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.

Keywords: dietary fiber; gut microbiota; hemicellulose; intestinal microbiota; prebiotics; probiotics.

FIG 1

Growth of B. longum subsp. longum JCM 1217 in media containing different carbon sources. The strain was cultured at 37°C in sugar-restricted basal medium supplemented with 2.0% different carbon sources under anaerobic conditions. The OD₆₀₀ was measured every 3 h for 48 h. Error bars indicate SD (n = 6).

FIG 2

Characterization of BlArafC (BLLJ_1852) and BlArafB (BLLJ_1853). (A) Domain structure of BlArafC and BlArafB. (B) SDS-PAGE of purified BlArafC. (C) SDS-PAGE of purified BlArafB. Lanes M, molecular size markers.

FIG 3

General properties of recombinant BlArafC and BlArafB. pNP-α-Araf was incubated with recombinant enzymes at 37°C. (A) Effect of pH on BlArafC. (B) Effect of pH on BlArafB. (C) Effect of temperature on BlArafC. (D) Effect of temperature on BlArafB. (E) Thermal stability of BlArafB at high temperatures. Error bars indicate SD (n = 3). For stability tests, enzymes were preincubated in each pH buffer for 16 h at 37°C or in sodium acetate buffer (pH 6.0) at each temperature for 1 h.

FIG 4

Substrate specificities of BlArafC and BlArafB. The substrates were incubated with recombinant BlArafC or BlArafB at 37°C overnight and analyzed by TLC. (A) Action on synthetic substrates. α-Araf, pNP-α-l-arabinofuranoside; α-Arap, pNP-α-l-arabinopyranoside; β-Xylp, pNP-β-xylopyranoside; β-Galp, pNP-β-galactopyranoside; Ara, standard l-arabinose; Xyl, standard d-xylose; Gal, standard d-galactose. (B) Action of BlArafC on natural substrates. AX, arabinoxylan; AG, arabinogalactan; AN, arabinan; Ara, standard l-arabinose. (C) Action of BlArafB on natural substrates. (D) Action of BlArafC and BlArafB on synthetic disaccharide substrates. α2, Arafα1,2Arafα1-OMe; α3, Arafα1,3Arafα1-OMe; α5, Arafα1,5Arafα1-OMe; Ara, standard l-arabinose. (E) Synergy effect of coincubation of BlArafC and BlArafB on arabinan degradation. Arabinan was incubated with either BlArafC or BlArafB, or both. The amount of released l-arabinose was determined and the hydrolysis (in percent) of arabinan is shown above the panel. Ara, standard l-arabinose. ND, not detected. 1-Butanol–acetic acid–water (2:1:1, by volume) was used as a developing solvent in the assays whose results are shown in panels A to C and E; chloroform-methanol-acetic acid (6:1:1, by volume) was used in the assay whose results are shown in panel D.

FIG 5

Effects of divalent metal ions on the activities of BlArafC and BlArafB. (A) Effects of metal ions on the activities of recombinant enzymes. The divalent metal ions were added at 5.0 mM. (B) Effects of various concentrations of EDTA on recombinant enzymes. (C) Restoration of enzyme activity by addition of divalent metal ions after treatment with EDTA. pNP-α-Araf was used as a substrate in all experiments. Error bars indicate SD (n = 3).

FIG 6

(A) Map of the hemicellulose-degrading gene cluster in B. longum subsp. longum JCM 1217. Gray boxes, glycosidases; striped boxes, sugar transporters; black boxes, transcriptional regulators; white boxes, hypothetical proteins. BlArafE (BLLJ_1850) and BlArafD (BLLJ_1851) have two GH43 domains, and the N-terminal GH43 domain of BlArafD belongs to an unclassified (UC) subfamily. (B) Conservation of the GH43 cluster in various B. longum strains. The scores indicated above the boxes indicate the percent identity of the amino acid sequences with those of B. longum subsp. longum JCM 1217. (C) Growth of B. longum subsp. longum JCM 1217 and B. longum NCC2705 in sugar-restricted basal medium supplemented with 1.0% arabinan or l-arabinose. The strains were cultured at 37°C under anaerobic conditions. Error bars indicate SD (n = 3).