Tailored-made biocatalysts that meet specific industrial or academic demands can be developed through enzyme engineering. By mutating one or several amino acids, the specificity or selectivity of enzymes can be altered to unlock novel and effective synthetic routes toward valuable compounds. Enzyme engineers typically target amino acids in the active site. However, residues at the entrance to the active site can constrain the movement of substrates and products in and out of the active site, which certainly contributes to specificity or selectivity as well. Despite their great potential, substrate access is typically studied in enzymes with access tunnels, but open crater-like active sites are regularly ignored. In this project, I will investigate the open entrance region of the glycoside hydrolase family GH13_18 to study the underlying mechanisms of substrate access evolvability and the co-evolution between entrance and active site. By drawing inspiration from natural evolution, fundamental knowledge gaps on the effect of mutations in the entrance to the active site will be filled in and novel engineering strategies will be developed. This will not only enlarge our knowledge of natural protein evolution but also our protein engineering toolbox.