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.     

Cloning and characterization of two thermostable xylanases from an alkaliphilic Bacillus firmus

Two genes encoding thermostable xylanases, named xyn10A and xyn11A, from an alkaliphilic Bacillus firmus were cloned and expressed in Escherichia coli. The E. coli harboring either gene showed clear zone with Congo red clearance assay on xylan plate. The Xyn10A and Xyn11A have molecular weights of 45 and 23kDa, respectively, and both show activities on xylan-zymogram. The xyn10A encodes 396 amino acid residues and is very similar to an alkaliphilic xylanase A from alkaliphilic Bacillus halodurans. The Xyn11A contains 210 amino acid residues and only one amino acid different from an endo-beta-1,4-xylanase from B. halodurans. From alignment of the amino acid sequences with other xylanases, Xyn10A and Xyn11A belong to family 10 and 11 glycosyl hydrolases, respectively. Both show activities over the pH range of 4-11 at 37 degrees C and over 80% activities at 70 degrees C. Interestingly both still retain over 70% activities after 16h preincubation at 62 degrees C.     

Direct cloning of genes encoding novel xylanases from the human gut

The aim of this study was to identify a novel 1,4-beta-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43,552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombinant Escherichia coli strain and characterized. This enzyme was optimally active at 40 degrees C and stable up to 50 degrees C at pH 6.5 and over the pH range 4.0-11.0 at 25 degrees C. In addition, 2 ORFs (ORF1 and ORF2) were identified upstream of xyn10A. These results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.     

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.     

Characteristics of adjacent family 6 acetylxylan esterases from Fibrobacter succinogenes and the interaction with the Xyn10E xylanase in hydrolysis of acetylated xylan

Acetylxylan esterase genes axe6A and axe6B located adjacent to one another on a Fibrobacter succinogenes chromosome have been separately cloned and their properties characterized. The corresponding esterases contained an N-terminal carbohydrate esterase family 6 catalytic domain (CD) and a C-terminal family 6 carbohydrate-binding module (CBM). The amino acid sequences of the CDs and CBMs were found to exhibit 52% and 40% amino acid similarity, respectively. The CDs of the two esterases exhibited the highest similarity to CDs of acetylxylan esterases: AxeA from the ruminal fungi Orpinomyces sp. and BnaA from Neocallimastix patriciarum. Axe6A and Axe6B were optimally active at neutral pH and had low K(m) values of 0.084 and 0.056 mmol x L(-1), respectively. Axe6A and Axe6B were shown to bind to insoluble cellulose and xylan and to soluble arabinoxylan. Axe6A deacetylated acetylated xylan at the same initial rate in the presence and absence of added Xyn10E xylanase from F. succinogenes, but the action of the xylanase on acetylated xylan was dependent upon the initial activity of Axe6A. The capacity of acetylxylan esterases to bind to plant cell wall polymers and to independently deacetylate xylan enabling xylanase to release xylooligo saccharides, documents the central role these enzymes have to improve access of F. succinogenes to cellulose.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a xylanase that contains a family 3 carbohydrate-binding module

Paenibacillus curdlanolyticus B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass.     

Characterization of thermostable Xyn10A enzyme from mesophilic Clostridium acetobutylicum ATCC 824

A thermostable xylanase gene, xyn10A (CAP0053), was cloned from Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the C. acetobutylicum xyn10A gene encoded a 318-amino-acid, single-domain, family 10 xylanase, Xyn10A, with a molecular mass of 34 kDa. Xyn10A exhibited extremely high (92%) amino acid sequence identity with Xyn10B (CAP0116) of this strain and had 42% and 32% identity with the catalytic domains of Rhodothermus marinus xylanase I and Thermoascus aurantiacus xylanase I, respectively. Xyn10A enzyme was purified from recombinant Escherichia coli and was highly active toward oat-spelt and Birchwood xylan and slightly active toward carboxymethyl cellulose, arabinogalactouronic acid, and various p-nitrophenyl monosaccharides. Xyn10A hydrolyzed xylan and xylooligosaccharides larger than xylobiose to produce xylose. This enzyme was optimally active at 60°C and had an optimum pH of 5.0. This is one of a number of related activities encoded on the large plasmid in this strain.     

Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes

Soil metagenomes represent an unlimited resource for the discovery of novel biocatalysts from soil microorganisms. Three large-inserts metagenomic DNA libraries were constructed from different grassland soil samples and screened for genes conferring cellulase or xylanase activity. Function-driven screening identified a novel cellulase-encoding gene (cel01) and two xylanase-encoding genes (xyn01 and xyn02). From sequence and protein domain analyses, Cel01 (831 amino acids) belongs to glycoside hydrolase family 9 whereas Xyn01 (170 amino acids) and Xyn02 (255 amino acids) are members of glycoside hydrolase family 11. Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases. Both Xyn01 and Xyn02 were most active at 60°C with high activities from 4 to 10 and optimal at pH 7 (Xyn01) and pH 6 (Xyn02). The cellulase gene, cel01, was expressed in E. coli BL21 and the recombinant enzyme (91.9 kDa) was purified. Cel01 exhibited high activity with soluble cellulose substrates containing β-1,4-linkages. Activity with microcrystalline cellulose was not detected. These data, together with the analysis of the degradation profiles of carboxymethyl cellulose and barley glucan indicated that Cel01 is an endo 1,4-β-glucanase. Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.     

Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, lambda gtWES lambda B, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated lambda FSX101 and lambda FSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from lambda FSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-beta-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the beta-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of beta-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan.     

