Degradation and Utilization of Xylans by the Rumen AnaerobePrevotella bryantii(formerlyP. ruminicolasubsp.brevis) B14

Freshly harvested whole cells from cultures ofP. bryantiiB₁4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantiiB₁4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation withP. bryantiishowed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobeRuminococcus flavefaciensgave little increase in overall pentose utilization suggesting that externalP. bryantiixylanases are as effective as the clonedR. flavefaciensenzymes in releasing products that can be utilised byP. bryantiicells. The xylanase system ofP. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

High genetic diversity and different distributions of glycosyl hydrolase family 10 and 11 xylanases in the goat rumen

Background

The rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.

Methodology/Principal Findings

We explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.

Conclusion/Significance

The numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.     

Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Fusarium graminearum xylanases show different functional stabilities,substrate specificities and inhibition sensitivities

When grown on arabinoxylan as the sole carbon source, the cereal phytopathogen Fusarium graminearum expresses four xylanases. Cloning and heterologous expression of the corresponding xylanase encoding genes and analysis of general biochemical properties, substrate specificities and inhibition sensitivities revealed some marked differences. XylA and XylB are glycoside hydrolase family (GH) 11 xylanases, while XylC and XylD belong to GH10. pH and temperature for optimal activity of the enzymes were between 6.0 and 7.0 and 40 °C, respectively. Interestingly, XylC displayed remarkable pH stability as it retained most of its activity even after pre-incubation at pH 1.0 and 13.0 for 120 min at room temperature. All xylanases hydrolysed xylotetraose, xylopentaose and xylohexaose, but to different extents, while only XylC and XylD hydrolysed xylotriose. The two GH10 xylanases released a higher percentage of smaller products from xylan and xylo-oligosaccharides than did their GH11 counterparts. Analysis of kinetic properties revealed that wheat arabinoxylan is the favoured XylC substrate while XylA and XylB prefer sparsely substituted oat spelt xylan. XylC and XylD were inhibited by xylanase inhibiting protein (XIP), while XylA and XylB were sensitive to Triticum aestivum xylanase inhibitor (TAXI). Because of its pH stability and preference for arabinoxylan, XylC is a valuable candidate for use in biotechnological applications.     

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.     

Structural Insights into the Specificity of Xyn10B from Paenibacillus barcinonensis and Its Improved Stability by Forced Protein Evolution

Paenibacillus barcinonensis is a soil bacterium bearing a complex set of enzymes for xylan degradation, including several secreted enzymes and Xyn10B, one of the few intracellular xylanases reported to date. The crystal structure of Xyn10B has been determined by x-ray analysis. The enzyme folds into the typical (β/α)₈ barrel of family 10 glycosyl hydrolases (GH10), with additional secondary structure elements within the β/α motifs. One of these loops -L7- located at the β7 C terminus, was essential for xylanase activity as its partial deletion yielded an inactive enzyme. The loop contains residues His²⁴⁹–Glu²⁵⁰, which shape a pocket opened to solvent in close proximity to the +2 subsite, which has not been described in other GH10 enzymes. This wide cavity at the +2 subsite, where methyl-2,4-pentanediol from the crystallization medium was found, is a noteworthy feature of Xyn10B, as compared with the narrow crevice described for other GH10 xylanases. Docking analysis showed that this open cavity can accommodate glucuronic acid decorations of xylo-oligosaccharides. Co-crystallization experiments with conduramine derivative inhibitors supported the importance of this open cavity at the +2 subsite for Xyn10B activity. Several mutant derivatives of Xyn10B with improved thermal stability were obtained by forced evolution. Among them, mutant xylanases S15L and M93V showed increased half-life, whereas the double mutant S15L/M93V exhibited a further increase in stability, showing a 20-fold higher heat resistance than the wild type xylanase. All the mutations obtained were located on the surface of Xyn10B. Replacement of a Ser by a Leu residue in mutant xylanase S15L can increase hydrophobic packing efficiency and fill a superficial indentation of the protein, giving rise to a more compact structure of the enzyme.     

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid

Treatment with rumen fluid improves methane production from non-degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community’s structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium, Diploplastron, and Eudiplodinium) and fungal (e.g., Neocallimastix, Orpinomyces, and Olpidium) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.     

