Characterization of genes fromThermoanaerobacterium thermosulfurigenes EM1 that encode two glycosyl hydrolases with conserved S-layer-like domains

Two genes fromThermoanaerobacterium thermosulfurigenes EM1 were identified which are predicted to encode a xylanase (XynA) and a polygalacturonate hydrolase (PglA). ThexynA gene has the potential to encode a 1234-amino acid product consisting of a signal peptide followed by a repeated domain, a xylanase family F domain, two cellulose-binding domains and a triplicated sequence at its C-terminus. The genepglA is predicted to encode a product of 1148 amino acids consisting of a signal sequence followed by a fibronectin type III-like domain (Fn3 domain), the catalytic domain, a Gly/Thr/Ser/Asn-rich segment and a triplicated domain. The triplicated segments at the C-termini of deduced XynA and PglA are about 95% identical to each other and to the S-layer-like domains of the previously characterized pullulanase (AmyB) from the same organism. In contrast, sequence comparisons revealed only distant amino acid sequence similarities between the fibronectin type III-like domains of PglA and AmyB fromT. thermosulfurigenes EM1.     

Mutagenesis of conserved charged amino acids in SLH domains of Thermoanaerobacterium thermosulfurigenes EM1 affects attachment to cell wall sacculi

SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial cell walls. The data presented here assign the conserved TRAE motif within SLH domains a key role for the binding. The charged amino acids arginine (R) or/and glutamic acid (E) were replaced via site-directed mutagenesis by different amino acids. Effects were visualized in an in vitro binding assay using native cell wall sacculi of Thermoanaerobacterium thermosulfurigenes EM1 and different variants of an SLH protein which consisted of the triplicate SLH domain of xylanase XynA of this bacterium and which was purified after expression in Escherichia coli. The results indicated (1) that the TRAE motif is critical for the binding function of SLH domains, (2) that a functional TRAE motif is necessary in all three domains, (3) that a least one (preferentially positively) charged amino acid in the TRAE motif is required for the functionality of the SLH domain, and (4) that the position of the negatively and positively charged amino acids is important. The finding that the cell wall of T. thermosulfurigenes EM1 contains pyruvate (4 μg mg⁻¹) is in agreement with the hypothesis that pyruvylated secondary cell wall polymers function as ligand for SLH domains.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.     

Cell wall of Thermoanaerobacterium thermosulfurigenes EM1: isolation of its components and attachment of the xylanase XynA

Thermoanaerobacterium thermosulfurigenes EM1 has a gram-positive type cell wall completely covered by a surface layer (S-layer) with hexagonal lattice symmetry. The components of the cell envelope were isolated, and the S-layer protein was purified and characterized. S-layer monomers assembled in vitro into sheets with the same hexagonal symmetry as in vivo. Monosaccharide analysis revealed that the S-layer is associated with fucose, rhamnose, mannosamine, glucosamine, galactose, and glucose. The N-terminal 31 amino acid residues of the S-layer protein showed significant similarity to SLH (S-layer homology) domains found in S-layer proteins of different bacteria and in the exocellular enzymes pullulanase, polygalacturonate hydrolase, and xylanase of T. thermosulfurigenes EM1. The xylanase from T. thermosulfurigenes EM1 was copurified with the S-layer protein during isolation of cell wall components. Since SLH domains of some structural proteins have been shown to anchor these proteins noncovalently to the cell envelope, we propose a common anchoring mechanism for the S-layer protein and exocellular enzymes via their SLH domains in the peptidoglycan-containing layer of T. thermosulfurigenes EM1. Received: 23 October 1998 / Accepted: 21 December 1998     

Identification of non-catalytic conserved regions in xylanases encoded by the xynB and xynD genes of the cellulolytic rumen anaerobe Ruminococcus flavefaciens

xynB is one of at least four genes from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that encode xylanase activity. The xynB gene is predicted to encode a 781-amino acid product starting with a signal peptide, followed by an amino-terminal xylanase domain which is identical at 89% and 78% of residues, respectively, to the amino-terminal xylanase domains of the bifunctional XynD and XynA enzymes from the same organism. Two separate regions within the carboxy-terminal 537 amino acids of XynB also show close similarities with domain B of XynD. These regions show no significant homology with cellulose- or xylan-binding domains from other species, or with any other sequences, and their functions are unknown. In addition a 30 to 32-residue threonine-rich region is present in both XynD and XynB. Codon usage shows a consistent pattern of bias in the three xylanase genes from R. flavefaciens that have been sequenced.     

