Bifidobacterial GH146 ß-L-arabinofuranosidase for the removal of ß1,3-L-arabinofuranosides on plant glycans.

L-Arabinofuranosides with ß-linkages are present in several plant molecules, such as arabinogalactan proteins (AGPs), extensin, arabinan, and rhamnogalacturonan-II. We previously characterized a ß-L-arabinofuranosidase from Bifidobacterium longum subsp. longum JCM 1217, Bll1HypBA1, which was found to belong to the glycoside hydrolase (GH) family 127. This strain encodes two GH127 genes and two GH146 genes. In the present study, we characterized a GH146 ß-L-arabinofuranosidase, Bll3HypBA1 (BLLJ_1848), which was found to constitute a gene cluster with AGP-degrading enzymes. This recombinant enzyme degraded AGPs and arabinan, which contain Araf-ß1,3-Araf structures. In addition, the recombinant enzyme hydrolyzed oligosaccharides containing Araf-ß1,3-Araf structures but not those containing Araf-ß1,2-Araf and Araf-ß1,5-Araf structures. The crystal structures of Bll3HypBA1 were determined at resolutions up to 1.7 Å. The monomeric structure of Bll3HypBA1 comprised a catalytic (α/α)6 barrel and two ß-sandwich domains. A hairpin structure with two ß-strands was observed in Bll3HypBA1, to extend from a ß-sandwich domain and partially cover the active site. The active site contains a Zn2+ ion coordinated by Cys3-Glu and exhibits structural conservation of the GH127 cysteine glycosidase Bll1HypBA1. This is the first study to report on a ß1,3-specific ß-L-arabinofuranosidase. KEY POINTS: • ß1,3-l-Arabinofuranose residues are present in arabinogalactan proteins and arabinans as a terminal sugar. • ß-l-Arabinofuranosidases are widely present in intestinal bacteria. • Bll3HypBA1 is the first enzyme characterized as a ß1,3-linkage-specific ß-l-arabinofuranosidase.

Bifidobacterial GH146 ß-l-Arabinofuranosidase (Bll4HypBA1) as the Last Enzyme for the Complete Removal of Oligoarabinofuranosides from Hydroxyproline-Rich Glycoproteins.

In plant cell walls, the hydroxyproline-rich glycoproteins (HRGPs) such as extensin contain oligoarabinofuranoside linked to a hydroxyproline (Hyp) residue. The mature arabinooligosaccharide was revealed to be a tetrasaccharide (α-l-Araf-(1→3)-ß-l-Araf-(1→2)-ß-l-Araf-(1→2)-ß-l-Araf, l-Araf4 ), whose linkages are targets of the bifidobacterial and Xanthomonas arabinooligosaccharide-degrading enzymes. The l-Araf4 motif was cleaved by GH43 α-l-arabinofuranosidase (Arafase) and converted to an l-Araf3 -linked structure. The latter is then cleaved by GH121 ß-l-arabinobiosidase (HypBA2), producing ß-l-Araf-(1→2)-l-Ara (ß-l-arabinobiose) and mono-ß-l-Araf linked to the HRGP backbone. In bifidobacteria, the ß-l-arabinobiose is then hydrolyzed by GH127 ß-l-Arafase (Bll1HypBA1), a mechanistically unique cysteine glycosidase. We recently identified the distantly related homologue from Xanthomonas euvesicatoria as GH146 ß-l-Arafase along with paralogues from Bifidobacterium longum, one of which, Bll4HypBA1 (BLLJ_0089), can degrade l-Araf1 -Hyp in a similar way to that of GH146. As the chemical synthesis of the extensin hydrophilic motif 1 a, which possesses three distinct linkages that connect four oligoAraf residues [Hyp(l-Arafn ) (n=4, 3, 1)], was achieved previously, we precisely monitored the step-wise enzymatic cleavage of 1 a in addition to that of potato lectin. The results unequivocally revealed that this enzyme specifically degrades the Hyp(l-Araf1 ) motif.

