Activity-based probes for dynamic characterisation of polysaccharide-degrading enzymes

Carbohydrate-active enzymes play essential roles in polysaccharide degradation, yet their biochemical characterisation remains challenging - especially in the face of rapidly expanding genomic and structural data. Standard annotations often overlook critical properties such as expression patterns, enzyme stability and substrate specificity, which are key to understanding function in biological and industrial contexts. Activity-based probes (ABPs) offer a direct solution by enabling selective detection of active enzymes within complex systems. This review focuses on ABPs for retaining glycosidases, tracing their development from early applications in medical diagnostics to emerging uses in biomass degradation. We examine recent advances in scaffold design - including fluorosugars, epoxides, aziridines and cyclic sulphates - and their utility in enzyme profiling, inhibitor discovery and biotechnology. Current ABPs remain limited: they cannot yet target inverting enzymes and other classes lacking nucleophilic residues, a gap that may be bridged through computational modelling and AI-guided probe development. Looking forward, integration of ABPs with enzyme engineering and design holds promise for unlocking new classes of biocatalysts tailored for industrial and biomedical use.