Journal
CHEMBIOCHEM
Volume 8, Issue 17, Pages 2125-2132Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200700481
Keywords
affinity fingerprinting; enzyme specificity; fluorescent probes; glycosidases; proteomics
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Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mecanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.
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