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Chembiochem. 2007 Nov 23;8(17):2125-32.

Specificity fingerprinting of retaining beta-1,4-glycanases in the Cellulomonas fimi secretome using two fluorescent mechanism-based probes.

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1
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., V6T 1Z1, Canada.

Abstract

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, mechanism-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.

PMID:
17952884
DOI:
10.1002/cbic.200700481
[Indexed for MEDLINE]

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