Substrate specificity of three recombinant α-L-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides

ArticleinBiochemical and Biophysical Research Communications 402(4):644-50 · October 2010with20 Reads
DOI: 10.1016/j.bbrc.2010.10.075 · Source: PubMed
Abstract
Bifidobacterium adolescentis possesses several arabinofuranosidases able to hydrolyze arabinoxylans (AX) and AX oligosaccharides (AXOS), the latter being bifidogenic carbohydrates with potential prebiotic properties. We characterized two new recombinant arabinofuranosidases, AbfA and AbfB, and AXH-d3, a previously studied arabinofuranosidase from B. adolescentis. AbfA belongs to glycoside hydrolase family (GH) 43 and removed arabinose from the C(O)2 and C(O)3 position of monosubstituted xylose residues. Furthermore, hydrolytic activity of AbfA was much larger towards substrates with a low amount of arabinose substitutions. AbfB from GH 51 only cleaved arabinoses on position C(O)3 of disubstituted xyloses, similar to GH 43 AXH-d3, making it to our knowledge, the first reported enzyme with this specificity in GH 51. AbfA acted synergistically with AbfB and AXH-d3. In combination with AXH-d3, it released 60% of arabinose from wheat AX. Together with recent studies on other AXOS degrading enzymes from B. adolescentis, these findings allowed us to postulate a mechanism for the uptake and hydrolysis of bifidogenic AXOS by this organism.
    • "However, up to now, no β-endoxylanases have been found in the genome of bifidobacteria. The only gene (i.e., BL1543) that was first annotated as a β-endoxylanase in B. longum NCC2705 (Schell et al., 2002 ) has shown to be an extracellular membraneassociated α-arabinofuranosidase (Lagaert et al., 2010Lagaert et al., , 2014 Rivière et al., 2014). For the complete utilization of AX, it is likely that most of the Bifidobacterium species require cooperation with β-endoxylanase-producing bacteria, such as Bacteroides and Roseburia species (Chassard et al., 2007; Dodd et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: With the increasing amount of evidence linking certain disorders of the human body to a disturbed gut microbiota, there is a growing interest for compounds that positively influence its composition and activity through diet. Besides the consumption of probiotics to stimulate favorable bacterial communities in the human gastrointestinal tract, prebiotics such as inulin-type fructans (ITF) and arabinoxylan-oligosaccharides (AXOS) can be consumed to increase the number of bifidobacteria in the colon. Several functions have been attributed to bifidobacteria, encompassing degradation of non-digestible carbohydrates, protection against pathogens, production of vitamin B, antioxidants, and conjugated linoleic acids, and stimulation of the immune system. During life, the numbers of bifidobacteria decrease from up to 90 % of the total colon microbiota in vaginally delivered breast-fed infants to < 5 % in the colon of adults and they decrease even more in that of elderly as well as in patients with certain disorders such as antibiotic-associated diarrhea, inflammatory bowel disease, irritable bowel syndrome, obesity, allergies, and regressive autism. It has been suggested that the bifidogenic effects of ITF and AXOS are the result of strain-specific yet complementary carbohydrate degradation mechanisms within cooperating bifidobacterial consortia. Except for a bifidogenic effect, ITF and AXOS also have shown to cause a butyrogenic effect in the human colon, i.e., an enhancement of colon butyrate production. Butyrate is an essential metabolite in the human colon, as it is the preferred energy source for the colon epithelial cells, contributes to the maintenance of the gut barrier functions, and has immunomodulatory and anti-inflammatory properties. It has been shown that the butyrogenic effects of ITF and AXOS are the result of cross-feeding interactions between bifidobacteria and butyrate-producing colon bacteria, such as Faecalibacterium prausnitzii (clostridial cluster IV) and Anaerostipes, Eubacterium and Roseburia species (clostridial cluster XIVa). These kinds of interactions possibly favor the co-existence of bifidobacterial strains with other bifidobacteria and with butyrate-producing colon bacteria in the human colon.
    Full-text · Article · Jun 2016
    • "able to release Araf substituents linked to the main chain. Depending on the linkage that is cleaved between the xylose and arabinose, as well as activity on either single or double substituted xylose units, different activity profiles can be distinguished for Arafases (Lagaert et al. 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: In this work, we present the first XOS degrading glycoside hydrolase from Weissella, WXyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa, and expressed in Escherichia coli. The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold β-propeller catalytic domain and a C-terminal β-sandwich domain of unknown function. WXyn43 hydrolyzed short (1–4)-β-d-xylooligosaccharides, with similar kcat/KM for xylobiose (X2) and xylotriose (X3) and clearly lower efficiency in xylotetraose (X4) conversion. WXyn43 displays the highest reported kcat for conversion of X3 (900 s−1 at 37°C) and X4 (770 s−1), and kcat for hydrolysis of X2 (907 s−1) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer–monomer hydrogen bonds, while the bonds creating dimer–dimer interactions only involved residues from the N-terminal domain. Docking of X2 and X3 in the active site shows interactions corresponding to subsites −1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X3 may be present.
    Article · Oct 2015
    • "The enzyme showed also activity on pNP-Araf, although much lower compared to the activity on AXs. The substrate specificity of the cloned enzyme indicates that it is an arabinofuranosidase belonging to the arabinoxylan arabinofuranohydrolase type (AXH) (Kormelink et al. 1991). We called the enzyme Abf43A. "
    [Show abstract] [Hide abstract] ABSTRACT: Arabinofuranosidase Abf43A from Bacillus sp. BP-7 is a newly discovered arabinoxylan arabinofuranohydrolase (AXH). It is a modular enzyme comprised of a GH43 catalytic domain and a carbohydrate-binding module of family CBM6. Recombinant Abf43A showed high activity on arabinoxylans, being rye arabinoxylan the preferred substrate on which the purified enzyme exhibited a K m of 10.6 ± 3.3 mg/ml and a V max of 29.2 ± 3.4 U/mg. Thin-layer chromatography analysis of hydrolysis products showed arabinose as the only sugar released by the enzyme from its substrates. The GH43 and CBM6 modules of the enzyme were individually cloned and expressed in Escherichia coli. While the isolated catalytic GH43 module did not show hydrolytic activity, the purified CBM6 bound to soluble arabinoxylan in affinity gel electrophoresis analysis. Evaluation of cooperative activity of arabinofuranosidase Abf43A with xylanases from families GH10, GH11, and GH30, (Xyn10A, Xyn11E, and Xyn30D from Paenibacillus barcinonensis) on arabinoxylan depolymerization revealed that the studied enzyme showed synergism with Xyn11E, a 2.54-fold increase in the amount of sugars released. On the contrary, Abf43A did not show synergism with the xylanases of families GH10 or GH30 evaluated. The enzyme characterized contributes to understanding the role of this class of enzymes in the catalytic depolymerization of arabinoxylans and their potential for the production of valuable xylooligosaccharides from these abundant plant polymers.
    Full-text · Article · Oct 2015
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