Mechanism of Cooperative Degradation of Gum Arabic Arabinogalactan Protein by Bifidobacterium longum Surface Enzymes.

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-ß1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and ß1,6-linked α-l-Rhap-(1→4)-ß-d-GlcpA 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 Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.

Novel 3-O-α-d-Galactosyl-α-l-Arabinofuranosidase for the Assimilation of Gum Arabic Arabinogalactan Protein in Bifidobacterium longum subsp. longum.

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052 and classified it into glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and ß-l-Arap-(1→3)-l-Ara from larch AGP, and the α-d-Galp-(1→3)-l-Ara release activity was found to be 594-fold higher than that of ß-l-Arap-(1→3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1→3)-l-Ara in B. longum Notably, when α-d-Galp-(1→3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the nonassimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1→3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type II AG degradative enzymes in B. longum The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longumIMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase carrier strains may affect GAfase noncarrier strains that express other type II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.

Degradation of plant arabinogalactan proteins by intestinal bacteria: characteristics and functions of the enzymes involved.

Arabinogalactan proteins (AGPs) are complex plant proteoglycans that function as dietary fiber utilized by human intestinal bacteria such as Bifidobacterium and Bacteroides species. However, the degradative mechanism is unknown because of the complexity of sugar chains of AGPs as well as variation among plant species and organs. Recently, AGP degradative enzymes have been characterized in Bifidobacterium and Bacteroides species. In this review, we summarize the characteristics and functions of AGP degradative enzymes in human intestinal bacteria.

Degradative enzymes for type II arabinogalactan side chains in Bifidobacterium longum subsp. longum.

Type II arabinogalactan (AG) is a soluble prebiotic fiber stimulating the proliferation of bifidobacteria in the human gut. Larch AG, which is comprised of type II AG, is known to be utilized as an energy source for Bifidobacterium longum subsp. longum (B. longum). We have previously characterized GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal) for the degradation of type II AG main chains in B. longum JCM1217. In this study, we characterized GH30_5 exo-ß-1,6-galactobiohydrolase (Bl1,6Gal) and GH43_22 α-L-arabinofuranosidase (BlArafA), which are degradative enzymes for type II AG side chains in cooperation with exo-ß-1,3-galactanase. The recombinant exo-ß-1,6-galactobiohydrolase specifically released ß-1,6-galactobiose (ß-1,6-Gal2) from the nonreducing terminal of ß-1,6-galactooligosaccharides, and the recombinant α-L-arabinofuranosidase released arabinofuranose (Araf) from α-1,3-Araf-substituted ß-1,6-galactooligosaccharides. ß-1,6-Gal2 was additively released from larch AG by the combined use of type II AG degradative enzymes, including Bl1,3Gal, Bl1,6Gal, and BlArafA. The gene cluster encoding the type II AG degradative enzymes is conserved in all B. longum strains, but not in other bifidobacterial species. The degradative enzymes for type II AG side chains are thought to be important for the acquisition of type II AG in B. longum.

Carbohydrate components in sweetpotato storage roots: their diversities and genetic improvement.

Carbohydrates are important components in sweetpotatoes in terms of both their industrial use and eating quality. Although there has been a narrow range of diversity in the properties of sweetpotato starch, unique varieties and experimental lines with different starch traits have been produced recently both by conventional breeding and genetic engineering. The diversity in maltose content, free sugar composition and textural properties in sweetpotato cultivars is also important for their eating quality and processing of storage roots. In this review, we summarize the current status of research on and breeding for these important traits and discuss the future prospects for research in this area.

Characterization of a novel ß-L-arabinofuranosidase in Bifidobacterium longum: functional elucidation of a DUF1680 protein family member.

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. Previously, we cloned and characterized the hypBA2 gene as a ß-L-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel ß-L-arabinofuranosidase that liberates L-arabinose from the L-arabinofuranose (Araf)-ß1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-338 is a critical residue for catalytic activity. This study provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the glycoside hydrolase family 127.

Bifidobacterium longum subsp. longum Exo-ß-1,3-Galactanase, an enzyme for the degradation of type II arabinogalactan.

