Expression Of Xylanase Research Articles

Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis.

BackgroundXylanase can replace chemical additives to improve the volume and sensory properties of bread in the baking. Suitable baking xylanase with improved yield will promote the application of xylanase in baking industry. The xylanase XYNZG from the Plectosphaerella cucumerina has been previously characterized by heterologous expression in Pichia pastoris. However, P. pastoris is not a suitable host for xylanase to be used in the baking process since P. pastoris does not have GRAS (Generally Regarded As Safe) status and requires large methanol supplement during the fermentation in most conditions, which is not allowed to be used in the food industry. Kluyveromyces lactis, as another yeast expression host, has a GRAS status, which has been successfully used in food and feed applications. No previous work has been reported concerning the heterologous expression of xylanase gene xynZG in K. lactis with an aim for application in baking.ResultsThe xylanase gene xynZG from the P. cucumerina was heterologously expressed in K. lactis. The recombinant protein XYNZG in K. lactis presented an approximately 19 kDa band on SDS-PAGE and zymograms analysis. Transformant with the highest halo on the plate containing the RBB-xylan (Remazol Brilliant Blue-xylan) was selected for the flask fermentation in different media. The results indicated that the highest activity of 115 U/ml at 72 h was obtained with the YLPU medium. The mass spectrometry analysis suggested that the hydrolytic products of xylan by XYNZG were mainly xylobiose and xylotriose. The results of baking trials indicated that the addition of XYNZG could reduce the kneading time of dough, increase the volume of bread, improve the texture, and have more positive effects on the sensory properties of bread.ConclusionsXylanase XYNZG is successfully expressed in K. lactis, which exhibits the highest activity among the published reports of the xylanase expression in K. lactis. The recombinant XYNZG can be used to improve the volume and sensory properties of bread. Therefore, the expression yield of recombinant XYNZG can be further improved through engineered strain containing high copy numbers of the XYNZG, and optimized fermentation condition, making bread-baking application possible.

Characterization and high expression of recombinant Ustilago maydis xylanase in Pichia pastoris.

A recombinant xylanase gene (rxynUMB) from Ustilago maydis 521 was expressed in Pichia pastoris, and the enzyme was purified and characterized. Phylogenetic analysis demonstrated that rxynUMB belongs to glycosyl hydrolase family 11. The Trp84, Trp95, Glu93, and Glu189 residues are proposed to be present at the active site. The apparent molecular mass of the recombinant xylananse was approximately 24 kDa, and the optimum pH and temperature were 4.3 and 50 °C, respectively. Xylanase activity was enhanced by 166 and 115% with Fe(2+) and Mn(2+), respectively. The biochemical properties of this recombinant xylanase suggest that it may be a useful candidate for a variety of commercial applications.

Cloning, expression, and characterization of a novel alkali-tolerant xylanase from alkaliphilic Bacillus sp. SN5.

A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry.

Secretory expression and characterization of two hemicellulases, xylanase, and β-xylosidase, isolated from Bacillus subtilis M015.

Microbial hydrolysis of lignocellulosic biomass is becoming increasingly important for the production of renewable biofuels to address global energy concerns. Hemicellulose is the second most abundant lignocellulosic biopolymer consisting of mostly xylan and other polysaccharides. A variety of enzymes is involved in complete hydrolysis of xylan into its constituent sugars for subsequent biofuel fermentation. Two enzymes, endo-β-xylanase and β-xylosidase, are particularly important in hydrolyzing the xylan backbone into xylooligosaccharides and individual xylose units. In this study, we describe the cloning, expression, and characterization of xylanase and β-xylosidase isolated from Bacillus subtilis M015 in Escherichia coli. The genes were identified to encode a 213 amino acid protein for xylanase (glycoside hydrolase (GH) family 11) and a 533 amino acid protein for β-xylosidase (GH family 43). Recombinant enzymes were produced by periplasmic-leaky E. coli JE5505 and therefore secreted into the supernatant during growth. Temperature and pH optima were determined to be 50 °C and 5.5–6 for xylanase and 35 °C and 7.0–7.5 for β-xylosidase using beech wood xylan and p-nitrophenyl-β-D-xylopyranoside as the substrates, respectively. We have also investigated the synergy of two enzymes on xylan hydrolysis and observed 90 % increase in total sugar release (composed of xylose, xylobiose, xylotriose, and xylotetraose) for xylanase/β-xylosidase combination as opposed to xylanase alone.

