Two Novel α- l -Arabinofuranosidases from Bifidobacterium longum subsp. longum Belonging to Glycoside Hydrolase Family 43 Cooperatively Degrade Arabinan

Abstract

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.

Plant Glycan Metabolism by Bifidobacteria

“…B. longum subsp. longum strains have been shown to grow on the pectic components arabinan and arabinogalactan ( O’Connell Motherway et al, 2011 ; Komeno et al, 2019 ). Arabinan consists of an α-1,5-linked L -arabinose backbone that can be mono- or di-substituted with either α-1,2-linked and/or α-1,3-linked L -arabinose ( Mohnen, 2008 ).…”

Characterization and functional analysis of two novel thermotolerant α-l-arabinofuranosidases belonging to glycoside hydrolase family 51 from Thielavia terrestris and family 62 from Eupenicillium parvum

“…The catalytic activities of ABFs have been shown to be affected by some divalent metal ions (Hu et al 2018;Kaur et al 2015;Komeno et al 2019). In our study, the activity of TtABF51A was suppressed by Cu 2+ , Zn 2+ , Mn 2+ , Fe 2+ , Ni 2+ , Co 2+ , and Ca 2+ at different levels.…”

Revisiting the Metabolic Capabilities of Bifidobacterium longum susbp. longum and Bifidobacterium longum subsp. infantis from a Glycoside Hydrolase Perspective

“…This cluster structure is present in the six genomes analyzed, although the genome of the strain F8 also contains other alpha-l-arabinofuranosidase enzymes, organized in different clusters including other sugar-actin enzymes nearby, such as beta-xylosidases and alpha-glucosidases (Figure 4). Indeed, arabinose and xylose can often be together in arabynoxylan structures [40,41], suggesting that those bifidobacteria possessing arabinose and xylose acting enzymes would have an additional advantage to metabolize this substrate.…”

“…Several papers have reported the involvement of Bifidobacterium alpha-l-arabinofuranosidases in the hydrolysis of arabinans, arabinoxylans, and arabinogalactans [40][41][42][43], and the prebiotic and/or bifidogenic activity of these substrates [44,45]. Since cereals are very rich in arabinan-containing sugar polymers, the fact that alpha-l-arabinosidases are over-represented in the subspecies longum, but are absent or scarcely present in infantis, could indicate the adaptation of the subspecies longum to fiber-rich vegetable substrates in an adult-like diet.…”

“…In this regard, it is worth noting that several authors have already demonstrated that B. longum subsp. longum representative strains are capable to degrade either arabinan or arabinoxylans [34,41,46]. Remarkably, among the bifidobacterial species with QPS status, l-arabinosidases and l-xylosidases appear to be restricted to B. longum subsp longum, suggesting that it represents a specific trait related to this bifidobactarial group that could be exploited in future biotechnological applications aimed at developing novel subspecies specific bifidogenic substrates and symbiotic formulations.…”