Identification of a novel cellulose-binding domain the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima

A segment of Thermotoga maritima strain MSB8 chromosomal DNA was isolated which encodes an endo-1,4-β-D-xylanase, and the nucleotide sequence of the xylanase gene, designated xynA, was determined. With a half-life of about 40 min at 90°C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059-amino-acid (?120 kDa) modular enzyme composed of an N-terminal signal peptide and five domains, in the order A1-A2-B-C1-C2. By comparison with other xylanases of family 10 of glycosyl hydrolases, the central ?340-amino-acid part (domain B) of XynA represents the catalytic domain. The N terminal ?150-amino-acid repeated domains (A1-A2) have no significant similarity to the C-terminal ?170-amino-acid repeated domains (C1-C2). Cellulose-binding studies with truncated XynA derivatives and hybrid proteins indicated that the C-terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding. C1 and C2 did not share amino acid sequence similarity with any other known cellulose-binding domain (CBD) and thus are CBDS of a novel type. Structurally related protein segments which are probably also CBDs were found in other multi-domain xylanolytic enzymes. Deletion of the N-terminal repeated domains or of all the non-catalytic domains resulted In substantially reduced tbermostability while a truncated xylanase derivative lacking the C-terminal tandem repeat was as thermostable as the full-length enzyme. It is argued that the multidomain organization of some enzymes may be one of the strategies adopted by thermophiles to protect their proteins against thermal denaturation.     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

Molecular Characterization of Genes Encoding a Novel ABC Transporter in Thermoanaerobacterium thermosulfurigenes EM1

The nucleotide sequence of two open reading frames (ORFs) from Thermoanaerobacterium thermosulfurigenes EM1 was determined that encode proteins with similarity to components of ATP-binding cassette (ABC) transport systems. Sequence analysis suggests that the deduced proteins AbcA and AbcB consist of an NH₂-terminal membrane-spanning domain and a COOH-terminal ATP-binding domain. The deduced proteins AbcA and AbcB showed highest similarity to proteins of the MsbA subfamily of ABC transporters. AbcA and AbcB probably function as a heterodimer. An ORF predicted to encode the primary sigma factor SigA was identified downstream of abcB. Received: 11 March 1997 / Accepted: 14 April 1997     

Purification,Characterization, and Molecular Cloning of Acidophilic Xylanase from Penicillium sp.40

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family11 xylanases from fungi.     

A bifunctional enzyme, with separate xylanase and beta(1,3-1,4)-glucanase domains, encoded by the xynD gene of Ruminococcus flavefaciens.

Adjacent regions of a Ruminococcus flavefaciens 17 DNA fragment were found to encode xylanase and beta(1,3-1,4)-glucanase activities. Sequencing of this fragment showed that both activities are encoded by a single 2,406-bp open reading frame corresponding to the xynD gene. The predicted product has a characteristic signal sequence that is followed by an amino-terminal domain related to family G xylanases, while the carboxyterminal domain is related to beta(1,3-1,4)-glucanases from several other bacterial species. These two domains are connected by a region of unknown function that consists of 309 amino acids and includes a 30-amino-acid threonine-rich sequence. A polypeptide having a molecular weight of approximately 90,000 and exhibiting xylanase and beta(1,3-1,4)-glucanase activities was detected in Escherichia coli cells carrying the cloned xynD gene. This is one of the first cases in which a microbial polysaccharidase has been shown to carry separate catalytic domains active against different plant cell wall polysaccharides within the same polypeptide. xynD is one of a family of related genes in R. flavefaciens that encode enzymes having multiple catalytic domains, and the amino terminus of XYLD exhibits a high degree of similarity with the corresponding regions of another xylanase, XYLA, which carries two different xylanase catalytic domains.     

Pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes): molecular analysis of the gene, composite structure of the enzyme, and a common model for its attachment to the cell surface.