High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

Background

In the hydrolysis of lignocellulosic materials, thermostable enzymes decrease the amount of enzyme needed due to higher specific activity and elongate the hydrolysis time due to improved stability. For cost-efficient use of enzymes in large-scale industrial applications, high-level expression of enzymes in recombinant hosts is usually a prerequisite. The main aim of the present study was to compare the biochemical and hydrolytic properties of two thermostable recombinant glycosyl hydrolase families 10 and 11 (GH10 and GH11, respectively) xylanases with respect to their potential application in the hydrolysis of lignocellulosic substrates.

Results

The xylanases from Nonomuraea flexuosa (Nf Xyn11A) and from Thermoascus aurantiacus (Ta Xyn10A) were purified by heat treatment and gel permeation chromatography. Ta Xyn10A exhibited higher hydrolytic efficiency than Nf Xyn11A toward birchwood glucuronoxylan, insoluble oat spelt arabinoxylan and hydrothermally pretreated wheat straw, and it produced more reducing sugars. Oligosaccharides from xylobiose to xylopentaose as well as higher degree of polymerization (DP) xylooligosaccharides (XOSs), but not xylose, were released during the initial hydrolysis of xylans by Nf Xyn11A, indicating its potential for the production of XOS. The mode of action of Nf Xyn11A and Ta Xyn10A on glucuronoxylan and arabinoxylan showed typical production patterns of endoxylanases belonging to GH11 and GH10, respectively.

Conclusions

Because of its high catalytic activity and good thermostability, T. aurantiacus xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas N. flexuosa xylanase shows more significant potential for the production of XOSs.     

Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.     

Thermostable xylanase10B from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC824

The Clostridium acetobutylicum xylanase gene xyn10B (CAP0116) was cloned from the type strain ATCC 824, whose genome was recently sequenced. The nucleotide sequence of C. acetobutylicum xyn10B encodes a 318-amino acid protein. Xyn10B consists of a single catalytic domain that belongs to family 10 of glycosyl hydrolases. The enzyme was purified from recombinant Escherichia coli. The Xyn10B enzyme was highly active toward birchwood xylan, oat-spelt xylan, and moderately active toward avicel, carboxymethyl cellulose, polygalacturonic acid, lichenan, laminarin, barley--glucan and various p-nitrophenyl monosaccharides. Xyn10B hydrolyzed xylan and xylooligosaccharides to produce xylobiose and xylotriose. The pH optimum of Xyn10B was 5.0, and the optimal temperature was 70°C. The enzyme was stable at 60°C at pH 5.0–6.5 for 1 h without substrate. This is one of a number of xylan-related activities encoded on the large plasmid in C. acetobutylicum ATCC 824.     

Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Purification of the alkaliphilic xylanases from Myceliophthora sp. IMI 387099 using cellulose-binding domain as an affinity tag

Ten xylanase isoforms produced by Myceliophthora sp. were characterized for their ability to bind to avicel. Three of the xylanases showing differential affinity for avicel were purified by column chromatography. The purified xylanase Xyl IIa, IIb and IIc showed molecular mass of 47, 41 and 30 kDa and pI of ∼3.5, 4.8 and 5.2, respectively. Xyl IIa was optimally active at pH 8.0 and temperature 70 °C, while Xyl IIb and IIc were optimally active at pH 9.0 and 60 °C and 7.0 and 80 °C, respectively. Xyl IIa and Xyl IIb showed higher stability under alkaline conditions (pH 9.0) and retained 80% of the original activity upto 1 h and 3 h respectively, at 50 °C. All three purified iso-xylanases showed enhanced activities in presence of Na⁺, Mg²⁺, Mn²⁺ and K⁺ ions, whereas, Zn²⁺ and Cu²⁺ showed negative effect on Xyl IIa. The activity of Xyl IIa increased in presence of reducing agents DTT and mercaptoethanol, however, SDS showed inhibitory effect. Kinetic studies showed that Xyl IIb and IIc degrade rye arabinoxylan, much more efficiently than oat spelt xylan, whereas, Xyl IIa showed much higher Kcat/Km value for birch wood xylan as compared to oat spelt xylan. The purified xylanases were apparently classified in family 10.     

The construction and characterization of two xylan-degrading chimeric enzymes

Degradation of xylan requires several enzymes. Two chimeric enzymes, xyln-ara and xyln-xylo, were constructed by linking the catalytic portion of a xylanase (xyln) to either an arabinofuranosidase (ara) or a xylosidase (xylo) with a flexible peptide linker. The recombinant parental enzymes and chimeras were produced in E. coli at high levels and purified for characterization of their enzymatic and kinetic properties as well as activities on natural substrates. The chimeras closely resemble the parental enzymes or their mixtures with regard to protein properties. They share similar temperature profiles and have similar catalytic efficiencies as the parental enzymes when assayed using substrates 4-nitrophenyl-alpha-L-arabinofuranoside or 2-nitrophenyl- beta-D-xylopyranoside. The chimeras also show unique enzymatic characteristics. In xylanase activity assays using Remazol Brilliant Blue-xylan, while the parental xylanase has a pH optimum of pH 8, the chimeras showed shifted pH optima as a consequence of significantly increased activity at pH 6 (the optimal pH for ara and xylo). Both chimeras exhibited additive effects of the parental enzymes when assayed at wide ranges of pH and temperatures. The xyln-xylo chimera had the same activities as the xyln/xylo mixture in hydrolyzing the natural substrates oat spelt xylan and wheat arabinoxylan. Compared to the xyln/ara mixture, the xyln-ara chimera released the same amounts of xylose from oat spelt xylan and approximately 30% more from wheat arabinoxylan at pH 6. Our results demonstrate the feasibility and advantages of generating bifunctional enzymes for the improvement of xylan bioconversion.     

A third xylanase from Trichoderma reesei PC-3-7

A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III. Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998     

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.