Purification of an almond emulsin fucosidase on Cibacron blue-sepharose and demonstration of its activity toward fucose-containing glycoproteins.

The almond emulsin fucosidase that specifically hydrolyzes fucose in alpha (1-3) linkage to N-acetylglucosamine has been purified 1250-fold. The purification procedure includes ion exchange chromatography on sulfopropyl-Sephadex C-25, gel filtration on Sephacryl S-200, and affinity chromatography on Cibacron blue-Sepharose 4B-CL. The molecular weight of the fucosidase was estimated by gel filtration as approximately 73,000. Enzyme activity was maximal at pH 5.3 in acetate buffer and was dependent on ionic strength; at least 0.1 M NaCl was necessary for optimal activity. The purified enzyme was free of beta-galactosidase activity toward the glycoprotein substrate [3H]galactosyl-asialotransferrin and did not release fucose from substrates containing fucose in alpha (1-6) linkage, (bovine IgG glycopeptides) or in alpha (1-2) linkage, (2'-fucosyllactose). The fucosidase displayed activity toward two glycoprotein substrates known to contain fucose in alpha (1-3) linkage. Extensive incubations resulted in the release of 83% and 43% of the total fucose of asialoorosomucoid and lactoferrin, respectively. The fucosidase did not release fucose from either the "slow" or the "fast" form of alpha 2-macroglobulin, suggesting the absence of fucosyl alpha (1-3) linkages on that glycoprotein.