Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28

► The catalytic acid/base residue in a glycoside hydrolase family 3 β-glucosidase was identified. ► Site-directed mutagenesis, enzyme kinetic analysis, and nucleophile rescue were used. ► Prediction of the corresponding residue among other GH3 enzymes is enabled. ► These results are crucial to understanding catalysis and active-site structure in GH3.