Arabinose-containing poly- or oligosaccharides are suitable carbohydrate sources for Bifidobacterium longum subsp. longum. However, their degradation pathways are poorly understood. In this study, we cloned and characterized the previously uncharacterized glycoside hydrolase family 43 (GH43) enzymes B. longum subsp. longum ArafC (BlArafC; encoded by BLLJ_1852) and B. longum subsp. longum ArafB (BlArafB; encoded by BLLJ_1853) from B. longum subsp. longum JCM 1217. Both enzymes exhibited α-l-arabinofuranosidase activity toward p-nitrophenyl-α-l-arabinofuranoside but no activity toward p-nitrophenyl-β-d-xylopyranoside. The specificities of the two enzymes for l-arabinofuranosyl linkages were different. BlArafC catalyzed the hydrolysis of α1,2- and α1,3-l-arabinofuranosyl linkages found on the side chains of both arabinan and arabinoxylan. It released l-arabinose 100 times faster from arabinan than from arabinoxylan but did not act on arabinogalactan. On the other hand, BlArafB catalyzed the hydrolysis of the α1,5-l-arabinofuranosyl linkage found on the arabinan backbone. It released l-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Coincubation of BlArafC and BlArafB revealed that these two enzymes are able to degrade arabinan in a synergistic manner. Both enzyme activities were suppressed with EDTA treatment, suggesting that they require divalent metal ions. The GH43 domains of BlArafC and BlArafB are classified into GH43 subfamilies 27 and 22, respectively, but show very low similarity (less than 15% identity) with other biochemically characterized members in the corresponding subfamilies. The B. longum subsp. longum strain lacking the GH43 gene cluster that includes BLLJ_1850 to BLLJ_1853 did not grow in arabinan medium, suggesting that BlArafC and BlArafB are important for assimilation of arabinan. IMPORTANCE We identified two novel α-l-arabinofuranosidases, BlArafC and BlArafB, from B. longum subsp. longum JCM 1217, both of which are predicted to be extracellular membrane-bound enzymes. The former specifically acts on α1,2/3-l-arabinofuranosyl linkages, while the latter acts on the α1,5-l-arabinofuranosyl linkage. These enzymes cooperatively degrade arabinan and are required for the efficient growth of bifidobacteria in arabinan-containing medium. The genes encoding these enzymes are located side by side in a gene cluster involved in metabolic pathways for plant-derived polysaccharides, which may confer adaptability in adult intestines.

Arabinose-containing poly-or oligosaccharides are suitable carbohydrate 14 sources for Bifidobacterium longum subsp. longum, though their degradation pathways are 15 poorly understood. In this study, we found that the gene expression levels of bllj_1852 and 16 bllj_1853 from B. longum subsp. longum JCM 1217 were enhanced in the presence of 17 arabinan. Both genes encode previously uncharacterized glycoside hydrolase (GH) family 18 43 enzymes. Subsequently, we cloned those genes and characterized the recombinant 19 enzymes expressed in Escherichia coli. Both enzymes exhibited -L-arabinofuranosidase 20 activity toward synthetic p-nitrophenyl glycoside, but the specificities for 21 L-arabinofuranosyl linkages were different. BLLJ_1852 catalyzed the hydrolysis of 1,2-22 and 1,3-L-arabinofuranosyl linkages found in the side chains of arabinan and arabinoxylan. 23 BLLJ_1852 released L-arabinose 100 times faster from arabinan than from arabinoxylan 24 but did not act on arabinogalactan. BLLJ_1853 catalyzed the hydrolysis of 25 1,5-L-arabinofuranosyl linkages found on the arabinan backbone. BLLJ_1853 released 26 L-arabinose from arabinan but not from arabinoxylan or arabinogalactan. Both enzyme 27 activities were largely suppressed with EDTA treatment, suggesting that they require 28 3 divalent metal ions. BLLJ_1852 was moderately activated in the presence of all divalent 29 cations tested, whereas BLLJ_1853 activity was inhibited by Cu 2+ . The GH43 domains of 30 BLLJ_1852 and BLLJ_1853 are classified into GH43 subfamilies 27 and 22, respectively, 31 but hardly share similarity with other biochemically characterized members in the 32 corresponding subfamilies. 33 IMPORTANCE We identified two novel -L-arabinofuranosidases from B. longum subsp. 34 longum JCM 1217 that act on different linkages in arabinan. These enzymes may be 35 required for efficient degradation and assimilation of arabinan in the probiotic 36 bifidobacteria. The genes encoding these enzymes are located side-by-side in a gene cluster 37 involved in metabolic pathways for plant-derived polysaccharides, which may confer 38 adaptability in adult intestines. 39 40 KEYWORDS dietary fiber, hemicellulose, gut microbiota, intestinal microbiota, prebiotics, 41 probiotics 42 43

Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Ara f and β1,6-linked α- l -Rha p -(1→4)-β- d -Glc p A residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Ara f of the side chain of gum arabic AGP.

Bifidobacterium longum subsp. infantis ATCC 15697 possesses five α-L-fucosidases, which have been previously characterized toward fucosylated human milk oligosaccharides containing α1,2/3/4-linked fucose [Sela et al.: Appl. Environ. Microbiol., 78, 795-803 (2012)]. In this study, two glycoside hydrolase family 29 α-L-fucosidases out of five (Blon_0426 and Blon_0248) were found to be 1,6-α-L-fucosidases acting on core α1,6-fucose on the N-glycan of glycoproteins. These enzymes readily hydrolyzed p-nitrophenyl-α-L-fucoside and Fucα1-6GlcNAc, but hardly hydrolyzed Fucα1-6(GlcNAcβ1-4)GlcNAc, suggesting that they de-fucosylate Fucα1-6GlcNAcβ1-Asn-peptides/ proteins generated by the action of endo-β-N-acetylglucosaminidase. We demonstrated that Blon_0426 can de-fucosylate Fucα1-6GlcNAc-IgG prepared from Rituximab using Endo-CoM from Cordyceps militaris. To generate homogenous non-fucosylated N-glycan-containing IgG with high antibodydependent cellular cytotoxicity (ADCC) activity, the resulting GlcNAc-IgG has a potential to be a good acceptor substrate for the glycosynthase mutant of Endo-M from Mucor hiemalis. Collectively, our results strongly suggest that Blon_0426 and Blon_0248 are useful for glycoprotein glycan remodeling.

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