Klonierung und Charakterisierung von Enzymen aus Aspergillus nidulans zur Analytik der Polysaccharide pflanzlicher Zellwände
Final Report Abstract
To facilitate analysis of plant cell wall polysaccharide structure and composition, we cloned 74 genes encoding polysaccharide-degrading enzymes from A. nidulans, A. fumigatus and N. crassa and expressed the genes as secreted proteins with c-terminal Myc and 6xhis tags. The clones of the enzymes are freely available from the fungal genetics stock center. Due to the histidine tag the enzymes can be readily purified from the medium by affinity chromatography. Because these enzymes can be produced without contaminating activities, they are very useful for the accurate analysis of most polysaccharides that are the constituents of plant cell walls. Enzymes are now available for the cleavage of most common plant cell wall polysaccharides such as cellulose, hemicellulose (glucans, xyloglucans, xylans and mannans) and pectins (polygalacturonic acid, xylogalacturonans and rhamnogalacturonan). The collection of enzymes will be a powerful tool in the analysis of polysaccharides complementing the commonly used techniques such as IR-spectroscopy, monosaccharide analysis and immunolabelling. By using the enzymes to hydrolyze specific polysaccharides, it is possible to selectively investigate one type of polymer at a time. Reproducible selective cleavage of specific polymers will facilitate and speed up the use of mass spectrometry and NMR for structural analysis. First results in the use of these enzymes in the analysis of Arabidopsis thaliana cell wall mutants irx8 and irx9 have demonstrated the strength of the collection. To our knowledge this is the first report of a collection of pure enzymes to this extent.
Publications
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Towards a System Approach to Understanding Plant Cell Walls. Science 306 (2004), 2206-2211
Somerville, C., Bauer, S., Brininstool, G. et al.
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Cloning, Expression, and Characterization of an Oligoxyloglucan Reducing End-specific Xyloglucanobiohydrolase from Aspergillus nidulans. Carbohydr Res 340 (2005), 2590-2597
Bauer, S., Vasu, P., Mort, A.J., Somerville, C.
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Development and Application of a Suite of Polysaccharide-degrading enzymes for Analyzing Plant Cell Walls. Proceed. Natl. Acad. Sci. 103 (2006), 11417-11422
Bauer, S., Vasu, P., Mort, A.J., Somerville, C.
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The Arabidopsis irregulär xylem8 Mutant is Deficient in Glucuronxylan and Homo gal acturonan, Which are Essential for Secondary Cell Wall Integrity. Plant Cell 19 (2007), 237-255
Persson, S., Caffall, K. H., Freshour, G., Hilley, M. T., Bauer, S., Poindexter, P., Hahn, M. G., Mohnen, D., Somerville, C.
