Annick Pollet

Structural determinants of the substrate specificities of xylanases from different glycoside hydrolase families.

Xylanases are of widespread importance in several food and non-food biotechnological applications. They degrade heteroxylans, a structurally heterogeneous group of plant cell wall polysaccharides, and other important components in various industrial processes. Because of the highly complex structures of heteroxylans, efficient utilization of xylanases in these processes requires an in-depth knowledge of their substrate specificity. A significant number of studies on the three-dimensional structures of xylanases from different glycoside hydrolase (GH) families in complex with the substrate provided insight into the different mechanisms and strategies by which xylanases bind and hydrolyze structurally different heteroxylans and xylo-oligosaccharides (XOS). Combined with reports on the hydrolytic activities of xylanases on decorated XOS and heteroxylans, major advances have been made in our understanding of the link between the three-dimensional structures and the substrate specificities of these enzymes. In this review, authors gave a concise overview of the structure–function relationship of xylanases from GH5, 8, 10, and 11. The structural basis for inter- and intrafamily variation in xylanase substrate specificity was discussed as are the implications for heteroxylan degradation.

Prebiotic effects and intestinal fermentation of cereal arabinoxylans and arabinoxylan oligosaccharides in rats depend strongly on their structural properties and joint presence

SCOPE Cereal arabinoxylan (AX) is one of the main dietary fibers in a balanced human diet. To gain insight into the importance of structural features of AX for their prebiotic potential and intestinal fermentation properties, a rat trial was performed. METHODS AND RESULTS A water unextractable AX-rich preparation (WU-AX, 40% purity), water extractable AX (WE-AX, 81% purity), AX oligosaccharides (AXOS, 79% purity) and combinations thereof were included in a standardized diet at a 5% AX level. WU-AX was only partially fermented in the ceco-colon and increased the level of butyrate and of butyrate producing Roseburia/E. rectale spp. Extensive fermentation of WE-AX and/or AXOS reduced the pH, suppressed relevant markers of the proteolytic breakdown and induced a selective bifidogenic response. Compared with WE-AX, AXOS showed a slightly less pronounced effect in the colon as its fermentation was virtually complete in the cecum. Combining WU-AX and AXOS caused a striking synergistic increase in cecal butyrate levels. WU-AX, WE-AX and AXOS together combined a selective bifidogenic effect in the colon with elevated butyrate levels, a reduced pH and suppressed proteolytic metabolites. CONCLUSION The prebiotic potential and fermentation characteristics of cereal AX depend strongly on their structural properties and joint presence.

β-Xylosidases and α-l-arabinofuranosidases: Accessory enzymes for arabinoxylan degradation

Arabinoxylan (AX) is among the most abundant hemicelluloses on earth and one of the major components of feedstocks that are currently investigated as a source for advanced biofuels. As global research into these sustainable biofuels is increasing, scientific knowledge about the enzymatic breakdown of AX advanced significantly over the last decade. This review focuses on the exo-acting AX hydrolases, such as α-arabinofuranosidases and β-xylosidases. It aims to provide a comprehensive overview of the diverse substrate specificities and corresponding structural features found in the different glycoside hydrolase families. A careful review of the available literature reveals a marked difference in activity between synthetically labeled and naturally occurring substrates, often leading to erroneous enzymatic annotations. Therefore, special attention is given to enzymes with experimental evidence on the hydrolysis of natural polymers.

A Simple and Accurate Method for Determining Wheat Grain Fructan Content and Average Degree of Polymerization

An improved method for the measurement of fructans in wheat grains is presented. A mild acid treatment is used for fructan hydrolysis, followed by analysis of the released glucose and fructose with high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Not only the amount of fructose set free from fructans but also the released glucose can be quantified accurately, allowing determination of the average degree of polymerization of fructans (DP(av)). Application of the mild acid treatment to different grain samples demonstrated that a correction should be made for the presence of sucrose and raffinose, but not for stachyose or higher raffinose oligosaccharides. The fructan content and DP(av) of spelt flour, wheat flour, and whole wheat flour were 0.6%, 1.2%, and 1.8% of the total weight and 4, 5, and 6, respectively. Validation experiments demonstrate that the proposed quantification method is accurate and repeatable and that also the DP(av) determination is precise.

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

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.

