The feasibility of transposing a successful mutation to a related enzyme was assessed using the fucosidase AlfC from Lactobacillus casei. Signals from α-L-Fucp-(1→6)-D-GlcNAc were insufficiently resolved from those of α-L-FucpOCNP, L-Fuc and D-GlcNAc in the H-2 and anomeric region to allow accurate monitoring of yield (Fig. ). Thus, compound formation and disappearance were monitored using the methyl group of L-Fuc (Fig. ). It is important to note that although progress curves established using the anomeric region were less precise, they were nevertheless consistent with those obtained when monitoring the methyl group (< 2% deviation for maximum yield assessment). Among the eight positions targeted by the conserved-sequence approach, the four yielding the best variants are spatially (within approximately 5 Å) and/or sequentially close to the catalytic residues 176 (acid/base, in cyan) and E298 (nucleophile, in deep blue). Accordingly, R69K, N175T, H240F/N and D297N (in green) are thought to impact the local H-bonding network and thus the pKa cycling that occurs during catalysis. Previously, the study of R69H revealed that R69 plays a vital role in the modulation of the ionization state and nucleophilic strength of E298. 11 N175 is thought to be involved in transition state stabilization. Therefore, modification of the interaction N175•••OH-2 of α-L-Araf unit (in orange), for example in N175T, might perturb the correct functioning of the two-step catalytic mechanism. Additionally, H240 could be involved in a putative water channel affecting water dynamics. Overall, the different mechanistic consequences of mutations at these four positions all translate into reduced water-mediated deglycosylation and/or increased lifetime of the covalent glycosyl-enzyme intermediate, which in turn favours sugar-mediated deglycosylation, hence transglycosylation. Therefore, all five single-mutants achieve the sought after result, namely tipping the T/H balance in favour of transglycosylation.