Multiple Regulatory Mechanisms Control the Expression of the Geobacillus stearothermophilus Gene for Extracellular Xylanase

Geobacillus stearothermophilus T-6 produces a single extracellular xylanase (Xyn10A) capable of producing short, decorated xylo-oligosaccharides from the naturally branched polysaccharide, xylan. Gel retardation assays indicated that the master negative regulator, XylR, binds specifically to xylR operators in the promoters of xylose and xylan-utilization genes. This binding is efficiently prevented in vitro by xylose, the most likely molecular inducer. Expression of the extracellular xylanase is repressed in medium containing either glucose or casamino acids, suggesting that carbon catabolite repression plays a role in regulating xynA. The global transcriptional regulator CodY was shown to bind specifically to the xynA promoter region in vitro, suggesting that CodY is a repressor of xynA. The xynA gene is located next to an uncharacterized gene, xynX, that has similarity to the NIF3 (Ngg1p interacting factor 3)-like protein family. XynX binds specifically to a 72-bp fragment in the promoter region of xynA, and the expression of xynA in a xynX null mutant appeared to be higher, indicating that XynX regulates xynA. The specific activity of the extracellular xylanase increases over 50-fold during early exponential growth, suggesting cell density regulation (quorum sensing). Addition of conditioned medium to fresh and low cell density cultures resulted in high expression of xynA, indicating that a diffusible extracellular xynA density factor is present in the medium. The xynA density factor is heat-stable, sensitive to proteases, and was partially purified using reverse phase liquid chromatography. Taken together, these results suggest that xynA is regulated by quorum-sensing at low cell densities.

A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology.

BackgroundThe search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis.ResultsXylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (β/α)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses.ConclusionsMpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.

High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein

A thermostable xylanase is encoded by xynA from fungus Thermomyces lanuginosus. The problem emerged from overexpression of xynA in Escherichia coli has been the formation of inclusion bodies. Here we describe the xynA was fused with the hyperosmotically inducible periplasmic protein of E. coli, OsmY. The fusion protein OsmY-xynA was expressed as almost all soluble form. The soluble expression level of fusion protein reached 98±6U/ml when cells containing pET-OsmY-xynA were expressed without IPTG induction at 37°C. The induction is probably due to auto-induction due to lactose in the medium (Studier (2005) [21]). The cells harboring pET-OsmY-xynA expressed an activity level about 24 times higher than that expressed from pET-20b-xynA. Xylanase activity was observed in the extracellular (36±1.3U/ml) and the periplasmic (42±4U/ml) when cells containing pET-OsmY-xynA were induced without IPTG addition. After the cold osmotic shock procedure followed by nickel affinity chromatography, the purified fusion protein showed a single band on SDS–PAGE gel with a molecular mass of 44kDa. The purified fusion enzyme exhibited the highest activity at 65°C and pH 6.0.

Expression pattern of cellulolytic and xylanolytic genes regulated by transcriptional factors XYR1 and CRE1 are affected by carbon source in Trichoderma reesei

Trichoderma reesei is the most important fungus for the industrial production of enzymes to biomass deconstruction. Most of the genes encoding cellulases and hemicellulases are regulated by the transcription factors CRE1 and XYR1. In this work, the regulation of 22 genes of cellulases and xylanases by these transcription factors was investigated under three different carbon sources. Analysis of gene expression and enzymatic profiles of CMCase, β-glucosidase, and xylanases showed different regulation that was depended of the carbon source in both Δxyr1 and Δcre1 mutants. In the presence of glucose, the majority of genes evaluated (82%) showed increased expression levels in the Δcre1 mutant compared to the parental QM9414 strain. In the Δxyr1 mutant, it was observed that expression of cellulase and xylanase genes was reduced compared to the parental QM9414 strain, when cultured in the presence of cellulose or sophorose. Interesting, in the presence of glucose, approximately 60% of the analyzed genes had increased expression in the Δxyr1 mutant compared to parental strain. Furthermore, no correlation between gene expression and the number of putative binding sites of XYR1 and CRE1 to promoter region of cellulolytic and xylanolytic studied genes was observed. Therefore, these results demonstrated that the regulation of cellulase and xylanase by the transcription factors CRE1 and XYR1 is influenced by different carbon sources.

