Expression Of Xylanase Research Articles

Comparative Quantitative Analysis of Gene Expression Profiles of Glycoside Hydrolase Family 10 Xylanases in the Sheep Rumen during a Feeding Cycle

Xylanase is a crucial hydrolytic enzyme that degrades plant polysaccharides in the rumen. To date, there is no information on the genetic composition and expression characteristics of ruminal xylanase during feeding cycles of ruminants. Here, the major xylanase of the glycoside hydrolase family 10 (GH 10) from the rumen of small-tail Han sheep was investigated during a feeding cycle. We identified 44 distinct GH 10 xylanase gene fragments at both the genomic and transcriptional levels. Comparison of their relative abundance showed that results from the evaluation of functional genes at the transcriptional level are more reliable indicators for understanding fluctuations in xylanase levels. The expression patterns of six xylanase genes, detected at all time points of the feeding cycle, were investigated; we observed a complex trend of gene expression over 24 h, revealing the dynamic expression of xylanases in the rumen. Further correlation analysis indicated that the rumen is a dynamic ecosystem where the transcript profiles of xylanase genes are closely related to ruminal conditions, especially rumen pH and bacterial population. Given the huge diversity and changing composition of enzymes over the entire rumen, this research provides valuable information for understanding the role of functional genes in the digestion of plant material.

Biofuel Self-Engineering

Plant Science![Figure][1] CREDIT: ADAPTED FROM B. SHEN ET AL., NAT. BIOTECHNOL. 30 (21 OCTOBER 2012) © 2012 NATURE PUBLISHING GROUP Biofuels could be a useful substitute for fossil fuels, but various aspects of the production process need improvement to increase yield and reduce costs. One approach would be to express processing enzymes in the growing plant itself. However, to avoid damage to the growing plant, the enzymes need to be held at bay until the time comes to convert cell walls into soluble sugars. Shen et al. have tackled this challenge in maize, for which xylanase can be used to break down cell walls into fermentable carbohydrates. Expression of xylanase in the plant alleviates problems of enzyme production and access to substrates, but at the same time produces problems in damaging plant growth, for example, yielding shriveled seeds. The xylanase was brought under control by adding an intein, a self-splicing peptide from the bacterium Thermus thermophilus . Use of a thermostable xylanase meant that treatment of the plant material containing the hybrid protein with temperatures in the range of 60° to 70°C resulted in removal of the inhibitory peptide and the generation of functional xylanase. With an optimized version of the hybrid xylanase-intein, transgenic plants showed normal development and normal seed set, and improved biomass conversion to glucose and xylose. Nat. Biotechnol. 30 , 10.1038/nbt.2402 (2012). [1]: pending:yes

Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing

Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall-degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.

Expression of a Trichoderma reesei β-1,4 endo-xylanase in tall fescue modifies cell wall structure and digestibility and elicits pathogen defence responses

An endo-xylanase from Trichoderma reesei (xyn2) has been expressed in tall fescue targeted to the vacuole, apoplast or Golgi, constitutively under the control of the rice actin promoter, and to the apoplast under the control of a senescence enhanced gene promoter. Constitutive xylanase expression in the vacuole, apoplast, and golgi, resulted in only a small number of plants with low enzyme activities and in reduced plant growth in apoplast, and golgi targeted plants. Constitutive expression in the apoplast also resulted in increased levels of cell wall bound hydroxycinnamic acid monomers and dimers, but no significant effect on cell wall xylose or arabinose content. In situ constitutive xylanase expression in the Golgi also resulted in increased ferulate dimers. However, senescence induced xylanase expression in the apoplast was considerably higher and did not affect plant growth or the level of monomeric hydroxycinnamic acids or lignin in the cell walls. These plants also showed increased levels of ferulate dimers, and decreased levels of xylose with increased levels of arabinose in their cell walls. While the release of cell wall hydroxycinnamic acids on self digestion was enhanced in these plants in the presence of exogenously applied ferulic acid esterase, changes in cell wall composition resulted in decreases in both tissue digestibility and cellulase mediated sugar release. In situ detection of H(2)O(2) production mediated by ethylene release in leaves of plants expressing apoplast xylanase could be leading to increased dimerisation. High-level xylanase expression in the apoplast also resulted in necrotic lesions on the leaves. Together these results indicate that xylanase expression in tall fescue may be triggering plant defence responses analogous to foliar pathogen attack mediated by ethylene and H(2)O(2).

