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

Division of Gene Technology, Katholieke Universiteit Leuven, Leuven, Belgium.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 10/2010; 402(4):644-50. DOI: 10.1016/j.bbrc.2010.10.075
Source: PubMed


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.

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    • "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). "
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    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.
    No preview · Article · Oct 2015 · Glycobiology
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    • "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. "
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    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 · Applied Microbiology and Biotechnology
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    • "The uptake and catabolism of XOS within bifidobacteria was recently proposed [37,60]. Comparative genomic of genes involved with XOS utilization within bifidobacteria (Figure 9) reflected a core gene structure of the XOS ABC transporter with a GH43 β-1,4-xylosidase (Balac_0517), while the occurrence of arabino-furanosidases, xylanases of GH8 and GH120 and carbohydrate esterases suggested more species and strain specific adaptation to utilize specific types of XOS e.g. "
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    ABSTRACT: Background Probiotic bifidobacteria in combination with prebiotic carbohydrates have documented positive effects on human health regarding gastrointestinal disorders and improved immunity, however the selective routes of uptake remain unknown for most candidate prebiotics. The differential transcriptomes of Bifidobacterium animalis subsp. lactis Bl-04, induced by 11 potential prebiotic oligosaccharides were analyzed to identify the genetic loci involved in the uptake and catabolism of α- and β-linked hexoses, and β-xylosides. Results The overall transcriptome was modulated dependent on the type of glycoside (galactosides, glucosides or xylosides) utilized. Carbohydrate transporters of the major facilitator superfamily (induced by gentiobiose and β-galacto-oligosaccharides (GOS)) and ATP-binding cassette (ABC) transporters (upregulated by cellobiose, GOS, isomaltose, maltotriose, melibiose, panose, raffinose, stachyose, xylobiose and β-xylo-oligosaccharides) were differentially upregulated, together with glycoside hydrolases from families 1, 2, 13, 36, 42, 43 and 77. Sequence analysis of the identified solute-binding proteins that determine the specificity of ABC transporters revealed similarities in the breadth and selectivity of prebiotic utilization by bifidobacteria. Conclusion This study identified the differential gene expression for utilization of potential prebiotics highlighting the extensive capabilities of Bifidobacterium lactis Bl-04 to utilize oligosaccharides. Results provide insights into the ability of this probiotic microbe to utilize indigestible carbohydrates in the human gastrointestinal tract.
    Full-text · Article · May 2013 · BMC Genomics
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