Cloning and heterologous expression of cellulose free thermostable xylanase from Bacillus brevis.

Xylanase gene isolated from Bacillus brevis was expressed in E. coli BL21. Sequencing of the gene (Gen Bank accession number: HQ179986) showed that it belongs to family 11 xylanases. The recombinant xylanase was predominantly secreted to culture medium and showed mesophilic nature (optimum activity at 55°C and pH 7.0). The cell free culture medium exhibited 30 IU/ml xylanse activity. The enzyme did not show any cellulose activity and was active under wide range of temperature (40°C to 80°C) and pH (4 to 9). The enzyme showed considerable thermo stability and regained over 90% of activity, when returned to 55°C after boiling for 5 min. These physiochemical properties of B. brevis xylanse show high potential of its applications in paper and pulp industry.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-20) contains supplementary material, which is available to authorized users.

Effect of Various Physiological Parameters and Different Carbon Sources on Cellulase and Xylanase Induction by Different Strains of Trichoderma Species

The recent interest in bioconversion of cellulosic wastes to value added chemicals has led to extensive studies on microorganisms capable of producing the enzyme. Cellulases are multi-enzyme complex proteins that catalyze the conversion of cellulose to glucose. Trichoderma is the main industrial source of cellulases and hemicellulases used to depolymerize plant biomass to simple sugars that are converted to chemical intermediates such as biofuels. The current study was carried out to isolate and characterize different Trichoderma species for successful applications. The strains were characterized for cellulase and xylanase assays to determine their extracellular hydrolytic activities. Expression of xylanase was induced maximally by xylan and repressed by glucose. Moreover, a comparative analysis was done between two different Trichoderma species which revealed that Trichoderma harzianum and Trichoderma harzianum Th-azad shows better enzyme activity than T. viride 01PP. Furthermore, optimal conditions for enzyme activity and induction of enzyme synthesis were also determined. The results revealed that the optimal pH, temperature and incubation time for cellulase production was found to be 5.5, 28° C and 120 h respectively. In the present study, we also evaluate the effects of wheat bran, cellulose powder as carbon sources for cellulase and xylanase production. The result showed that the enzyme production was highest with wheat bran as sole source ofcarbon.

Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production

BackgroundThe soft rot ascomycetal fungus Trichoderma reesei is utilized for industrial production of secreted enzymes, especially lignocellulose degrading enzymes. T. reesei uses several different enzymes for the degradation of plant cell wall-derived material, including 9 characterized cellulases, 15 characterized hemicellulases and at least 42 genes predicted to encode cellulolytic or hemicellulolytic activities. Production of cellulases and hemicellulases is modulated by environmental and physiological conditions. Several regulators affecting the expression of cellulase and hemicellulase genes have been identified but more factors still unknown are believed to be present in the genome of T. reesei.ResultsWe have used transcriptional profiling data from T. reesei cultures in which cellulase/hemicellulase production was induced by the addition of different lignocellulose-derived materials to identify putative novel regulators for cellulase and hemicellulase genes. Based on this induction data, supplemented with other published genome-wide data on different protein production conditions, 28 candidate regulatory genes were selected for further studies and they were overexpressed in T. reesei. Overexpression of seven genes led to at least 1.5-fold increased production of cellulase and/or xylanase activity in the modified strains as compared to the parental strain. Deletion of gene 77513, here designated as ace3, was found to be detrimental for cellulase production and for the expression of several cellulase genes studied. This deletion also significantly reduced xylanase activity and expression of xylan-degrading enzyme genes. Furthermore, our data revealed the presence of co-regulated chromosomal regions containing carbohydrate-active enzyme genes and candidate regulatory genes.ConclusionsTranscriptional profiling results from glycoside hydrolase induction experiments combined with a previous study of specific protein production conditions was shown to be an effective method for finding novel candidate regulatory genes affecting the production of cellulases and hemicellulases. Recombinant strains with improved cellulase and/or xylanase production properties were constructed, and a gene essential for cellulase gene expression was found. In addition, more evidence was gained on the chromatin level regional regulation of carbohydrate-active enzyme gene expression.

