Expression, Purification and Characterization of Human -L-Fucosidase

-L-Fucosidases (EC 3.2.1.51) are exo-glycosidases. On the basis of the multi-alignment of amino acid sequence, -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 -L-fucosidase results in Fucosidosis due to the accumulation of fucose-con-taining 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 bio-logical function of -L-fucosidases may relate to this abnormal cell physiology. Although the gene of hu-man -L-fucosidase (h-fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a solu-ble and active from. We report herein that, with carefully control on the growing condition, an active hu-man -L-fucosidases (h-Fuc) was successfully expressed in Escherichia coli for the first time. After a se-ries 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 incuba-tion 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. TheK