The complete pullulanase gene (amyB) from Thermoanaerobacterium thermosulfurigenes EM1 was cloned in Escherichia coli, and the nucleotide sequence was determined. The reading frame of amyB consisted of 5,586 bp encoding an exceptionally large enzyme of 205,991 Da. Sequence analysis revealed a composite structure of the pullulanase consisting of catalytic and noncatalytic domains. The N-terminal half of the protein contained a leader peptide of 35 amino acid residues and the catalytic domain, which included the four consensus regions of amylases. Comparison of the consensus regions of several pullulanases suggested that enzymes like pullulanase type II from T. thermosulfurigenes EM1 which hydrolyze alpha-1,4- and alpha-1,6-glycosidic linkages have specific amino acid sequences in the consensus regions. These are different from those of pullulanases type I which only cleave alpha-1,6 linkages. The C-terminal half, which is not necessary for enzymatic function, consisted of at least two different segments. One segment of about 70 kDa contained two copies of a fibronectin type III-like domain and was followed by a linker region rich in glycine, serine, and threonine residues. At the C terminus, we found three repeats of about 50 amino acids which are also present at the N-termini of surface layer (S-layer) proteins of, e.g., Thermus thermophilus and Acetogenium kivui. Since the pullulanase of T. thermosulfurigenes EM1 is known to be cell bound, our results suggest that this segment serves as an S-layer anchor to keep the pullulanase attached to the cell surface. Thus, a general model for the attachment of extracellular enzymes to the cell surface is proposed which assigns the S-layer a new function and might be widespread among bacteria with S-layers. The triplicated S-layer-like segment is present in several enzymes of different bacteria. Upstream of amyB, another open reading frame, coding for a hypothetical protein of 35.6 kDa, was identified. No significant similarity to other sequences available in DNA and protein data bases was found.     

Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure

One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.     

In Thermoanaerobacterium thermosulfurigenes EM1 S-Layer Homology Domains Do Not Attach to Peptidoglycan

Three exocellular enzymes of Thermoanaerobacterium thermosulfurigenes EM1 possess a C-terminal triplicated sequence related to a domain of bacterial cell surface proteins (S-layer proteins). At least one copy of this sequence, named the SLH (for S-layer homology) domain, is also present at the N terminus of the S-layer protein of this bacterium. The hypothesis that SLH domains serve to anchor proteins to the cell surface was investigated by using the SLH domain-containing xylanase. This enzyme was isolated from T. thermosulfurigenes EM1, and different forms with and without SLH domains were synthesized in Escherichia coli. The interaction of these proteins with isolated components of the cell envelope was determined to identify the attachment site in the cell wall. In addition, a polypeptide consisting of three SLH domains and the N terminus of the S-layer protein of T. thermosulfurigenes EM1 were included in these studies. The results indicate that SLH domains are necessary for the attachment of these proteins to peptidoglycan-containing sacculi. Extraction of the native sacculi with hydrofluoric acid led to the conclusion that not peptidoglycan but accessory cell wall polymers function as the adhesion component in the cell wall. Our results provide further evidence that attachment of proteins via their SLH domains represents an additional mode to display polypeptides on the cell surfaces of bacteria.     

Molecular characterization of xynX, a gene encoding a multidomain xylanase with a thermostabilizing domain from Clostridium thermocellum

A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined. The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa). Sequence analysis of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain. A deletion mutant of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability. In terms of half-life at 70 °C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme, XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain. The optimum temperature of XynX-C was about 5–10 °C lower than that of XynX-TC. Received: 12 January 2000 / Received revision: 24 April 2000 / Accepted: 1 May 2000     

The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor

The members of the interleukin-6-type family of cytokines interact with receptors that have a modular structure and are built of several immunoglobulin-like and fibronectin type III-like domains. These receptors have a characteristic cytokine receptor homology region consisting of two fibronectin type III-like domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 (IL-6) initially binds to its cognate alpha-receptor and subsequently to a homodimer of the signal transducer receptor gp130. The IL-6 receptor (IL-6R) consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane-proximal fibronectin-like domain (IL-6R-D3) accounts for more than 90% of the binding energy to IL-6. Here, we present the solution structure of the IL-6R-D3 domain solved by multidimensional heteronuclear NMR spectroscopy.     

