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The α-1,3/4- l -fucosidases (EC 3.2.1.111; GH29) Bb AfcB from Bifidobacterium bifidum and Cp Afc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto- N -fucopentaose II (LNFP II) through regioselective transfucosylation of lacto- N- tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering Bb AfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of Bb AfcB with the corresponding 17-aminoacid α-helical loop of Cp Afc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT Bb AfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4- l -fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ± 2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT Bb AfcB (14 ± 0.3%) and comparable to that obtained with Cp Afc2 (50 ± 8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto- N -neotetraose (LNnT) could be enriched with LNFP III. [ABSTRACT FROM AUTHOR]

α- l -Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α- l -Fucosidases, glycoside hydrolases for catalyzing the removal of α- l -fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α- l -fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α- l -fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α- l -fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α- l -fucosidases. [ABSTRACT FROM AUTHOR]