Expression of a Recombinant Endo-β-1,4-Xylanase in <i>Pichia pastoris</i> and its Application in Degradation of Corn Stalk

The optimization of xylanase expression by recombinantPichia pastoriswere carried out in this study. Several factors were evaluated and the conclusion were as follow: the optimal conditions were in shake flask cultivation with the rotate speed 180 r/min using BMGY medium with initial pH 7.0, initial OD6001.0, 0.1% histidine, 0.05% tween80. 1.0% methanol was added into the culture every 24 h. The xylanase activity was up to 1527 U/mL at the optimal conditions after 15 days. The optimum pH and temperature were pH5.0 and 50°C. The recombinant xylanase was stable over a pH range of 2.0-8.0. The optimal conditions of degradation were as below: after 20 h, with the pH 5.0 and temperature 45°C, 2.0% of substrate concentration, 100 U/mL of enzyme dosage and 0.05% of tween80 concentration,the degradation of xylan was the best, which indicating great potential in the bioconversion of lignocellulosic waste to xylooligosaccharide.

Expression and Characterization of Recombinant GH11 Xylanase from Thermotolerant Streptomyces sp. SWU10

Xylans are major hemicellulose components of plant cell wall which can be hydrolyzed by xylanolytic enzymes. Three forms of endo-β-1,4-xylanases (XynSW1, XynSW2A, and XynSW2B) produced by thermotolerant Streptomyces sp. SWU10 have been reported. In the present study, we described the expression and characterization of the fourth xylanase enzyme from this bacteria, termed XynSW3. The gene containing 726 bp was cloned and expressed in Escherichia coli. The recombinant enzyme (rXynSW3) was purified from cell-free extract to homogeneity using Ni-affinity column chromatography. The apparent molecular mass of rXynSW3 was 48 kDa. Amino acid sequence analysis revealed that it belonged to a xylanase of glycoside hydrolase family 11. The optimum pH and temperature for enzyme activity were 5.5-6.5 and 50 °C, respectively. The enzyme was stable up to 40 °C and in wide pH ranges (pH 0.6-10.3). Xylan without arabinosyl side chain is the most preferable substrate for the enzyme. By using birch wood xylan as substrate, rXynSW3 produced several oligosaccharides in the initial stage of hydrolysis, and their levels increased with time, demonstrating that the enzyme is an endo-acting enzyme. The major products were xylobiose, triose, and tetraose. The rXynSW3 can be applied in several industries such as food, textile, and biofuel industries, and waste treatment.

Introduction of silent mutations in a codon-optimized xylanase (xynB) results in enhanced protein expression in HEK293A cells

Xylanase is used extensively to improve feed conversion rates to enhance the performance of poultry and pigs. By expressing xylanase in simple-stomached animals, new breeds of genetically modified animals with enhanced feed conversion rates may be obtained. However, expression of heterologous proteins derived from lower organisms in mammalian cells is usually inefficient. When common codons of a ''one amino acid-one codon"-optimized xylanase from Streptomyces olivaceoviridis were replaced with rare codons, xylanase expression in human embryonic kidney 293A cells increased by 1.4- to 2.3-fold as determined by flow cytometry, western blot and enzymatic activity assay. Quantitative RT-PCR assay indicated that the enhanced expression could not be attributed to altered mRNA levels. This study provides an alternative strategy for improving expression levels of heterologous proteins in mammalian cells, which is potentially helpful for generating genetically modified animals with enhanced feed conversion ability.

Sda1, a Cys2-His2 zinc finger transcription factor, is involved in polyol metabolism and fumonisin B1 production in Fusarium verticillioides.