Type II arabinogalactan (AG-II) is a suitable carbohydrate source for Bifidobacterium longum subsp. longum, but the degradative enzymes have never been characterized. In this study, we characterized an exo-ß-1,3-galactanase, BLLJ_1840, belonging to glycoside hydrolase family 43 from B. longum subsp. longum JCM1217. The recombinant BLLJ_1840 expressed in Escherichia coli hydrolyzed ß-1,3-linked galactooligosaccharides but not ß-1,4- and ß-1,6-linked galactooligosaccharides. The enzyme also hydrolyzed larch wood arabinogalactan (LWAG), which comprises a ß-1,3-linked galactan backbone with ß-1,6-linked galactan side chains. The kcat/Km ratio of dearabinosylated LWAG was 24-fold higher than that of ß-1,3-galactan. BLLJ_1840 is a novel type of exo-ß-1,3-galactanase with a higher affinity for the ß-1,6-substituted ß-1,3-galactan than for nonsubstituted ß-1,3-galactan. BLLJ_1840 has 27% to 28% identities with other characterized exo--1,3-galactanases from bacteria and fungi. The homologous genes are conserved in several strains of B. longum subsp. longum and B. longum subsp. infantis but not in other bifidobacteria. Transcriptional analysis revealed that BLLJ_1840 is intensively induced with BLLJ_1841, an endo-ß-1,6-galactanase candidate, in the presence of LWAG. This is the first report of exo-ß-1,3-galactanase in bifidobacteria, which is an enzyme used for the acquisition of AG-II in B. longum subsp. longum.

Hyaluronidase-inhibiting Polysaccharide from Caulerpa lentillifera.

Algal sulfated polysaccharides are known to be effective hyaluronidase inhibitors. We evaluated hyaluronidase inhibitory activity of sulfated polysaccharide (SP) from Caulerpa lentillifera. Results showed that SP with IC50 of 163 µg/mL appears to allosterically inhibit the hyaluronidase activity. Main sugar composition and sulfate content of SP was estimated to be Gal, Glc, Xyl, Man, uronic acids, and sulfate in the weight percent of 27.7: 28.9: 14.6: 22.5: 3.4: 21.7. We modified the SP by desulfation and partial hydrolysis with trifluoroacetic acid (TFA) to investigate the effect of sulfate content and molecular weight on inhibition. Hyaluronidase inhibitory activity of desulfated SP, 0.1 M TFA-hydrolyzed SP and 0.5 M TFA-hydrolyzed SP were significantly lower than that of native SP, revealing that sulfate content or molecular weight is important for hyaluronidase inhibition.

Fractionation and characterization of cell wall polysaccharides from the brown alga Cladosiphon okamuranus.

Brown algae contain a polysaccharide-rich cell wall, mainly composed of alginate and fucoidan which have been extensively studied for their individual structure and bioactivities. Particularly, the cell wall of Cladosiphon okamuranus is rich in fucoidan rather than alginate. However, little is known about its arrangement or interlinking with other polysaccharides such as cellulose in the cell wall. To determine its structure in detail, the cell wall was sequentially fractionated into five fractions: hot water (HW), ammonium oxalate, hemicellulose-I (HC-I), HC-II, and cellulose. Almost 80% of the total cell wall recovered from alcohol insoluble residue in C. okamuranus consisted of HW and HC-I, which mainly contained fucoidan composed of fucose, glucuronic acid, and sulfate in molar ratios of 1.0:0.3:0.9 and 1.0:0.2:0.3, respectively. Methylation analysis revealed that fucoidan in HW and HC-I structurally differed in terms of content of sulfate, and sugar residue which was 1,4-linked xylose and 1,4-linked fucose. Small angle X-ray scattering measurements also showed distinct conformational differences between HW and HC-I. These structural heterogeneities of fucoidan may be related to their localization, and fucoidan in HC-I may be involved in reinforcing cell wall structure by cross-linking to cellulose.

