Efficient Production and Purification of Extracellular 1,2-α-L-Fucosidase ofBacillussp. K40T

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Abstract
When Bacillus sp. K40T was cultured in the presence of L-fucose, 1,2-alpha-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200,000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5.5-7.0 and was stable at pH 6.0-9.0. The enzyme hydrolyzed the alpha-(1-->2)- L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I 2)-O-beta-D-galactose-(1-->3)-N-acetyl-O-beta-D- glucosamine-(1-->3)-O-beta-D-galactose-(1-->4)-D-glucose>, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze alpha-(1-->3)-, alpha-(1-->4)- and alpha-(1-->6)-L-fucosidic linkages in LNF-III 4)[O- alpha-L-fucose-(1-->3)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose>, LNF-II 3)[O-alpha-L- fucose-(1-->4)-]-N-acetyl-O-beta-D-glucosamine-(1-->3)-O-beta-D- galactose-(1-->4)-D-glucose> or 6-O-alpha-L-fucopyranosyl-N-acetylglucosamine.

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