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

A xylanase gene (xyn10A) was cloned from Bacillus sp. SN5 and expressed in Escherichia coli. It encoded a 348-residue polypeptide of ~45kDa. The deduced amino acid sequence had 68% identity with the endo-1,4-beta-xylanase from Paenibacillus lactis 154 that belonged to family 10 of the glycoside hydrolases. Purified recombinant Xyn10A had maximum activity at 40°C and pH 7.0, with the specific activity of 105U/mg and a Km of 0.6mg/ml for beechwood xylan. Xyn10A retained more than 80% activity between 25 and 45°C and 29% activity at 5°C. It exhibited the highest activity (134%) in 0.5M NaCl and still retained 90% activity in 2.5M NaCl. It retained about 87% activity after incubation in 2M NaCl for 24h. The cold-active and halo-tolerant properties of Xyn10A make it promising for application in the food industry, especially in the processing of saline food and sea food.

Cloning, expression, and characterization of an alkaline thermostable GH11 xylanase from Thermobifida halotolerans YIM 90462T

A xylanase gene (thxyn11A) from the Thermobifida halotolerans strain YIM 90462(T) was cloned and expressed in Escherichia coli. The open reading frame (ORF) of thxyn11A has 1,008 bp encoding a mature xylanase with a high degree of similarity (80 %) to the xylanase from Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111. This enzyme (Thxyn11A) also possesses a glycosyl hydrolases family 11 (GH11) domain and a high isoelectric point (pI = 9.1). However, Thxyn11A varies from most GH11 xylanases, due to its large molecular mass (34 kDa). Recombinant Thxyn11A demonstrated a strong pH and temperature tolerance with a maximum activity at pH 9.0 and 70 °C. Xylotriose, the end-product of xylan hydrolysis by Thxyn11A, serves as a catalyst for hemicellulose pretreatment in industrial applications and can also function as a food source or supplement for enterobacteria. Due to its attractive biochemical properties, Thxyn11A may have potential value in many commercial applications.

Production of soluble recombinant proteins in Escherichia coli: Effects of process conditions and chaperone co-expression on cell growth and production of xylanase

In this study, effects of temperature, inducer concentration, time of induction and co-expression of molecular chaperones (GroEL–GroES and DnaKJE), on cell growth and solubilization of model protein, xylanases, were investigated. The yield of soluble xylanases increased with decreasing cultivation temperature and inducer level. In addition, co-expression of DnaKJE chaperone resulted in increased soluble xylanases though the time of induction of chaperone and target protein had a bearing on this yield. A combination of chaperone co-expression and partial induction resulted in ∼40% (in DnaKJE) and 33% (in GroEL–GroES) of total xylanase yield in soluble fraction. However, the conditions for maximum yield of soluble r-XynB and maximum % soluble expression of r-XynB were different. Higher expression of soluble xylanases in a scalable semi-synthetic medium showed potential of the process for soluble enzyme production.

Characterization of the novel xylanase from the thermophilic Geobacillus thermodenitrificans JK1

Thermophilic strain JK1 was isolated from compost using xylan as a single carbon source. On the basis of 16S rRNA gene phylogenetic analysis and spo0A gene sequence similarity analysis, strain JK1 was identified as Geobacillus thermodenitrificans strain. During the exponential culture growth, the strain JK1 was found to produce the single xylan degrading enzyme -45 kDa in size. Xylose was not an inducer of this xylanase. Cloning, expression and characterization of the recombinant xylanase were performed. Xylanase of G. thermodenitrificans JK1 was cellulase-free; pH and temperature optimums were found to be 6.0 and 70 degrees C, respectively. The metal ions Na+, K+, Ca2+, and Co2+ showed partial inhibition of the activity, while Mn2+ had slight stimulating effect on the enzymatic activity. Recombinant xylanase was thermostable over the temperature range of 55-70 degrees C. It presented the highest stability after incubation at 55 degrees C for 60 min showing 84% residual activity. 50% residual activity was revealed after incubation at 60 degrees C for 60 min as well as at 65 and 70 degrees C for 30 min. Results of the thermostability experiments showed xylanase of JK1 having quite low thermostability when compared with the respective enzymes of the other geobacilli.

Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters

BackgroundsThe fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II.ResultsThe transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase) and eno (encoding enolase) genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter) and 1.52 g/L (with the eno promoter), approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively.ConclusionsThis work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters.

