Purification and Characterization of α -L-Fucosidase from Bacillus circulans Grown on Porcine Gastric Mucin

Bacillus circulans isolated from soil was found to produce two types of a-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM), but not from artificial substrates, including p-nitrophenyl and methyl α -L-fucosides, while the other acted not on PGM but on p-nitrophenyl α -L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The a-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285, 000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave a-(l→ 2)-, (l→ 3)-, and (l→ 4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze α -(l→ 6)-L-fucosic linkages in 6-O-α -L-fucopyranosyl-iV-acetylgluco-samine and intact bovine thyroglobulin.