis method. Based on PAD responses and sugar concentrations for each fraction, a series of response factors were developed that can be used to quantify xylooligosaccharides of DP from 2 to 14 without standards. The resulting approach can profile xylooligosaccharides and help gain new insights into biomass deconstruction.
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.
Cellulose microfibril angles and cell-wall polymers in different wood types of Pinus radiata.
Brennan, M., McLean, J. P., Altaner, C. M., Ralph, J. & Harris, P. J. (2012). Cellulose, 19(4), 1385-1404.
Four corewood types were examined from sapling trees of two clones of Pinus radiata grown in a glasshouse. Trees were grown either straight to produce normal corewood, tilted at 45° from the vertical to produce opposite corewood and compression corewood, or rocked to produce flexure corewood. Mean cellulose microfibril angle of tracheid walls was estimated by X-ray diffraction and longitudinal swelling measured between an oven dry and moisture saturated state. Lignin and acetyl contents of the woods were measured and the monosaccharide compositions of the cell-wall polysaccharides determined. Finely milled wood was analysed using solution-state 2D NMR spectroscopy of gels from finely milled wood in DMSO-d6/pyridine-d5. Although there was no significant difference in cellulose microfibril angle among the corewood types, compression corewood had the highest longitudinal swelling. A lignin content >32% and a galactosyl residue content >6% clearly divided severe compression corewood from the other corewood types. Relationships could be drawn between lignin content and longitudinal swelling, and between galactosyl residue content and longitudinal swelling. The 2D NMR spectra showed that the presence of H-units in lignin was exclusive to compression corewood, which also had a higher (1→4)- β-D-galactan content, defining a unique composition for that corewood type.
Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus.
Zhang, J. & Viikari, L. (2012). Bioresource Technology, 117, 286-291.
The effects of xylo-oligosaccharides (XOS) and xylose on the hydrolytic activities of cellulases, endoglucanase II (EGII, originating from Thermoascus aurantiacus), cellobiohydrolase I (CBHI, from T. aurantiacus), and cellobiohydrolase II (CBHII, from Trichoderma reesei) on Avicel and nanocellulose were investigated. After the addition of XOS, the amounts of cellobiose, the main product released from Avicel and nanocellulose by CBHI, decreased from 0.78 and 1.37 mg/ml to 0.59 and 1.23 mg/ml, respectively. During hydrolysis by CBHII, the amounts of cellobiose released from the substrates were almost cut in half after the addition of XOS. Kinetic experiments showed that xylobiose and xylotriose were competitive inhibitors of CBHI. The results revealed that the strong inhibition of cellulase by XOS can be attributed to the inhibitory effect of XOS especially on cellobiohydrolase I. The results indicate the necessity to totally hydrolyze xylo-oligosaccharides into the less inhibitory product, xylose, to increasing hydrolytic efficiency.
Cloning, expression and characterization of a glycoside hydrolase family 39 xylosidase from Bacillus halodurans C-125.
Wagschal, K., Franqui-Espiet, D., Lee, C. C., Robertson, G. H. & Wong, D. W. (2008). Biotechnology for Fuels and Chemicals, 146(1-3), 69-78.
The gene encoding a glycoside hydrolase family 39 xylosidase (BH1068) from the alkaliphile Bacillus halodurans strain C-125 was cloned with a C-terminal His-tag, and the recombinant gene product termed BH1068(His)6 was expressed in Escherichia coli. Of the artificial substrates tested, BH1068(His)6 hydrolyzed nitrophenyl derivatives of β-D-xylopyranose, α-L-arabinofuranose, and α-L-arabinopyranose. Deviation from Michaelis-Menten kinetics at higher substrate concentrations indicative of transglycosylation was observed, and Kcat and Km values were measured at both low and high substrate concentrations to illuminate the relative propensities to proceed along this alternate reaction pathway. The pH maximum was 6.5, and under the conditions tested, maximal activity was at 47°C, and thermal instability occurred above 45°C. BH1068(His)6 was inactive on arabinan, hydrolyzed xylooligosaccharides, and released only xylose from oat, wheat, rye, beech, and birch arabinoxylan, and thus, can be classified as a xylosidase with respect to natural substrate specificity. The enzyme was not inhibited by up to 200 mM xylose. The oligomerization state was tetrameric under the size-exclusion chromatography conditions employed.
Purification and characterization of a thermophilic xylanase from the brown-rot fungus Gloeophyllum trabeum.
Ritschkoff, A. C., Buchert, J. & Viikari, L. (1994). Journal of Biotechnology, 32(1), 67-74.
A xylanase produced by the brown-rot fungus, Gloeophyllum trabeum, was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration. The enzyme had an isoelectric point of 5.0 and molecular mass of 39–42 kDa, respectively. The xylanase appeared to prefer the most substituted glucurono-xylan (DMSO-xylan) as substrate and exhibited a pH optimum of 4.0 and a temperature optimum of 80°C after 30 min incubation. Approximately 22% of the activity remained after 2 h incubation at 70°C and the half-life of xylanase at 60°C was 24 h. The xylanase also showed β-glucanase activity with barley β-glucan as substrate as side activity. The xylanase of G. trabeum was very tolerant to inhibitors. Among the various inhibitors studied, only 10 mM AlCl3 was found to inhibit the xylanase activity.
Purification and characterization of a neutral endoxylanase from Aspergillus nidulans.
