Specificity Fingerprinting of Retaining (5-1,4-Glycanases in the Cellulomonas fimi Secretome Using Two Fluorescent Mechanism-Based Probes

摘要

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 letoining celluloses and xylanases were pa-pored.The specificity-based covolent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures.Using the two probes and mass spectrometry,the seaetomes of the bio-mass-converting hocterium Cellulomonas fimi,under induction by diffeient 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 biomassdegrvding organisms.