Molecular detection and diversity of xylanase genes in alpine tundra soil

Xylan is a major polysaccharide in plant cell walls, and its degradation is mainly conducted by microbial xylanases in nature. To explore the xylanase diversity in the environment, two sets of degenerate primers were designed based on the microbial xylanase sequences in Pfam database of glycosyl hydrolase (GH) family 10 and 11 and were used to amplify objective gene fragments directly from the alpine tundra soil DNA of the Tianshan Mountains, China. Ninety-six distinct GH 10 and 31 GH 11 xylanase gene fragments were retrieved, and most of them have low identities with known sequences in GenBank. Based on phylogenetic analysis, all of the GH 10 xylanase sequences fell into six clusters and were related to xylanases from Actinobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Firmicutes, and Acidobacteria. Three clusters of GH 11 xylanase sequences were established, and two of them were related with enzymes from fungi. These results indicated the diversity of xylanase genes in this cold environment. Four xylanolytic strains were isolated from the soil, and GH 10 xylanase gene fragments were cloned using the same primers. A full-length gene was obtained and expressed in Escherichia coli, and the recombinant enzyme showed some cold-related characteristics. Our study provides an efficient molecular approach to study xylanase in complex environments and casts an insight into the diversity and distribution of xylanases in a cold environment, which is very meaningful to understand their roles in xylan degradation in nature.     

High Phylogenetic Diversity of Glycosyl Hydrolase Family 10 and 11 Xylanases in the Sediment of Lake Dabusu in China

Soda lakes are one of the most stable naturally occurring alkaline and saline environments, which harbor abundant microorganisms with diverse functions. In this study, culture-independent molecular methods were used to explore the genetic diversity of glycoside hydrolase (GH) family 10 and GH11 xylanases in Lake Dabusu, a soda lake with a pH value of 10.2 and salinity of 10.1%. A total of 671 xylanase gene fragments were obtained, representing 78 distinct GH10 and 28 GH11 gene fragments respectively, with most of them having low homology with known sequences. Phylogenetic analysis revealed that the GH10 xylanase sequences mainly belonged to Bacteroidetes, Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while the GH11 sequences mainly consisted of Actinobacteria, Firmicutes and Fungi. A full-length GH10 xylanase gene (xynAS10-66) was directly cloned and expressed in Escherichia coli, and the recombinant enzymes showed high activity at alkaline pH. These results suggest that xylanase gene diversity within Lake Dabusu is high and that most of the identified genes might be novel, indicating great potential for applications in industry and agriculture.     

Characterization of a Family GH5 Xylanase with Activity on Neutral Oligosaccharides and Evaluation as a Pulp Bleaching Aid

A new bacterial xylanase belonging to family 5 of glycosyl hydrolases was identified and characterized. The xylanase, Xyn5B from Bacillus sp. strain BP-7, was active on neutral, nonsubstituted xylooligosaccharides, showing a clear difference from other GH5 xylanases characterized to date that show a requirement for methyl-glucuronic acid side chains for catalysis. The enzyme was evaluated on Eucalyptus kraft pulp, showing its effectiveness as a bleaching aid.The catabolic breakdown of xylan is a critical step in the recycling of carbon in nature and has been targeted as a subject of intense research as a renewable energy resource as well as for bioconversion of plant biomass into high-added-value products (21, 29, 37, 40). Biodegradation of xylan is a complex process that requires the coordinate action of several enzymes, among which xylanases (1,4-β-D-xylan xylanohydrolase; EC 3.2.1.8), cleaving internal linkages on the β-1,4-xylose backbone, play a key role.Most known xylanases are grouped into glycoside hydrolase (GH) families 10 and 11 (CAZy [Carbohydrate-Active enZYmes] database) (17), although a few xylanases have recently been ascribed to glycoside hydrolase families 5, 7, 8, and 43 (8, 9, 24). Among xylanases not grouped in the typical families GH10 and GH11, only two xylanases belonging to family GH5 have been biochemically characterized in detail. The enzymes XynA from Erwinia chrysanthemi (18, 39) and XynC from Bacillus subtilis (32) hydrolyze glucuronoxylan to branched xylooligosaccharides. The activity of Erwinia chrysanthemi XynA has also been evaluated on other substrates containing xylose, showing an absolute requirement for methyl-glucuronic substitutions. In this way, only methyl-glucuronic acid-branched oligosaccharides can be cleaved by XynA, whereas linear xylooligosaccharides or arabinoxylans are not cleaved by this enzyme (39). This type of xylanases must play an important role in complementing the action of GH10 and GH11 enzymes during depolymerization of glucuronoxylans in lignocellulosic fibers.Xylanases are widely used in the pulp industry to enhance the effectiveness of bleaching agents, thereby reducing the generation of toxic wastes (adsorbable organic halogens; AOX) (1, 38). Several reports have evaluated the effectiveness of family GH10 and GH11 xylanases on the bleaching process, showing that GH11 xylanases usually display better performance (7, 12), although there are many other factors that contribute to the bleach-boosting effect of a xylanase, such as the source of the pulp and the pulping process itself (6, 11). Besides their contribution to the increase in brightness, an innovative aspect of the application of xylanases is their contribution to the removal of hexenuronic acids (HexA) produced during the kraft cooking process, which can accelerate the brightness reversion (yellowing tendency) of paper (35). However, it remains to be known if all xylanases are capable of removing HexAs and/or enhancing bleachability.Bacillus sp. strain BP-7 is a xylanolytic strain isolated from agricultural soils (25). It shows a multiple enzymatic system for xylan degradation, including a GH11 xylanase cloned and characterized previously (13). In this work, we describe the identification and cloning of a second xylanase from the strain, belonging to the GH5 family. The enzyme hydrolyzes linear xylooligosaccharides, clearly differing from the two GH5 xylanases characterized up to date. The new enzyme has been tested on Eucalyptus pulps, showing good performance as a bleaching aid. The results obtained suggest an important role for the enzyme in xylan degradation and indicate the potential of this xylanase for biotechnological applications in the bioconversion of glucuronoxylan-containing biomass.     

