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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 glyco-conjugates. 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.