Strategies for GH functional annotation: toward harmonized and predictive CAZymology

BACKGROUND: The functional annotation and biochemical characterization of glycoside hydrolases (GHs) for polysaccharides and oligosaccharides deconstruction are a major challenge. Here, we traced the transition from traditional "activity first" approaches, based on biochemical assays, to modern "sequence first" discovery methods enabled by modern genomics. RESULTS: We reported 169 glycosidase activities, the substrates, and the techniques used for the characterization of GHs. We highlight the challenges posed by the diversity of GH families including overlapping substrate specificities, heterogeneous assay conditions, and inconsistent reporting standards. To address these issues, we compiled a list of substrates for each GH family's glycosidase activities as a practical guide to serve as an initial framework for characterizing newly identified GHs. CONCLUSIONS: The adoption of standardized substrates and protocols, uniform reporting practices, and community-driven data sharing initiatives provides a practical roadmap toward harmonized GH functional annotation and predictive enzymology while acknowledging current limitations in data standardization and reporting heterogeneity.