Specificity Fingerprinting of Retaining β-1,4-Glycanases in the Cellulomonas fimi Secretome Using Two Fluorescent Mechanism-Based Probes

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 β-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 β-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.