Action of different types of endoxylanases on eucalyptus xylan in situ

Most studies of the mode of action of industrially important endoxylanases have been done on alkali extracted-plant xylan. In just few cases, the native form of the polysaccharide, acetylated xylan, was used as a substrate. In this work action of xylanases belonging to three glycoside hydrolase families, GH10, GH11, and GH30 was investigated on acetylglucuronoxylan directly in hardwood cell walls. Powdered eucalyptus wood was used as xylanase substrate. Enzyme-generated fragments were characterized by TLC, MALDI ToF MS, and NMR spectroscopy. All three xylanases generated from eucalyptus wood powder acetylated xylooligosaccharides. Those released by GH10 enzyme were the shortest, and those released by GH30 xylanase were of the largest diversity. For GH30 xylanase the 4-O-methyl-D-glucuronic acid (MeGlcA) side residues function as substrate specificity determinants regardless the acetylation of the neighboring hydroxyl group. Much simpler xylooligosaccharide patterns were observed when xylanases were applied in combination with carbohydrate esterase family 6 acetylxylan esterase. In the presence of the esterase, all aldouronic acids remained 3-O-acetylated on the xylopyranosyl (Xylp) residue substituted with MeGlcA. The 3-O-acetyl group, in contrast to the acetyl groups of otherwise unsubstituted Xylp residues, does not affect the mode of action of endoxylanases, but contributes to recalcitrance of the acidic xylan fragments. The results confirm importance of acetylxylan esterases in microbial degradation of acetylated hardwood glucuronoxylan. They also point to still unresolved question of efficient enzymatic removal of the 3-O-acetyl group on MeGlcA-substituted Xylp residues negatively affecting the saccharification yields.

     

Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from Cellulomonas pachnodae

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.     

Sequencing and Expression of Additional Xylanase Genes from the Hyperthermophile Thermotoga maritima FjSS3B.1

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking–PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87°C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.     

Two New Xylanases with Different Substrate Specificities from the Human Gut Bacterium Bacteroides intestinalis DSM 17393

