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 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

Gut microbiota modulation with long-chain corn bran arabinoxylan in adults with overweight and obesity is linked to an individualized temporal increase in fecal propionate

“…In our study, the dominant effects of arabinoxylan were directed toward two bacterial species, B. longum and P. copri, while nine additional OTUs showed smaller increases, including three Bacteroides species (e.g., B. ovatus, B. plebeius, and Bacteroides sp.). This high degree of specificity toward B. longum over other Bifidobacterium species is in agreement with other studies testing long-chain arabinoxylans [29][30][31] and genomic analyses that showed that genes encoding arabinoxylan-degrading glycosidase (e.g., βxylosidase and α-arabinofuranosidase) are conserved only among B. longum strains [32,33]. In contrast to the species-specific enrichment of B. longum, arabinoxylan enriched several species within the phylum Bacteroidetes that possess the genetic and functional traits necessary for accessing arabinoxylan [34][35][36][37][38].…”

“…and one member in CARG2 (an unclassified Lachnospiraceae), suggesting syntrophic relationships. B. longum has been shown to be a primary degrader of arabinoxylans [32,33] that is able to cleave the complex arabinoxylan structure by soluble arabinoxylan-degrading glycosidase [48,49]. This degradation could release xylan and AXOS (or even xylose, arabinose, and galactose) to xylanutilizing Bacteroides species like B. plebeius [36,37,40] and putative secondary fermenters like B. obeum and Subdoligranulum sp.…”

Identification of a Key Enzyme for the Hydrolysis of β-(1→3)-Xylosyl Linkage in Red Alga Dulse Xylooligosaccharide from Bifidobacterium Adolescentis

“…GH43_27 from B. longum sp. (BLLJ_1852) has shown an arabinandegrading exo-1,2-1,3-α-l-arabinofuranosidase [40] and the study showed that the activity of arabinose releasing from arabinoxylan of BLLJ_1852 was 0.62-fold of pNP-Ara, suggesting that the character did not agree with the high DX3 hydrolysis activity. In addition, they also mentioned that the subfamily 27 possessed various substrate specific enzymes.…”

“…GH43 is a large family containing 37 subfamilies, and many studies have been performed with the understanding of the relationship between Bifidobacterium sp. and XOS [39][40][41][42][43][44][45]. To find the key enzymes for DX3 hydrolysis, the enzymes in GH43 were evaluated.…”

Multiple Transporters and Glycoside Hydrolases Are Involved in Arabinoxylan-Derived Oligosaccharide Utilization in Bifidobacterium pseudocatenulatum

“…longum) are predominant (8,9). Genomic analysis and culture-based assays suggest that the latter two taxa are able to utilize plant-derived carbohydrates (10)(11)(12). Thus, plant-derived carbohydrates could be a driving factor in bifidobacterial colonization in the adult gut.…”