Fernández-Espinar, M. T., Piñaga, F., Sanz, P., Ramón, D. & Vallés, S. (1993). FEMS Microbiology Letters, 113(2), 223-228.
A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a Km of 4.2 mg oat spelt xylan per ml and a Vmax of 710 μmol min-1 (mg protein) -1.
Inverting character of family GH115 α-glucuronidases.
Kolenová, K., Ryabova, O., Vršanská, M. & Biely, P. (2010). FEBS Letters, 584(18), 4063-4068.
α-Glucuronidases of glycoside hydrolase family 115 of the xylose-fermenting yeast Pichia stipitis and wood-destroying fungus Schizophyllum commune liberate 4-O-methyl-D-glucuronic acid residues from aldouronic acids and glucuronoxylan. The specific activities of both enzymes depended on polymerization degree of the acidic xylooligosaccharides and were inhibited by linear β-1,4-xylooligosaccharides. These results suggest interaction of the enzyme with several xylopyranosyl residues of the xylan main chain. Using 1H NMR spectroscopy and reduced aldopentaouronic acid (MeGlcA3Xyl4-ol) as a substrate, it was found that both enzymes are inverting glycoside hydrolases releasing 4-O-methyl-D-glucuronic acid (MeGlcA) as its β-anomer.
Crystallographic analysis shows substrate binding at the -3 to+ 1 active-site subsites and at the surface of glycoside hydrolase family 11 endo-1,4-β-xylanases.
Vandermarliere, E., Bourgois, T. M., Rombouts, S., Van Campenhout, S., Volckaert, G., Strelkov, S. V., Delcour, J. A., Rabijns, A. & Courtin, C. (2008). Biochem. J, 410, 71-79.
GH 11 (glycoside hydrolase family 11) xylanases are predominant enzymes in the hydrolysis of heteroxylan, an abundant structural polysaccharide in the plant cell wall. To gain more insight into the protein–ligand interactions of the glycone as well as the aglycone subsites of these enzymes, catalytically incompetent mutants of the Bacillus subtilis and Aspergillus niger xylanases were crystallized, soaked with xylo-oligosaccharides and subjected to X-ray analysis. For both xylanases, there was clear density for xylose residues in the -1 and -2 subsites. In addition, for the B. subtilis xylanase, there was also density for xylose residues in the -3 and +1 subsite showing the spanning of the -1/+1 subsites. These results, together with the observation that some residues in the aglycone subsites clearly adopt a different conformation upon substrate binding, allowed us to identify the residues important for substrate binding in the aglycone subsites. In addition to substrate binding in the active site of the enzymes, the existence of an unproductive second ligand-binding site located on the surface of both the B. subtilis and A. niger xylanases was observed. This extra binding site may have a function similar to the separate carbohydrate-binding modules of other glycoside hydrolase families.
Purification and regulation of the synthesis of a β-xylosidase from Aspergillus nidulans.
Kumar, S. & Ramón, D. (1996). FEMS Microbiology Letters, 135(2‐3), 287-293.
β-xylosidase (EC 18.104.22.168) has been purified from Aspergillus nidulans mycelium grown on oat-spelt xylan as sole carbon source. Its pH optimum for activity was found to be 5.0 and the optimum temperature was 50°C. Its molecular mass was estimated by gel filtration to be 180000. Using p-nitrophenyl-β-D-xylopyranoside as substrate, the Km and Vmax values have been found to be 1.1 mM and 25.6 μmol min-1(mg protein) -1, respectively. Enzyme activity was inhibited by Hg2+, Ag2+, and Cu2+ at a concentration of 1 × 10-3 M. The synthesis of β-xylosidase in A. nidulans is strongly induced by arabinose and xylose and is subject to carbon catabolite repression mediated by the creA gene product.
Preparation of arabinoxylobiose from rye xylan using family 10 Aspergillus aculeatus endo-1, 4-β-D-xylanase.
Rantanen, H., Virkki, L., Tuomainen, P., Kabel, M., Schols, H. & Tenkanen, M. (2007). Carbohydrate Polymers, 68(2), 350-359.
Commercial xylanase preparation Shearzyme®, which contains the glycoside hydrolase family 10 endo-1,4-β-D-xylanase from Aspergillus aculeatus, was used to prepare short-chain arabinoxylo-oligosaccharides (AXOS) from rye arabinoxylan (AX). A major AXOS was formed as a hydrolysis product. Longer AXOS were also produced as minor products. The pure GH10 xylanase from A. aculeatus was used as a comparison to ensure that the formed AXOS were consequence of the endoxylanase‘s function instead of some side enzymes present in Shearzyme. The major AXOS was purified and the structure confirmed with various analysis methods (TLC, HPAEC-PAD, MALDI-TOF-MS, and one- and two-dimensional NMR spectroscopy with nano-probe) as α-L-Araf-(1→3)- β-D-Xylp-(1→4)-D-Xylp (arabinoxylobiose). This is the first report on 13C NMR data of pure arabinoxylobiose. The yield of arabinoxylobiose was 12% from the quantified hydrolysis products. In conclusion, GH10 endoxylanase from A. aculeatus is thus able to cut efficiently the xylosidic linkage next to the arabinofuranosyl-substituted xylose unit which is not typical for all the GH10 endoxylanases. Interestingly, pure A. aculeatus xylanase showed notably activity towards p-nitrophenyl-β-D-xylopyranose. In previously studies longer AXOS have been produced with Shearzyme but the formation of short-chain AXOS by A. aculeatus GH10 xylanase has not been studied before.