The ubiquitous ascomycete Fusarium verticillioides causes ear rot and stalk rot of maize, both of which reduce grain quality and yield. Additionally, F. verticillioides produces the mycotoxin fumonisin B1 (FB1) during infection of maize kernels, and thus potentially compromises human and animal health. The current knowledge is fragmentary regarding the regulation of FB1 biosynthesis, particularly when considering interplay with environmental factors such as nutrient availability. In this study, SDA1 of F. verticillioides, predicted to encode a Cys-2 His-2 zinc finger transcription factor, was shown to play a key role in catabolizing select carbon sources. Growth of the SDA1 knock-out mutant (Δsda1) was completely inhibited when sorbitol was the sole carbon source and was severely impaired when exclusively provided mannitol or glycerol. Deletion of SDA1 unexpectedly increased FB1 biosynthesis, but reduced arabitol and mannitol biosynthesis, as compared to the wild-type progenitor. Trichoderma reesei ACE1, a regulator of cellulase and xylanase expression, complemented the F. verticillioides Δsda1 mutant, which indicates that Ace1 and Sda1 are functional orthologs. Taken together, the data indicate that Sda1 is a transcriptional regulator of carbon metabolism and toxin production in F. verticillioides.

Characterization, cloning and functional expression of novel xylanase from Thermomyces lanuginosus SS-8 isolated from self-heating plant wreckage material

Extracellular cellulase free xylanase from Thermomyces lanuginosus sp. SS-8, isolated from self heating plant wreckage material was identified as β-1,4-endo-xylanase precursor, a monomer of 21.3kDa with no carbohydrate residue. This xylanase retained 80% activity at 60°C for 96h, was active at a wide pH range of 3-11 and uniquely hydrolyzed xylan to xylose without production of xylo-oligosaccharides. Gene xynSS8 encoding xylanase from T. lanuginosus SS-8 was cloned and functionally expressed in Escherichia coli XL1 Blue using pTZ57R/T plasmid and xynSS8/pQE-9 expression vector construct respectively. Gene xynSS8 was of 777bp and deduced amino acid sequence was a mature xylanase of 258 amino acids. XynSS8 has extra 33 amino acids compared to its nearest homolog and was thermo-alkali tolerant as that of native protein. The xylanase could degrade pulp and release substantial chromophoric materials and lignin derived compounds indicating its effective utility in pulp bleaching. Novel characteristics of the enzyme may contribute to its wide industrial usage. This is first report of cloning and functional expression of the novel xylanase from T. lanuginosus SS-8.

Mutation of the Xylanase regulator 1 causes a glucose blind hydrolase expressing phenotype in industrially used Trichoderma strains

BackgroundTrichoderma reesei is an organism involved in degradation of (hemi)cellulosic biomass. Consequently, the corresponding enzymes are commonly used in different types of industries, and recently gained considerable importance for production of second-generation biofuel. Many industrial T. reesei strains currently in use are derived from strain Rut-C30, in which cellulase and hemicellulase expression is released from carbon catabolite repression. Nevertheless, inducing substances are still necessary for a satisfactory amount of protein formation.ResultsHere, we report on a T. reesei strain, which exhibits a very high level of xylanase expression regardless if inducing substances (e.g. D-xylose, xylobiose) are used. We found that a single point mutation in the gene encoding the Xylanase regulator 1 (Xyr1) is responsible for this strong deregulation of endo-xylanase expression and, moreover, a highly elevated basal level of cellulase expression. This point mutation is localized in a domain that is common in binuclear zinc cluster transcription factors. Only the use of sophorose as inducer still leads to a slight induction of cellulase expression. Under all tested conditions, the formation of cbh1 and cbh2 transcript level strictly follows the transcript levels of xyr1. The correlation of xyr1 transcript levels and cbh1/cbh2 transcript levels and also their inducibility via sophorose is not restricted to this strain, but occurs in all ancestor strains up to the wild-type QM6a.ConclusionsEngineering a key transcription factor of a target regulon seems to be a promising strategy in order to increase enzymes yields independent of the used substrate or inducer. The regulatory domain where the described mutation is located is certainly an interesting research target for all organisms that also depend so far on certain inducing conditions.

