ß-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 ß-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.

Analysis of storage and structural carbohydrates in developing wheat (Triticum aestivum L.) grains using quantitative analysis and microscopy.

In this paper, the content of all major carbohydrates and the spatial distribution of starch, arabinoxylan and ß-glucan in developing wheat kernels (Triticum aestivum L. var. Homeros) from anthesis until maturity were studied. By combining information from microscopy and quantitative analysis, a comprehensive overview on the changes in storage and structural carbohydrates in developing grains was obtained. In the phase of cell division and expansion, grains were characterized by a rapid accumulation of water and high concentrations of the water-soluble carbohydrates fructan, sucrose, glucose and fructose. During the grain filling phase, starch, protein, ß-glucan and arabinoxylan accumulated, while during grain maturation and desiccation, only a loss of moisture took place. The comprehensive approach of this study allowed finding correlations, which are discussed within the context of grain development. Particular attention was given to the transient presence of high fructan concentrations, which was associated with the most striking compositional changes during grain development.

A versatile and colorful screening tool for the identification of arabinofuranose-acting enzymes.

Selecting wall-nibblers: Three 4-nitrocatechol derivatives were designed to facilitate high-throughput screening of arabinofuranose hydrolases, enzymes that typically digest plant cell walls. The designed compounds can be used in solid and liquid media, and, importantly, one allows the specific detection of AXH-d, a specialized enzyme that only releases L-arabinose from disubstituted D-xylosyl moieties.

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.

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.

Evaluation of the xylan breakdown potential of eight mesophilic endoxylanases.

In biomass degradation using simultaneous saccharification and fermentation (SSF), there is a need for efficient biomass degrading enzymes that can work at lower temperatures suitable for yeast fermentation. As xylan is an important lignocellulosic biomass constituent, this study aimed at investigating the possible differences in xylan breakdown potential of endoxylanases using eight different endoxylanases at conditions relevant for SSF. Both solubilising and degrading capacities of the endoxylanases were investigated using water-insoluble and water-soluble oat spelt xylan as model substrates for biomass xylan. Results showed that selecting for combinations of endoxylanases that are efficient at solubilising xylan on the one hand and degrading it to large extent on the other hand, coupled to high specific activities, seems the best option for complete xylan breakdown in lignocellulosic biomass conversion using SSF.

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.

Characterization of two ß-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 ß-xylosidases, XylB and XylC, with different substrate specificities from Bifidobacterium adolescentis. XylB is a novel ß-xylosidase that belongs to the recently introduced glycoside hydrolase family 120. In contrast to most reported ß-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 ß-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.

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 K(d) 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.

Crystallization and preliminary X-ray analysis of a cold-active endo-ß-1,4-D-xylanase from glycoside hydrolase family 8.

Endo-ß-1,4-D-xylanases are used in a multitude of industrial applications. Native crystals of a cold-adapted xylanase from glycoside hydrolase family 8 were obtained by the vapour-diffusion technique. The crystals belonged to space group I222, with unit-cell parameters a=46.6, b=110.8, c=150.2 Šat 100 K, and diffracted to 2.7 Šresolution at a synchrotron source. The asymmetric unit is likely to contain one molecule, with a VM of 2.07 Å3 Da(-1), corresponding to a solvent content of ∼40%.

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.

Functional analysis of glycoside hydrolase family 8 xylanases shows narrow but distinct substrate specificities and biotechnological potential.

The potential of glycoside hydrolase family (GH) 8 xylanases in biotechnological applications is virtually unexplored. Therefore, the substrate preference and hydrolysis product profiles of two GH8 xylanases were evaluated to investigate their activities and substrate specificities. A GH8 xylanase from an uncultured bacterium (rXyn8) shows endo action but very selectively releases xylotriose from its substrates. It has a higher activity than the Pseudoalteromonas haloplanktis GH8 endo-xylanase (PhXyl) on xylononaose and smaller xylo-oligosaccharides. PhXyl preferably degrades xylan substrates with a high degree of polymerization. It is sterically more hindered by arabinose substituents than rXyn8, producing larger end hydrolysis products. The specificities of rXyn8 and PhXyl differ completely from these of the previously described GH8 xylanases from Bifidobacterium adolescentis (BaRexA) and Bacillus halodurans (BhRex). As reducing-end xylose-releasing exo-oligoxylanases, they selectively release xylose from the reducing end of small xylo-oligosaccharides. The findings of this study show that GH8 xylanases have a narrow substrate specificity, but also one that strongly varies between family members and is distinct from that of GH10 and GH11 xylanases. Structural comparison of rXyn8, PhXyl, BaRexA, and BhRex showed that subtle amino acid changes in the glycon as well as the aglycon subsites probably form the basis of the observed differences between GH8 xylanases. GH8 xylanases, therefore, are an interesting group of enzymes, with potential towards engineering and applications.

