The purification and characterization of alpha-L-fucosidase from the hepatopancreas of Octopus vulgaris.

Abstract

We have isolated and purified, by affinity chromatography with Agarose-epsilon-amino-caproyl-fucosamine, an alpha-L-fucosidase [alpha-L-fucoside fucohydrolase EC 3.2.1.51] from the hepatopancreas of Octopus vulgaris. In the purified fraction only fucosidase activity could be detected. However, two protein bands, one major (about 95 per cent) and one minor (about 5 per cent), were evident on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing also revealed two activities, pI 8.1 (major) and pI 7.3 (minor). Under denaturing conditions the molecular weight of the major band was estimated to be 52,000 while that of the minor one was 43,000. Only one major activity peak with an apparent molecular weight of 70,000--75,000 was detected by gel filtration chromatography. The enzyme has two optimal pH values, and the relative activities are temperature-dependent; one optimum is at pH 5.5 +/- 0.2 and the other at pH 3.0 +/- 0.2. We found that the enzyme has a maximum activity at about 70 degrees C, but 50 per cent of the enzyme was inactivated at 70 degrees C after 5 min. The purified enzyme, using p-nitrophenyl-L-fucoside as substrate, has a specific activity of 38.9 units/mg of protein, Km of 3.58 x 10(-4) M and Vmax of 65 mumol/min/mg of protein. alpha-L-Fucose acts as a competitive inhibitor, with a K1 of 1.2 x 10(-3) M. alpha-L-Fucosidase released radioactive fucose from cellular glycopeptides, but no detectable free fucose was released fom 5 natural substrates.