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.
Oligosaccharides obtained by enzymatic hydrolysis of birch kraft pulp xylan: analysis by capillary zone electrophoresis and mass spectrometry.
Rydlund, A. & Dahlman, O. (1997). Carbohydrate Research, 300(2), 95-102.
Neutral and acidic oligosaccharides were obtained from an unbleached birch kraft pulp by treatment with a Trichoderma reesei endoxylanase pI 9 and subsequently characterized using capillary zone electrophoresis (CZE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The borate complexes of unsaturated acidic oligosaccharides having a 4-deoxy-β-L-threo-hex-4-enopyranosyluronic acid (4ΔUA) residue linked to a β-D-(1 → 4)-xylooligosaccharide backbone were separated by CZE and detected by their UV absorption at 232 nm without prior derivatization. Pre-column derivatization with the chromophore 6-aminoquinoline (6-AQ) followed by CZE in alkaline borate buffer using detection based on absorption at 245 nm was used in the case of neutral xylosaccharides. Furthermore, MALDI-TOF-MS was employed to determine the molecular masses of both unsaturated and saturated acidic oligosaccharides. The acidic oligosaccharides released upon endoxylanase treatment of the birch kraft pulp were a (4ΔUA)- β-D-xylotetraose, a (4ΔUA)-β-D-xylopentaose, a (4-O-methyl-α-D-glucurono)-β-D-xylotetraose and a (4-O-methyl-α-D-glucurono)-β-D-xylopentaose. Analysis after enzymatic hydrolysis with β-xylosidase and α-glucuronidase from Trichoderma reesei strongly indicated that the uronic acid residue in these acidic oligosaccharides was linked to the D-xylose unit adjacent to the non-reducing D-xylose unit. The neutral xylosaccharides obtained after endoxylanase treatment of the pulp sample were D-xylose, β-(1-4)-D-xylobiose and β-(1-4)-D-xylotriose.
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.
Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.
Su, X., Han, Y., Dodd, D., Moon, Y. H., Yoshida, S., Mackie, R. I. & Cann, I. K. O. (2013). Applied and Environmental Microbiology, 79(5), 1481-1490.
Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ~6.5) and temperature (75 to ~90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated kcat values of ~8,000 and ~4,500 s-1, respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.
Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation.
Koutaniemi, S., van Gool, M. P., Juvonen, M., Jokela, J., Hinz, S. W., Schols, H. A. & Tenkanen, M. (2013). Journal of Biotechnology, 168(4), 684-692.
Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally.
Isolation of α-glucuronidase enzyme from a rumen metagenomic library.
Lee, C. C., Kibblewhite, R. E., Wagschal, K., Li, R. & Orts, W. J. (2012). The Protein Journal, 31(3), 206-211.
α-Glucuronidase enzymes play an essential role in the full enzymatic hydrolysis of hemicellulose. Up to this point, all genes encoding α-glucuronidase enzymes have been cloned from individual, pure culture strains. Using a high-throughput screening strategy, we have isolated the first α-glucuronidase gene (rum630-AG) from a mixed population of microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The RUM630-AG enzyme had optimum activity at pH 6.5 and 40°C. When birchwood xylan was used as substrate, the RUM630-AG functioned synergistically with an endoxylanase enzyme to hydrolyze the substrate.
Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus.
Rosa, L., Ravanal, M. C., Mardones, W. & Eyzaguirre, J. (2013). Fungal Biology, 117(5), 380-387.
The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 184.108.40.206) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91 725 and shows 99% identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100 000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37°C and pH 4.5–5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.
Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module.
Valenzuela, S. V., Diaz, P., & Pastor, F. J. (2012). Applied and environmental microbiology, 78(11), 3923-3931.
Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a Km of 14.72 mg/ml and a kcatvalue of 1,510 min-1. The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)8 barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.
Isolation and characterization of a novel GH67 α-glucuronidase from a mixed culture.
Lee, C. C., Kibblewhite, R. E., Wagschal, K., Li, R., Robertson, G. H. & Orts, W. J. (2012). Journal of Industrial Microbiology & Biotechnology, 39(8), 1245-1251.
Hemicelluloses represent a large reservoir of carbohydrates that can be utilized for renewable products. Hydrolysis of hemicellulose into simple sugars is inhibited by its various chemical substituents. The glucuronic acid substituent is removed by the enzyme α-glucuronidase. A gene (deg75-AG) encoding a putative α-glucuronidase enzyme was isolated from a culture of mixed compost microorganisms. The gene was subcloned into a prokaryotic vector, and the enzyme was overexpressed and biochemically characterized. The DEG75-AG enzyme had optimum activity at 45°C. Unlike other α-glucuronidases, the DEG75-AG had a more basic pH optimum of 7–8. When birchwood xylan was used as substrate, the addition of DEG75-AG increased hydrolysis twofold relative to xylanase alone.
Isolation and divalent-metal activation of a β-xylosidase, RUM630-BX.
Jordan, D. B., Braker, J. D., Wagschal, K., Stoller, J. R. & Lee, C. C. (2016). Enzyme and microbial technology, 82, 158-163.
The gene encoding RUM630-BX, a β-xylosidase/arabinofuranosidase, was identified from activity-based screening of a cow rumen metagenomic library. The recombinant enzyme is activated as much as 14-fold (kcat) by divalent metals Mg2+ , Mn2+ and Co2+ but not by Ca2+, Ni2+, and Zn2+. Activation of RUM630-BX by Mg2+ (t0.5 144 s) is slowed two-fold by prior incubation with substrate, consistent with the X-ray structure of closely related xylosidase RS223-BX that shows the divalent-metal activator is at the back of the active-site pocket so that bound substrate could block its entrance. The enzyme is considerably more active on natural substrates than artificial substrates, with activity (kcat/Km) of 299 s-1 mM-1 on xylotetraose being the highest reported.