Expression,Purification and Characterization of Human α‐l‐Fucosidase

α‐l ‐Fucosidases (EC 3.2.1.51) are exo‐glycosidases. On the basis of the multi‐alignment of amino acid sequence, α‐l ‐fucosidases were classified into two families of glycoside hydrolases, GH‐29 and GH‐95. They are responsible for the removal of l ‐fucosyl residues from the non‐reducing end of glycoconjugates. Deficiency of α‐l ‐fucosidase results in Fucosidosis due to the accumulation of fucose‐containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of α‐l ‐fucosidases may relate to this abnormal cell physiology. Although the gene of human α‐l ‐fucosidase (h‐fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human α‐l ‐fucosidases (h‐Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion‐exchange and gel‐filtration chromatographic purification, the recombinant h‐Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS‐PAGE (～50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h‐Fuc was stable up to 55 °C with incubation at pH 6.8 for 2 h; the optimum temperature for h‐Fuc is approximately 55 °C. The enzyme was stable at pH 2.5–7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The K_m and k_cat values of the recombinant h‐Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s^?1, respectively. The study of pH‐dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h‐Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h‐Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two‐step, double displacement mechanism.