RNA interference with carbon catabolite repression in Trichoderma koningii for enhancing cellulase production

The cellulase and xylanase genes of filamentous Trichoderma fungi exist under carbon catabolite repression mediated by the regulator carbon catabolite repressor (CREI). Our objective was to find the role of CREI in a cellulase-hyperproducing mutant of Trichoderma koningii, and address whether enzyme production can be further improved by silencing the cre1 gene. cre1 partially silenced strains were constructed to improve enzyme production in T. koningii YC01, a cellulase-hyperproducing mutant. Silencing of cre1 resulted in derepression of cellulase gene expression in glucose-based cultivation. The cre1 interference strain C313 produced 2.1-, 1.4-, 0.8-, and 0.8-fold higher amounts of filter paper activity, β-1,4-exoglucanase activity (ρ-nitrophenyl-β-d-cellobioside as substrate), β-1,4-endoglucanase activity (sodium carboxymethyl cellulose as substrate), and xylanase activity, respectively, than the control strain, suggesting that silencing of cre1 resulted in enhanced enzyme production capability. In addition, downregulation of cre1 resulted in elevated expression of another regulator of xylanase and cellulase expression, xyr1, indicating that CREI also acted as a repressor of xyr1 transcription in T. koningii under inducing conditions. These results show that RNAi is a feasible method for analyzing the regulatory mechanisms of gene expression and improving xylanase and cellulase productivity in T. koningii.

Gene Cloning, Expression and Characterization of a Novel Xylanase from the Marine Bacterium, Glaciecola mesophila KMM241

Marine xylanases are rather less studied compared to terrestrial xylanases. In this study, a new xylanase gene, xynB, was cloned from the marine bacterium, Glaciecola mesophila KMM241, and expressed in Escherichia coli. xynB encodes a multi-domain xylanase XynB of glycoside hydrolase (GH) family 8. The recombinant XynB comprises an N-terminal domain (NTD) with unknown function and a catalytic domain, which is structurally novel among the characterized xylanases of GH family 8. XynB has the highest identity (38%) to rXyn8 among the characterized xylanases. The recombinant XynB showed maximal activity at pH 6–7 and 35 °C. It is thermolabile and salt-tolerant. XynB is an endo-xylanase that demands at least five sugar moieties for effective cleavage and to hydrolyze xylohexaose and xylopentaose into xylotetraose, xylotriose and xylobiose. NTD was expressed in Escherichia coli to analyze its function. The recombinant NTD exhibited a high binding ability to insoluble xylan and avicel and little binding ability to chitosan and chitin. Since the NTD shows no obvious homology to any known carbohydrate-binding module (CBM) sequence in public databases, XynB may contain a new type of CBM.

Expression of a bacterial xylanase in Trichoderma reesei under the egl2 and cbh2 glycosyl hydrolase gene promoters

Expression vectors were constructed for Trichoderma reesei using the promoters, secretion signals and the modular structure of the efficiently expressed and secreted cellulase enzymes EGL2 (Cel5A) and CBH2 (Cel6A) as a prelude to establishing a platform where a gene of interest can be expressed under several promoters simultaneously. The designs featured (i) EGL2sigpro (egl2 promoter and secretion signal), (ii) EGL2cbmlin (egl2 promoter, secretion signal, EGL2 cellulose binding module and linker), (iii) CBH2sigpro (cbh2 promoter and secretion signal) and (iv) CBH2cbmlin (cbh2 promoter, secretion signal, CBH2 cellulose binding module and linker). Recombinant vectors were introduced individually into the high protein-secreting T. reesei RUT-C30 strain to generate single-promoter transformants expressing the Dictyoglomus thermophilum xynB gene that encodes a thermophilic xylanase enzyme (XynB). Ten transformants producing XynB representing each of the four different types of vectors were selected for further testing and the highest XynB production was achieved from a transformant containing 1-2copies of the EGL2cbmlin vector. Best xylanase producers did not show any particular pattern in terms of the number of gene copies and their mode of integration into the chromosomal DNA. Transformants generated with the cbmlin-type vectors produced multiple forms of XynB which were decorated with various N- and O-glycans. One of the O-glycans was identified as hexuronic acid, whose presence had not been observed previously in the glycosylation patterns of T. reesei.