alpha-L-Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. alpha-L-Fucosidases, glycoside hydrolases for catalyzing the removal of alpha-L-fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an alpha-L-fucosidase named as Alfl_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alfl_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alfl_Wf with 6xHis tag was expressed in E. coli and showed alpha-L-fucosidase activity. Sequence annotation revealed that Alfl_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alfl_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 Alfl_Wf was capable in hydrolyzing alpha 1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alfl_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel alpha-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 alpha-L-fucosidases. (C) 2016 Elsevier Inc. All rights reserved.