Xylan is an abundant plant cell wall polysaccharide and is a dominant component of dietary fiber. Bacteria in the distal human gastrointestinal tract produce xylanase enzymes to initiate the degradation of this complex heteropolymer. These xylanases typically derive from glycoside hydrolase (GH) families 10 and 11; however, analysis of the genome sequence of the xylan-degrading human gut bacterium Bacteroides intestinalis DSM 17393 revealed the presence of two putative GH8 xylanases. In the current study, we demonstrate that the two genes encode enzymes that differ in activity. The xyn8A gene encodes an endoxylanase (Xyn8A), and rex8A encodes a reducing-end xylose-releasing exo-oligoxylanase (Rex8A). Xyn8A hydrolyzed both xylopentaose (X₅) and xylohexaose (X₆) to a mixture of xylobiose (X₂) and xylotriose (X₃), while Rex8A hydrolyzed X₃ through X₆ to a mixture of xylose (X₁) and X₂. Moreover, rex8A is located downstream of a GH3 gene (xyl3A) that was demonstrated to exhibit β-xylosidase activity and would be able to further hydrolyze X₂ to X₁. Mutational analyses of putative active site residues of both Xyn8A and Rex8A confirm their importance in catalysis by these enzymes. Recent genome sequences of gut bacteria reveal an increase in GH8 Rex enzymes, especially among the Bacteroidetes, indicating that these genes contribute to xylan utilization in the human gut.     

Cloning and characterization of a xylanase,KRICT PX1 from the strain Paenibacillus sp. HPL-001

The KRICT PX1 gene (GB: FJ380951) consisting of 996 bp encoding a protein of 332 amino acids (38.1 kDa) from the recently isolated Paenibacillus sp. strain HPL-001 (KCTC11365BP) has been cloned and expressed in Escherichia coli. The xylanase KRICT PX1 showed high activity on birchwood xylan, and was active over a pH range of 5.0 to 11.0, with two optima at pH 5.5 and 9.5 at 50 °C with K_m value of 5.35 and 3.23, respectively. The xylanase activity was not affected by most salts, such as NaCl, LiCl, KCl, NH₄Cl, CaCl₂, MgCl₂, MnCl₂, and CsCl₂ at 1 mM, but affected by CuSO₄, ZnSO₄, and FeCl₃. One mM of EDTA, 2-mercaptoethanol, and PMSF did not affect the xylanase activity. TLC analysis of the catalyzed products after reaction with birchwood xylan revealed that xylobiose was the major product with smaller amounts of xylotriose and xylose. A similarity analysis of the amino acids in KRICT PX1 resulted 72% identity with xylanase from Geobacillus stearothermophilus (GB: ZP_03040360), 70% identity with intracellular xylanase from an uncultured bacterium (GB: AAP51133), 68% identity with endo-1-4-xylanse from Paenibacillus sp. (GB: ZP_02847150). In addition, the amino acid alignment of KRICT PX1 with glycosyl hydralase (GH) family 10 xylanases revealed a high degree of homology in highly conserved regions including the catalytic sites, and this was confirmed through PROSITE scan. These results imply that KRICT PX1 is a new xylanase gene, and this alkaline xylanase belongs to GH family 10.     

Structural and biochemical analysis of Cellvibrio japonicus xylanase 10C: how variation in substrate-binding cleft influences the catalytic profile of family GH-10 xylanases

Microbial degradation of the plant cell wall is the primary mechanism by which carbon is utilized in the biosphere. The hydrolysis of xylan, by endo-beta-1,4-xylanases (xylanases), is one of the key reactions in this process. Although amino acid sequence variations are evident in the substrate binding cleft of "family GH10" xylanases (see afmb.cnrs-mrs.fr/CAZY/), their biochemical significance is unclear. The Cellvibrio japonicus GH10 xylanase CjXyn10C is a bi-modular enzyme comprising a GH10 catalytic module and a family 15 carbohydrate-binding module. The three-dimensional structure at 1.85 A, presented here, shows that the sequence joining the two modules is disordered, confirming that linker sequences in modular glycoside hydrolases are highly flexible. CjXyn10C hydrolyzes xylan at a rate similar to other previously described GH10 enzymes but displays very low activity against xylooligosaccharides. The poor activity on short substrates reflects weak binding at the -2 subsite of the enzyme. Comparison of CjXyn10C with other family GH10 enzymes reveals "polymorphisms" in the substrate binding cleft including a glutamate/glycine substitution at the -2 subsite and a tyrosine insertion in the -2/-3 glycone region of the substrate binding cleft, both of which contribute to the unusual properties of the enzyme. The CjXyn10C-substrate complex shows that Tyr-340 stacks against the xylose residue located at the -3 subsite, and the properties of Y340A support the view that this tyrosine plays a pivotal role in substrate binding at this location. The generic importance of using CjXyn10C as a template in predicting the biochemical properties of GH10 xylanases is discussed.