In Vitro Fermentation of Arabinoxylan Oligosaccharides and Low Molecular Mass Arabinoxylans with Different Structural Properties from Wheat (Triticum aestivum L.) Bran and Psyllium (Plantago ovata Forsk) Seed Husk

Ball milling was used for producing complex arabinoxylan oligosaccharides (AXOS) and low molecular mass arabinoxylans (AX) from wheat bran, pericarp-enriched wheat bran, and psyllium seed husk. The arabinose to xylose ratio of the samples produced varied between 0.14 and 0.92, and their average degree of polymerization (avDP) ranged between 42 and 300. Their fermentation for 48 h in an in vitro system using human colon suspensions was compared to enzymatically produced wheat bran AXOS with an arabinose to xylose ratio of 0.22 and 0.34 and an avDP of 4 and 40, respectively. Degrees of AXOS fermentation ranged from 28% to 50% and were lower for the higher arabinose to xylose ratio and/or higher avDP materials. Arabinose to xylose ratios of the unfermented fractions exceeded those of their fermented counterparts, indicating that molecules less substituted with arabinose were preferably fermented. Xylanase, arabinofuranosidase, and xylosidase activities increased with incubation time. Enzyme activities in the samples containing psyllium seed husk AX or psyllium seed husk AXOS were generally higher than those in the wheat bran AXOS preparations. Fermentation gave rise to unbranched short-chain fatty acids. Concentrations of acetic, propionic, and butyric acids increased to 1.9-2.6, 1.9-2.8, and 1.3-2.0 times their initial values, respectively, after 24 h incubation. Results show that the human intestinal microbiota can at least partially use complex AXOS and low molecular mass AX. The tested materials are thus interesting physiologically active carbohydrates.

Crystallographic and activity-based evidence for thumb flexibility and its relevance in glycoside hydrolase family 11 xylanases

Enzyme intramolecular mobility and conformational changes of loops in particular play a significant role in biocatalysis. In this respect, the highly conserved thumb loop of glycoside hydrolase family (GH) 11 xylanases is an intriguing and characteristic structural element, of which the true dynamic nature and function in catalysis is still unknown. Crystallographic analysis of the structure of a Bacillus subtilis xylanase A mutant, found as a dimer in an asymmetric unit, revealed that the thumb region can adopt an extended conformation, which is stabilized in the crystal lattice through intermolecular contacts. In contrast to the closed thumb conformation of GH11 xylanases and the previously observed small conformational changes upon substrate binding, a relocation of the tip of the thumb of more than 15 Å was observed. Site‐directed mutagenesis of five thumb residues, including putative hinge point residues, and enzyme kinetics assays showed that Arg112, Asn114, and Thr126 play a role in the open‐close thumb movement. Replacement of Arg112 by glycine or proline caused a strong decrease of turnover numbers and elevated Michaelis constants on xylan. Mutant N114P hindered thumb movement, provoking a fourfold decrease of turnover numbers and a sharp rise in Michaelis constants, whereas the proline mutant of Thr126 displayed an increase in specific activity. The observation that extensive thumb opening is possible combined with the kinetic data suggests that the thumb plays a crucial role in both binding of substrate and release of product from the active site. Proteins 2009.

Characterization of two β-xylosidases from Bifidobacterium adolescentis and their contribution to the hydrolysis of prebiotic xylooligosaccharides

Xylooligosaccharides have strong bifidogenic properties and are increasingly used as a prebiotic. Nonetheless, little is known about the degradation of these substrates by bifidobacteria. We characterized two recombinant β-xylosidases, XylB and XylC, with different substrate specificities from Bifidobacterium adolescentis. XylB is a novel β-xylosidase that belongs to the recently introduced glycoside hydrolase family 120. In contrast to most reported β-xylosidases, it shows only weak activity on xylobiose and prefers xylooligosaccharides with a degree of polymerization above two. The remaining xylobiose is efficiently hydrolyzed by the second B. adolescentis β-xylosidase, XylC, a glycoside hydrolase of family 43. Furthermore, XylB releases more xylose from arabinose-substituted xylooligosaccharides than XylC (30% and 20%, respectively). The different specificities of XylB, XylC, and the recently described reducing-end xylose-releasing exo-oligoxylanase RexA show how B. adolescentis can efficiently degrade prebiotic xylooligosaccharides.

Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis

ABSTRACT The family 8 glycoside hydrolase (RexA) from Bifidobacterium adolescentis was expressed in Escherichia coli. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the B. adolescentis breakdown of prebiotic xylooligosaccharides.

Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases

The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site‐directed mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. Modification of the SBS resulted in an up to three‐fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates. Weakening of the SBS increased Kd values by up to 70‐fold in binding affinity tests using natural substrates. The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such structural elements can increase the efficiency of these single domain enzymes on their natural substrates.