Assimilation of arabinogalactan side chains with novel 3-O-ß-L-arabinopyranosyl-α-L-arabinofuranosidase in Bifidobacterium pseudocatenulatum.

Aim: Dietary plant fibers affect gut microbiota composition; however, the underlying microbial degradation pathways are not fully understood. We previously discovered 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase), a glycoside hydrolase family 39 enzyme involved in the assimilation of side chains of arabinogalactan protein (AGP), from Bifidobacterium longum subsp. longum (B. longum) JCM7052. Although GAfase homologs are not highly prevalent in the Bifidobacterium genus, several Bifidobacterium strains possess the homologs. To explore the differences in substrate specificity among the homologs, a homolog of B. longum GAfase in Bifidobacterium pseudocatenulatum MCC10289 (MCC10289_0425) was characterized. Methods: Gum arabic, larch, wheat AGP, and sugar beet arabinan were used to determine the substrate specificity of the MCC10289_0425 protein. An amino acid replacement was introduced into GAfase to identify a critical residue that governs the differentiation of substrate specificity. The growth of several Bifidobacterium strains on ß-L-arabinopyranosyl disaccharide and larch AGP was examined. Results: MCC10289_0425 was identified to be an unprecedented 3-O-ß-L-arabinopyranosyl-α-L-arabinofuranosidase (AAfase) with low GAfase activity. A single amino acid replacement (Asn119 to Tyr) at the catalytic site converted GAfase into AAfase. AAfase releases sugar source from AGP, thereby allowing B. pseudocatenulatum growth. Conclusion: Bifidobacteria have evolved several homologous enzymes with overlapping but distinct substrate specificities depending on the species. They have acquired different fitness abilities to respond to diverse plant polysaccharide structures.

Identification and characterization of endo-α-, exo-α-, and exo-ß-D-arabinofuranosidases degrading lipoarabinomannan and arabinogalactan of mycobacteria.

The cell walls of pathogenic and acidophilic bacteria, such as Mycobacterium tuberculosis and Mycobacterium leprae, contain lipoarabinomannan and arabinogalactan. These components are composed of D-arabinose, the enantiomer of the typical L-arabinose found in plants. The unique glycan structures of mycobacteria contribute to their ability to evade mammalian immune responses. In this study, we identified four enzymes (two GH183 endo-D-arabinanases, GH172 exo-α-D-arabinofuranosidase, and GH116 exo-ß-D-arabinofuranosidase) from Microbacterium arabinogalactanolyticum. These enzymes completely degraded the complex D-arabinan core structure of lipoarabinomannan and arabinogalactan in a concerted manner. Furthermore, through biochemical characterization using synthetic substrates and X-ray crystallography, we elucidated the mechanisms of substrate recognition and anomer-retaining hydrolysis for the α- and ß-D-arabinofuranosidic bonds in both endo- and exo-mode reactions. The discovery of these D-arabinan-degrading enzymes, along with the understanding of their structural basis for substrate specificity, provides valuable resources for investigating the intricate glycan architecture of mycobacterial cell wall polysaccharides and their contribution to pathogenicity.

Characterization of a novel ß-L-Arabinofuranosidase in Bifidobacterium longum: functional elucidation of A DUF1680 family member.

Pfam DUF1680 (PF07944) is an uncharacterized protein family conserved in many species of bacteria, actinomycetes, fungi, and plants. In a previous article, we cloned and characterized the hypBA2 gene as a ß-l-arabinobiosidase in Bifidobacterium longum JCM 1217. In this study, we cloned a DUF1680 family member, the hypBA1 gene, which constitutes a gene cluster with hypBA2. HypBA1 is a novel ß-l-arabinofuranosidase that liberates l-arabinose from the l-arabinofuranose (Araf)-ß1,2-Araf disaccharide. HypBA1 also transglycosylates 1-alkanols with retention of the anomeric configuration. Mutagenesis and azide rescue experiments indicated that Glu-366 is a critical residue for catalytic activity. This report provides the first characterization of a DUF1680 family member, which defines a new family of glycoside hydrolases, the GH family 127.

