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Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2--L-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University,¹ Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kitashirakawa, Sakyo-ku, Kyoto 606-8502,² Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011,³ The Noguchi Institute, Itabashi-ku, Tokyo 173-0003, Japan⁴

Received 24 December 2003/ Accepted 28 April 2004

A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the -(12) linkage of 2'-fucosyllactose, and a gene encoding 1,2--L-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal -(12)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2'-fucosyllactose was determined to be inversion by using ¹H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

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