Purification and Characterization of an α-L-Fucosidase from Pomacea canaliculata

An α-L-fucosidase (EC 3.2.1.51) was isolated from the hepatopancreas of Pomacea canals culata. The enzyme was purified 285-fold from the crude enzyme extract by procedures involving first heat treatment, ammonium sulfate fractionation, second heat treatment, and chromatography on DEAE-Sepharose, hydroxylapatite, and L-fucosylamine-CH-Sepharose. When assayed by using p-nitrophenyl glycosides as substrates, the final preparation was free from other glycosidase activities and gave a single protein band which corresponded to α-L-fucosidase activity on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be 260,000 by Sephacryl 5-300 column chromatography. The enzyme has two optimum pH values, 2.5 and 5.0, and the apparent Km value and the maximum velocity for p-nitrophenyl α-L-fucoside at both pH were calculated to be 0.45 mM and 1.46 μmol/min/mg of protein, respectively. The enzyme was shown to hydrolyze the Fucα1 → 2Gal, the Fucα1 → 4GlcNAc, and the Fucα1 → 6GIcNAc linkages, but hardly acts on the Fucα1 → 3GlcNAc linkage in various oligosaccharides.