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.
Enzyme structure dynamics of xylanase I from Trichoderma longibrachiatum.
Uzuner, U., Shi, W., Liu, L., Liu, S., Dai, S. Y. & Yuan, J. S. (2010). BMC Bioinformatics, 11(Suppl 6), S12.
Background: Enzyme dynamics has recently been shown to be crucial for structure-function relationship. Among various structure dynamics analysis platforms, HDX (hydrogen deuterium exchange) mass spectrometry stands out as an efficient and high-throughput way to analyze protein dynamics upon ligand binding. Despite the potential, limited research has employed the HDX mass spec platform to probe regional structure dynamics of enzymes. In particular, the technique has never been used for analyzing cell wall degrading enzymes. We hereby used xylanase as a model to explore the potential of HDX mass spectrometry for studying cell wall degrading enzymes. Results: HDX mass spectrometry revealed significant intrinsic dynamics for the xylanase enzyme. Different regions of the enzymes are differentially stabilized in the apo enzyme. The comparison of substrate-binding enzymes revealed that xylohexaose can significantly stabilize the enzyme. Several regions including those near the reaction centres were significantly stabilized during the xylohexaose binding. As compared to xylohexaose, xylan induced relatively less protection in the enzyme, which may be due to the insolubility of the substrate. The structure relevance of the enzyme dynamics was discussed with reference to the three dimensional structure of the enzyme. HDX mass spectrometry revealed strong dynamics-function relevance and such relevance can be explored for the future enzyme improvement. Conclusion: Ligand-binding can lead to the significant stabilization at both regional and global level for enzymes like xylanase. HDX mass spectrometry is a powerful high-throughput platform to identify the key regions protected during the ligand binding and to explore the molecular mechanisms of the enzyme function. The HDX mass spectrometry analysis of cell wall degrading enzymes has provided a novel platform to guide the rational design of enzymes.
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.
Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1.
Santos, C. R., Meza, A. N., Hoffmam, Z. B., Silva, J. C., Alvarez, T. M., Ruller, R., Giesel, G. M., Verli, H., Squina, F. M., Prade, R. A. & Murakami, M. T. (2010). Biochemical and Biophysical Research Communications, 403(2), 214-219.
Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the −1 and −2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20°C, and exclusively xylobiose at 90°C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.
Glycosynthase Activity of Geobacillus stearothermophilus GH52 β-Xylosidase: Efficient Synthesis of Xylooligosaccharides from α-D‐Xylopyranosyl Fluoride through a Conjugated Reaction.
Ben‐David, A., Bravman, T., Balazs, Y. S., Czjzek, M., Schomburg, D., Shoham, G. & Shoham, Y. (2007). ChemBioChem, 8(17), 2145-2151.
Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 β-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using α-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of α-D-xylopyranosyl fluoride, providing mainly α-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis–Menten behavior, with kinetic constants of 1.3 s-1 and 2.2 mm for Kcat and KM(acceptor)
, respectively, and a Kcat/KM(acceptor)
value of 0.59 s-1 mm-1. When the β-xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable α-D-xylopyranosyl fluoride as the sole substrate.
An attempt to identify the low molecular feruloylated oligosaccharides in beer.
Szwajgier, D., Waśko, A., Zapp, J. & Targoński, Z. (2007). Journal of the Institute of Brewing, 113(2), 185-195.
Ferulic acid (4-hydroxy-3-methoxycinnamic acid), predominantly in ester form in arabinoxylan chains, is the main phenolic acid present in barley and malt. Only about 1% of the total ferulic acid in barley is present in the free form. A number of previous works concerned the contents of free and esterified ferulic acid in a broad range of popular beers, but there is little information about the possible composition of feruloylated oligosaccharides in beers. The aim of this preliminary work was to purify the feruloylated oligosaccharides from lager beers (by the means of preparative chromatography) followed by composition elucidation using TLC, HPLC with RI or UV detection and 1H-NMR. Indeed, the qualitative analyses of isolated fractions from beer revealed that the fractions contained ferulic acid in the ester form (as proven after mild alkaline hydrolysis). It was also shown that molecular masses of oligosaccharides present in the purified beer fractions were similar to the masses of arabinose and xylooligosaccharides in the range of xylose to xylohexaose. Although a number of purified beer samples contained oligosaccharides of higher molecular masses, these were not further characterized. Taking under consideration the presented results, it can be concluded that beer can be a good source of feruloylated oligosaccharides, significant in the context of human health benefits.
Simultaneous production of xylooligosaccharides and antioxidant compounds from sugarcane bagasse via enzymatic hydrolysis.
Mandelli, F., Brenelli, L. B., Almeida, R. F., Goldbeck, R., Wolf, L. D., Hoffmam, Z. B., Ruller, R., Rocha, G. J. M., Mercadante, A. Z. & Squina, F. M. (2014). Industrial Crops and Products, 52, 770-775.
Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial residues such as sugarcane bagasse, which is the major by-product of the sugarcane industry. Due to its abundant availability and despite the complex chemical composition, it can be considered an ideal substrate for microbial processes for the production of value-added products. In the present study we evaluated the enzymatic production of xylooligosaccharides (XOS) and antioxidant compounds from sugarcane bagasse using XynZ from Clostridium thermocellum, a naturally chimeric enzyme comprising activities of xylanase and feruloyl esterase along with a carbohydrate binding module (CBM6). In order to reveal the biotechnological potential of XynZ, the XOS released after enzymatic hydrolysis using different substrates were characterized by capillary electrophoresis and quantified by high performance anion exchange chromatography. In parallel, the antioxidant capacity related to the release of phenolic compounds was also determined. The results indicated noteworthy differences regarding the amount of XOS and antioxidant phenolic compounds produced, as well as the XOS profile, functions of the pre-treatment method employed. The ability of XynZ to simultaneously produce xylooligosaccharides, natural probiotics, phenolic compounds and antioxidant molecules from natural substrates such as sugarcane bagasse demonstrated the biotechnological potential of this enzyme. Production of value-added products from agro-industrial residues is of great interest not only for advancement in the biofuel field, but also for pharmaceutical and food industries.