Cloning,expression and characterization of glycoside hydrolase family 11 endoxylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> ARA

An endoxylanase gene, xynA, was cloned from Bacillus pumilus ARA and expressed in Escherichia coli. The open reading frame of the xynA gene was 687 bp encoding a signal peptide and a mature xylanase with a molecular mass of 23 kDa. The enzyme was categorized as a glycosyl hydrolase family 11 member based on the sequence analysis of the putative catalytic domain. The recombinant XynA (Bpu XynA) was purified to homogeneity by Ni–NTA and ion exchange chromatography on DEAE–Sepharose FF. The enzyme exhibited highest activity at pH 6.6 and 50°C. The purified Bpu XynA was stable for at least 2 h at 45°C, and retained over 50% residual activity after being incubated at 60°C for 1 h. The activity of the xylanase was not significantly affected by metal ions and EDTA. The K _m and K _cat /K _m of Bpu XynA for oat-spelt xylan were 5.53 mg/ml and 10.14 ml/mg s at 50°C and pH 6.6. The main product of hydrolysis by Bpu XynA was xylooligosaccharide. The results revealed that the consumption of grass xylan by B. pumilus ARA depended on the synergistic reactions of Bpu XynA and Bpu arabinosidase, and that a typical GH11 xylanase e.g. Tla XynA had capability to remove the side chain of xylan. The properties Bpu XynA make it promising for application in the production of Bifidobacterium growth-promoting factors and in feed industry.     

Cloning,expression and characterization of a novel cold-active and halophilic xylanase from Zunongwangia profunda

A new xylanase gene (xynA) from the marine microorganism Zunongwangia profunda was identified to encode 374 amino acid residues. Its product (XynA) showed the highest identity (42.78 %) with a xylanase from Bacillus sp. SN5 among the characterized xylanases. XynA exhibited the highest activity at pH 6.5 and 30 °C, retaining 23 and 38 % of the optimal activity at 0 and 5 °C, respectively. XynA was not only cold active, but also halophilic, and both its activity and thermostability could be significantly increased by NaCl, showing the highest activity (180 % of the activity) at 3 M NaCl and retaining nearly 100 % activity at 5 M NaCl, compared to the absence of NaCl. In the presence of 3 M NaCl, the k _cat/K _m value of XynA exhibited a 3.41-fold increase for beechwood xylan compared to no added NaCl, and the residual activity of XynA increased from 23 % (no added NaCl) to 58 % after 1 h incubation at 45 °C. This may be the first report concerning a cold-adapted xylanase from a non-halophilic species that displays the highest activity at a NaCl concentration range from 3 to 5 M. The features of cold activity and salt tolerance suggest the potential application of XynA in the food industry and bioethanol production from marine seaweeds.     

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.     

Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant ceil wall hydroiases

Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non-catalytic cellulose-binding domain (CBD). To establish whether CBDs are common features of plant cell-wall hydroiases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of M_r 68000. The deduced primary sequence of XYLD was confirmed by the size (64kDa) and N-terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endo-acting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an /V-terminal signal peptide followed by a 190-residue domain that exhibited significant homology to Family-G xylanases. Truncated derivatives of xynD, encoding the W-terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190-residue N-terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90-residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197-amino-acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/prolineor novel glycine-rich linker regions. Although full-length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C-terminal CBD hydrolysed xylan but did not bind to cellulose. Fusion of the C-terminal domain to glutathione-Stransferase generated hybrid proteins that bound to crystalline cellulose, but not to amorphous cellulose or xylan. The location of CBDs in a C. fimi xylanase indicates that domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell-wall hydrolases. The role of the NodB homologue in XYLD is less certain.     

Purification, characterization, and molecular cloning of acidophilic xylanase from penicillium sp.40

Penicillum sp. 40, which can grow in an extremely acidic medium at pH 2.0 was screened from an acidic soil. This fungus produces xylanases when grown in a medium containing xylan as a sole carbon source. A major xylanase was purified from the culture supernatant of Penicillium sp. 40 and designated XynA. The molecular mass of XynA was estimated to be 25,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. XynA has an optimum pH at 2.0 and is stable in pH 2.0-5.0. Western blot analysis using anit-XynA antibody showed that XynA was induced by xylan and repressed by glucose. Also, its production was increased by an acidic medium. The gene encoding XynA (xynA) was isolated from the genomic library of Penicillium sp. 40. The structural part of xynA was found to be 721 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynA was interrupted by a single intron which was 58 bp in size and encoded 221 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynA had a signal peptide composed of 31 amino acids. The molecular mass caliculated from the deduced amino acid sequence of XynA is 20,713. This is lower than that estimated by gel electrophoresis, suggesting that XynA is a glycoprotein. The predicted amino acid sequence of XynA has strong similarity to other family xylanases from fungi.