The two successful H80F and W130H mutations were transposed to another fucosidase from Lactobacillus casei, AlfC, which is reported to synthesize α-L-Fucp-(1→6)-D-GlcNAc . Although AlfB and AlfC are only distantly related (below 30% identity), analysis of AlfC-H87F and AlfC-W137H revealed that these also procured increased disaccharide yields in reactions containing equimolar (8 mM) amounts of α-L-FucpOCNP and D-GlcNAc (Figs. 5, S8 and S9). Significantly, this result illustrates how successful mutations generated in one enzyme can be transposed to other GHs without the need to perform further analyses. In addition to the reduction of the sequence space and increased relevance, mutations transfer is a key advantage of targeting conserved residues. GH51, α-L-arabinofuranosidase TxAbf. Five-carbon furanose rings are notoriously more flexible and thermodynamically less stable than their six-carbon counterparts, meaning that unlike pyranoses, furanoses can interconvert between different ring conformations. This reflects similar energy states for the different conformations , a point that complicates the organic synthesis of furanosides. As catalysis mediated by any given GH involves a specific conformational itinerary for the donor substrate, the lowered energy barriers between furanoside conformations implies that furanosidase-catalysed reactions display altered mechanisms when compared to those catalysed by pyranosidases. Therefore, it is of interest to investigate to which extent the GH engineering approach described herein applies to furanosidases. The hydrolytic α-L-arabinofuranosidase from Thermobacillus xylanilyticus (TxAbf) belonging to the GH51 family was used as a model furanosidase. Displaying inherent ability to perform transfuranosylation , TxAbf has been the target of several studies aimed at improving its transglycosylation capability . All eight mutants generated in this study showed improved ability to synthesize arabinoxylo-oligosaccharides (AXOS). Compared to TxAbf-WT (9% yield), AXOS yields for the variants were in the range 19-82% (Fig. and Table ), with R69K, N175T, D297N and H240F/N being the best performers (37-82% overall yields). The transglycosylation activity (specific activity in transglycosylation mode, SAT, Table ) of the mutants was lower (0.1-66%) than that of the WT enzyme. It is noteworthy that R69K-, H240F-and D297N-catalysed transglycosylation reactions required relatively low amounts of catalyst (1.8-8 μg•mL -1 , i.e. 32-140 nM; Fig. ). . b Protein concentrations used to obtain the yields within 15 h are given in µg•mL -1 and nM (in brackets). Right, NMR monitoring of transglycosylation yield progress for TxAbf-WT and two of its more significant mutants.