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family.

Extensin is a glycoprotein that is rich in hydroxyprolines linked to ß-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel ß-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-ß1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara(4)-Hyp) or Araf-ß1,2-Araf-ß-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked ß-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.

Characterization of a GH36 α-D-Galactosidase Associated with Assimilation of Gum Arabic in Bifidobacterium longum subsp. longum JCM7052.

We recently characterized a 3-O-α-D-galactosyl-α-L-arabinofuranosidase (GAfase) for the release of α-D-Gal-(1→3)-L-Ara from gum arabic arabinogalactan protein (AGP) in Bifidobacterium longum subsp. longum JCM7052. In the present study, we cloned and characterized a neighboring α-galactosidase gene (BLGA_00330; blAga3). It contained an Open Reading Frame of 2151-bp nucleotides encoding 716 amino acids with an estimated molecular mass of 79,587 Da. Recombinant BlAga3 released galactose from α-D-Gal-(1→3)-L-Ara, but not from intact gum arabic AGP, and a little from the related oligosaccharides. The enzyme also showed the activity toward blood group B liner trisaccharide. The specific activity for α-D-Gal-(1→3)-L-Ara was 4.27- and 2.10-fold higher than those for melibiose and raffinose, respectively. The optimal pH and temperature were 6.0 and 50 °C, respectively. BlAga3 is an intracellular α-galactosidase that cleaves α-D-Gal-(1→3)-L-Ara produced by GAfase; it is also responsible for a series of gum arabic AGP degradation in B. longum JCM7052.

Inhibition of the expression of the starch synthase II gene leads to lower pasting temperature in sweetpotato starch.

The sweetpotato cultivar Quick Sweet (QS) with a lower pasting temperature of starch is a unique breeding material, but the biochemical background of this property has been unknown. To assess the physiological impact of the reduced isoform II activity of starch synthase (SSII) on the starch properties in sweetpotato storage root, transgenic sweetpotato plants with reduced expressions of the SSII gene were generated and evaluated. All of the starches from transgenic plants showed lower pasting temperatures and breakdown measured by a Rapid Visco Analyzer. The pasting temperatures in transgenic plants were approximately 10-15 degrees C lower than in wild-type plants. Distribution of the amylopectin chain length of the transgenic lines showed marked differences compared to that in wild-type plants: more chains with degree of polymerization (DP) 6-11 and fewer chains with DP 13-25. The starch granules from the storage root of transgenic plants showed cracking on the hilum, while those from wild-type plants appeared to be typical sweetpotato starch. In accordance with these observations, the expression of SSII in the storage roots of the sweetpotato cultivar with low pasting temperature starch (QS) was notably lower than in cultivars with normal starch. Moreover, nucleotide sequence analysis suggested that most of the SSII transcripts in the cultivar with low pasting temperature starch were inactive alleles. These results clearly indicate that the activity of SSII in sweetpotato storage roots, like those in other plants, affects the pasting properties of starch through alteration of the amylopectin structure.

Characterization of a GH36 ß-L-Arabinopyranosidase in Bifidobacterium adolescentis.

ß-L-Arabinopyranosidases are classified into the glycoside hydrolase family 27 (GH27) and GH97, but not into GH36. In this study, we first characterized the GH36 ß-L-arabinopyranosidase BAD_1528 from Bifidobacterium adolescentis JCM1275. The recombinant BAD_1528 expressed in Escherichia coli had a hydrolytic activity toward p-nitrophenyl (pNP)-ß-L-arabinopyranoside (Arap) and a weak activity toward pNP-α-D-galactopyranoside (Gal). The enzyme liberated L-arabinose efficiently not from any oligosaccharides or polysaccharides containing Arap-ß1,3-linkages, but from the disaccharide Arap-ß1,3-L-arabinose. However, we were unable to confirm the in vitro fermentability of Arap-ß1,3-Ara in B. adolescentis strains. The enzyme also had a transglycosylation activity toward 1-alkanols and saccharides as acceptors.