Bifidobacteria are predominant bacteria present in the intestines of breast-fed infants and offer important health benefits for the host. Human milk oligosaccharides are one of the most important growth factors for bifidobacteria and are frequently fucosylated at their non-reducing termini. Previously, we identified 1,2-alpha-l-fucosidase (AfcA) belonging to the novel glycoside hydrolase (GH) family 95, from Bifidobacterium bifidum JCM1254 (Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K. 2004. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-l-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 186:4885-4893). Here, we identified a gene encoding a novel 1,3-1,4-alpha-l-fucosidase from the same strain and termed it afcB. The afcB gene encodes a 1493-amino acid polypeptide containing an N-terminal signal sequence, a GH29 alpha-l-fucosidase domain, a carbohydrate binding module (CBM) 32 domain, a found-in-various-architectures (FIVAR) domain and a C-terminal transmembrane region, in this order. The recombinant enzyme was expressed in Escherichia coli and was characterized. The enzyme specifically released alpha1,3- and alpha1,4-linked fucosyl residues from 3-fucosyllactose, various Lewis blood group substances (a, b, x, and y types), and lacto-N-fucopentaose II and III. However, the enzyme did not act on glycoconjugates containing alpha1,2-fucosyl residue or on synthetic alpha-fucoside (p-nitrophenyl-alpha-l-fucoside). The afcA and afcB genes were introduced into the B. longum 105-A strain, which has no intrinsic alpha-l-fucosidase. The transformant carrying afcA could utilize 2'-fucosyllactose as the sole carbon source, whereas that carrying afcB was able to utilize 3-fucosyllactose and lacto-N-fucopentaose II. We suggest that AfcA and AfcB play essential roles in degrading alpha1,2- and alpha1,3/4-fucosylated milk oligosaccharides, respectively, and also glycoconjugates, in the gastrointestinal tracts.

Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Gal␤1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Gal␤1,3GlcNAc␤1,3Gal␤1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified ␤-linked lacto-Nbiose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Gal␤1-3GlcNAc) and galacto-N-biose (GNB; Gal␤1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K d values of 0.087 and 0.010 M, respectively, and the binding process is enthalpydriven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.Bifidobacteria are Gram-positive anaerobes naturally present in the dominant colonic microbiota and have been considered to be beneficial for human health. As probiotic agents, Bifidobacteria can prevent or alleviate infectious diarrhea through their effects on the immune system and promote the host resistance to colonization by pathogens (1). Carbon source compounds, including oligofructose, inulin, and raffinose, are used as food additives (prebiotics) to selectively promote the growth of Bifidobacteria in the gut (2, 3). Bifidobacteria predominate the intestinal flora of breastfed infants (4, 5), whereas bottle-fed infants do not show rapid colonization of these organisms (6). It has been widely accepted that oligosaccharides other than lactose in human milk (human milk oligosaccharides, HMOs) 4 play a key role in the growth of Bifidobacteria in the gut (7,8). However, it remains unknown what structure, in HMOs, constitutes the bifidus factor responsible for increasing the bifidobacterial population. Human milk is reported to contain more than 100 kinds of oligosaccharides, the building blocks of which are the following three basic core disaccharides: lactose (Gal␤1-4Glc), lacto-N-biose I (LNB; Gal␤1-3GlcNAc), and N-acetyllactosamine (LacNAc; Gal␤1-4GlcNAc). These oligosaccharides are often modified by sialic acid and/or L-fucose (7). Lacto-N-tetraose (LNT; Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc), which is formed by a ␤1-3 linkage between LNB and lactose, and fucosylated derivative (Fuc␣1-2Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc) are the major components of HMOs (9 -11).Recently, we found that Bifidobacterium longum JCM1217 has a unique metabolic pathway specific for LNB and galacto-N-biose (GNB, Gal␤1-3GalNAc), and we presented the hypothesis that the LNB residue in HMOs is the bifidus factor in breastfed infants (12). B...