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.

Mutagenesis and subsite mapping underpin the importance for substrate specificity of the aglycon subsites of glycoside hydrolase family 11 xylanases.

Glycoside hydrolase family (GH) 11 xylanase A from Bacillus subtilis (BsXynA) was subjected to site-directed mutagenesis to probe the role of aglycon active site residues with regard to activity, binding of decorated substrates and hydrolysis product profile. Targets were those amino acids identified to be important by 3D structure analysis of BsXynA in complex with substrate bound in the glycon subsites and the +1 aglycon subsite. Several aromatic residues in the aglycon subsites that make strong substrate-protein interactions and that are indispensable for enzyme activity, were also important for the specificity of the xylanase. In the +2 subsite of BsXynA, Tyr65 and Trp129 were identified as residues that are involved in the binding of decorated substrates. Most interestingly, replacement of Tyr88 by Ala in the +3 subsite created an enzyme able to produce a wider variety of hydrolysis products than wild type BsXynA. The contribution of the +3 subsite to the substrate specificity of BsXynA was established more in detail by mapping the enzyme binding site of the wild type xylanase and mutant Y88A with labelled xylo-oligosaccharides. Also, the length of the cord - a long loop flanking the aglycon subsites of GH11 xylanases - proved to impact the hydrolytic action of BsXynA. The aglycon side of the active site cleft of BsXynA, therefore, offers great potential for engineering and design of xylanases with a desired specificity.

Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI-IA and TAXI-IIA.

Triticum aestivum xylanase inhibitor (TAXI)-type inhibitors are active against microbial xylanases from glycoside hydrolase family 11, but the inhibition strength and the specificity towards different xylanases differ between TAXI isoforms. Mutational and biochemical analyses of TAXI-I, TAXI-IIA and Bacillus subtilis xylanase A showed that inhibition strength and specificity depend on the identity of only a few key residues of inhibitor and xylanase [Fierens K et al. (2005) FEBS J 272, 5872-5882; Raedschelders G et al. (2005) Biochem Biophys Res Commun335, 512-522; Sorensen JF & Sibbesen O (2006) Protein Eng Des Sel 19, 205-210; Bourgois TM et al. (2007) J Biotechnol 130, 95-105]. Crystallographic analysis of the structures of TAXI-IA and TAXI-IIA in complex with glycoside hydrolase family 11 B. subtilis xylanase A now provides a substantial explanation for these observations and a detailed insight into the structural determinants for inhibition strength and specificity. Structures of the xylanaseinhibitor complexes show that inhibition is established by loop interactions with active-site residues and substrate-mimicking contacts in the binding subsites. The interaction of residues Leu292 of TAXI-IA and Pro294 of TAXI-IIA with the -2 glycon subsite of the xylanase is shown to be critical for both inhibition strength and specificity. Also, detailed analysis of the interaction interfaces of the complexes illustrates that the inhibition strength of TAXI is related to the presence of an aspartate or asparagine residue adjacent to the acid/base catalyst of the xylanase, and therefore to the pH optimum of the xylanase. The lower the pH optimum of the xylanase, the stronger will be the interaction between enzyme and inhibitor, and the stronger the resulting inhibition.

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 A 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.

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

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.

Unprocessed barley aleurone endo-beta-1,4-xylanase X-I is an active enzyme.

Endo-beta-1,4-xylanase X-I is a major hydrolase produced by the aleurone tissue of germinating barley grain. It was previously reported that this cytosolic enzyme is synthesized as an inactive precursor which is proteolytically processed to active forms upon its programmed cell death dependent release. We here demonstrate, however, that the precursor form of X-I is an active enzyme. Purified recombinant precursor X-I was characterised with respect to its molecular weight, iso-electric point and temperature and pH activity and stability. Analysis of the hydrolysis products showed that it is an endo-acting enzyme which has the striking ability to release xylose from both polymeric xylan as well as from small xylo-oligosaccharides. The implications of these findings in relation to the putative role of the N-terminal propeptide as a carbohydrate binding module and the possible consequences for the way X-I fulfils its role in the germination process, are discussed.