In summary, this research integrates synthetic organic chemistry, enzymology, and molecular modelling to advance the structural and mechanistic understanding of carbohydrate-active enzymes. The findings provide fundamental insights into enzyme specificity, catalysis, and inhibition, thereby contributing to the development of improved biocatalysts for industrial processes and novel inhibitors for therapeutic applications. Key contributions include the characterization of α-amylases, β-amylase, and DispersinB, as well as the development of novel isothermal titration calorimetry and HPLC-based assay methodologies for monitoring enzyme activity and inhibition kinetics.