Combined transcriptome and proteome analysis of Bifidobacterium animalis subsp. lactis BB-12 grown on xylo-oligosaccharides and a model of their utilization.
Gilad, O., Jacobsen, S., Stuer-Lauridsen, B., Pedersen, M. B., Garrigues, C. & Svensson, B. (2010). Applied and Environmental Microbiology, 76(21), 7285-7291.
Recent studies have demonstrated that xylo-oligosaccharides (XOS), which are classified as emerging prebiotics, selectively enhance the growth of bifidobacteria in general and of Bifidobacterium animalis subsp. lactis strains in particular. To elucidate the metabolism of XOS in the well-documented and widely used probiotic strain B. animalis subsp. lactis BB-12, a combined proteomic and transcriptomic approach was applied, involving DNA microarrays, real-time quantitative PCR (qPCR), and two-dimensional difference gel electrophoresis (2D-DIGE) analyses of samples obtained from cultures grown on either XOS or glucose. The analyses show that 9 of the 10 genes that encode proteins predicted to play a role in XOS catabolism (i.e., XOS-degrading and -metabolizing enzymes, transport proteins, and a regulatory protein) were induced by XOS at the transcriptional level, and the proteins encoded by three of these (β-D-xylosidase, sugar-binding protein, and xylose isomerase) showed higher abundance on XOS. Based on the obtained results, a model for the catabolism of XOS in BB-12 is suggested, according to which the strain utilizes an ABC (ATP-binding cassette) transport system (probably for oligosaccharides) to bind XOS on the cell surface and transport them into the cell. XOS are then degraded intracellularly through the action of xylanases and xylosidases to D-xylose, which is subsequently metabolized by the D-fructose-6-P shunt. The findings obtained in this study may have implications for the design of a synbiotic application containing BB-12 and the XOS used in the present study.
Three members of the Arabidopsis glycosyltransferase family 8 are xylan glucuronosyltransferases.
Rennie, E. A., Hansen, S. F., Baidoo, E. E. K., Hadi, M. Z., Keasling, J. D. & Scheller, H. V. (2012). Plant Physiology, 159(4), 1408-1417.
Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell walls that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of β(1,4)-linked xylose residues substituted with α(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a Km for UDP-GlcA of 165 µM. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan α-glucuronosyltransferases.
Capillary electrophoresis with detection by laser-induced fluorescence.
Mort, A. & Wu, X. (2011). The Plant Cell Wall Methods in Molecular Biology, 715(1), 93-102.
The importance of capillary zone electrophoresis (CZE) has been increasing in use for: structural analysis of plant cell walls, characterization of enzymes that degrade polysaccharides, and profiling of oligosaccharides to characterize cell wall mutants. CZE with laser-induced fluorescence detection provides high separation efficiencies, high speed analysis, with extremely small sample requirements. Here, we describe the instrumentation we use and the methods for attaching fluorescent labels to oligosaccharides so that they can be detected.
Enzyme-aided alkaline extraction of oligosaccharides and polymeric xylan from hardwood kraft pulp.
Hakala, T. K., Liitiä, T. & Suurnäkki, A. (2013). Carbohydrate Polymers, 93(1), 102-108.
In this paper we describe the effect of enzyme treatments on the production of polymeric xylan, oligosaccharides and hemicellulose lean pulp by alkaline extraction of bleached hardwood kraft pulp. Enzyme treatments were carried out before one or in between two subsequent alkaline extractions by purified Trichoderma reesei xylanase and endoglucanase II (Cel 5a) as well as by a commercial monocomponent endoglucanase (FibreCareR). Without enzyme pre-treatment 61% and 7% of the pulp xylan was extracted in high purity in the first and second alkaline stage, respectively. Higher molecular mass xylan was obtained in the second than in the first alkaline extraction. Xylanase treatment before alkaline extraction hydrolyzed up to 12% of xylan to xylooligosaccharides. According to our results, preparation of polymeric xylan, and/or oligosaccharides as well as hemicellulose lean pulp with cellulose content of 93–94%, is possible by enzyme-aided alkaline extraction process.
Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-β-D-xylanase 10B in complex with xylohexaose.
Najmudin, S., Pinheiro, B. A., Romao, M. J., Prates, J. A. M. & Fontes, C. M. G. A. (2008). Acta Crystallogr Section F: Structural Biology and Crystallization Communications, 64(8), 715-718.
The cellulosome of Clostridium thermocellum is a highly organized multi-enzyme complex of cellulases and hemicellulases involved in the hydrolysis of plant cell-wall polysaccharides. The bifunctional multi-modular xylanase Xyn10B is one of the hemicellulase components of the C. thermocellum cellulosome. The enzyme contains an internal glycoside hydrolase family 10 catalytic domain (GH10) and a C-terminal family 1 carbohydrate esterase domain (CE1). The N-terminal moiety of Xyn10B (residues 32-551), comprising a carbohydrate-binding module (CBM22-1) and the GH10 E337A mutant, was crystallized in complex with xylohexaose. The crystals belong to the trigonal space group P3221 and contain a dimer in the asymmetric unit. The crystals diffracted to beyond 2.0 Å resolution.