Thermobifida fuscaLam81A is a single domain family-81 β-1,3-endoglucanase, but no structure is known for this family. Site-directed mutagenesis of 14 conserved residues chosen from sequence alignments was used to identify those with critical roles in catalysis, binding or substrate specificity. Mutant enzymes were assayed for their ability to bind and hydrolyze substrates with various glycosyl linkages. Residues D422, E499 and E503 were candidates for the catalytic acid or catalytic base, and E499 was shown to be the catalytic base by azide rescue. F425 was shown to have a major role in substrate binding possibly mediated by aromatic ring stacking with the sugar substrate. In addition, mutation of D424 to histidine altered the substrate specificity by increasing the rate of cleavage of mixed-linkage β-glucan and carboxymethyl-cellulose, 60- and 16-fold, respectively, over the wild-type enzyme.