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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.Hide Abstract
Verhertbruggen, Y., Marcus, S. E., Haeger, A., Verhoef, R., Schols, H. A., McCleary, B. V., McKee, L., Gilbert, H. J. & Knox, J. P. (2009). The Plant Journal, 59(3), 413-425.
Plant cell walls are constructed from a diversity of polysaccharide components. Molecular probes directed to structural elements of these polymers are required to assay polysaccharide structures in situ, and to determine polymer roles in the context of cell wall biology. Here, we report on the isolation and the characterization of three rat monoclonal antibodies that are directed to 1,5-linked arabinans and related polymers. LM13, LM16 and LM17, together with LM6, constitute a set of antibodies that can detect differing aspects of arabinan structures within cell walls. Each of these antibodies binds strongly to isolated sugar beet arabinan samples in ELISAs. Competitive-inhibition ELISAs indicate the antibodies bind differentially to arabinans with the binding of LM6 and LM17 being effectively inhibited by short oligoarabinosides. LM13 binds preferentially to longer oligoarabinosides, and its binding is highly sensitive to arabinanase action, indicating the recognition of a longer linearized arabinan epitope. In contrast, the binding of LM16 to branched arabinan and to cell walls is increased by arabinofuranosidase action. The presence of all epitopes can be differentially modulated in vitro using glycoside hydrolase family 43 and family 51 arabinofuranosidases. In addition, the LM16 epitope is sensitive to the action of β-galactosidase. Immunofluorescence microscopy indicates that the antibodies can be used to detect epitopes in cell walls, and that the four antibodies reveal complex patterns of epitope occurrence that vary between organs and species, and relate both to the probable processing of arabinan structural elements and the differing mechanical properties of cell walls.Hide Abstract
A novel neutral and thermophilic endoxylanase from Streptomyces ipomoeae efficiently produced xylobiose from agricultural and forestry residues.
Xian, L., Li, Z., Tang, A. X., Qin, Y. M., Li, Q. Y., Liu, H. B. & Liu, Y. Y. (2019). Bioresource Technology, 285, 121293.
Endoxylanases capable of producing high ratios of xylobiose from agricultural and forestry residues in neutral and high temperature conditions are attractive for the prebiotic and alternative sweetener industries. In this study, a putative glycosyl hydrolase gene from Streptomyces ipomoeae was cloned and expressed in Escherichia coli. The recombinant enzyme, named as SipoEnXyn10A, hydrolyzed beechwood xylan in endo-action mode releasing xylobiose as its main end product. It was most active at pH 6.5 and 75-80°C and showed remarkable stability at 65°C. The xylobiose yield from 10 g corncob and moso bamboo reached 1.123 ± 0.021 and 0.229 ± 0.005 g, respectively, at pH 6.5 and 70°C, which was higher than other reports using the same material. Moreover, high ratios of xylobiose in the xylose-based product of about 85% were obtained from corncob, moso bamboo sawdust, cassava stem and Chinese fir sawdust. These results demonstrated that SipoEnXyn10A has potential for industrial application.Hide Abstract