Hydrolytic enzyme activity of EN113 standard basidiomycetes in the fermentation of lignocellulosic material and wood colonization

Abstract The white-rot fungus Trametes versicolor and brown-rot fungi Coniophora puteana, Gloeophyllum trabeum, and Postia placenta have a potential to produce a range of hydrolytic enzymes participating in the degradation of lignocellulosic materials. In the colonization of untreated and thermally modified wood blocks wheat bran-containing medium (WBA) greatly favored the secretion of xylanase by C. puteana and both endoglucanase and xylanase expression by T. versicolor, although rather higher carboxymethyl cellulase activity of C. puteana was observed on the malt extract-based medium (malt extract-containing agar (MEA). Coniophora puteana and T. versicolor are able to actively colonize and degrade the untreated pine and birch wood blocks. The mycelium applied on blocks in the form of pellets was more aggressive in wood degradation in comparison with the colonized mycelium. WBA favored the dry matter loss during the initial stages (10–20 days) of pine wood colonization; however, MEA further promoted more rapid wood degradation. The main finding was that the thermal treatment of birch wood was accompanied by a low degree of fungal colonization and increased durability against the fungal attack in spite of the high cellulase and xylanase activities. Thermal modification of wood was not toxic to the fungi because it did not disturb the secretion of the hydrolytic enzymes involved in wood degradation. The thermally modified hollocellulose is probably less susceptible to the enzymes involved in the degradation of polysaccharides or less accessible to hydrolytic enzymes due to the change of the cell wall structure.

SYNONYMOUS CONDON USAGE BIAS AND OVEREXPRESSION OF A SYNTHETIC xynB GENE FROM Aspergillus niger NL-1 IN Pichia pastoris

To further improve the expression level of recombinant xylanase in Pichia pastoris, the xynB gene, encoding the mature peptide from Aspergillus niger NL-1, was designed and synthesized based on the synonymous condon bias of P. pastoris and optimized G+C content. 155 nucleotides were changed, and the GC content decreased from 57.7% to 43.6%. The synthetic xynB was inserted into the pPICZaA and then integrated into P. pastoris GS115. The activity of the recombinant xylanase reached 1414.7 U/mL, induced with 0.8% methanol after 14-day cultivation at a temperature of 28oC in shake flasks, which was 267% higher than that of the native gene. Furthermore, the maximum xylanase activity of 20424.2 U/mL was obtained by high-density fermentation in a 5-L fermenter, which was the highest xylanase expression in P. pastoris yet reported. The recombinant xylanase had its optimal activity at a pH of 5.0 and temperature of 50oC. The recombinant xylanase was stable over a pH range of 4.5 to 8.0. Thus, this report provides an industrial means to produce the recombinant xylanase in P. pastoris.

Attenuated Secretion of the Thermostable Xylanase xynB from Pichia pastoris Using Synthesized Sequences Optimized from the Preferred Codon Usage in Yeast

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.

High-level expression of a hyperthermostable Thermotoga maritima xylanase in Pichia pastoris by codon optimization

The second xylanase gene (xynB) from the hyperthermophilic Thermotoga maritima was optimized according to the codon usage of Pichia pastoris and expressed in P. pastoris. The optimized gene (xynBop) shared 77.8% of nucleotide sequence identity with that of native gene. A total of 232 nucleotides were changed and the G + C ratio was simultaneously increased from 42.7% to 43.1%. The recombinant xylanase (XynBop) was secreted into the culture medium that reached a total extracellular protein concentration of 10.1 g l−1 with an activity of 40,020 U ml−1 in 5-l fermentor culture. The recombinant enzyme was optimally active at pH 5.5 and at 100 °C, respectively. The secreted expression level makes the enzyme a good candidate for hyperthermostable xylanase production.

Induction of xylanase in thermophilic fungi Scytalidium thermophilum and Sporotrichum thermophile

Regulation of xylanase production in two thermophilic fungi Scytalidium thermophilum and Sporotrichum thermophile was investigated. The expression of xylanase was found to be inducible in both the cases. Various carbon sources were tested so as to identify the inducers. Soy flour and oat spelt xylan induced maximum level of xylanase in Scytalidium thermophilum and Sporotrichum thermophile respectively. Induction of xylanase in Scytalidium thermophilum led to simultaneous induction of cellulase. The zymography of enzyme preparations revealed that different carbon sources caused differential expression of multiple isoforms of xylanase in Scytalidium thermophilum, but same isoforms were expressed by Sporotrichum thermophile irrespective of the carbon source used.

Evaluation of the catalase promoter for expressing the alkaline xylanase gene (alx) in Aspergillus niger

Aspergillus niger represents a promising host for the expression of recombinant proteins, but only a few expression systems are available for this organism. In this study, the inducible catalase promoter (PcatR) from A. niger was characterized. For this, constructs were developed and checked for the expression of the alkaline xylanase gene transcriptionally fused under the cat R promoter. Two versions of the catalase (catR) promoter sequence from A. niger (P(cat300,) P(cat924)) were isolated and tested for their ability to drive expression of the alkaline xylanase (alx) gene. P(cat924) showed better efficiency (more than 10-fold increase in AlX activity compared to P(cat300)) under the optimized culture conditions. Induction of the catR promoter with 0.20% H(2)O(2) and 1.5% CaCO(3) in the culture medium, further increased expression of AlX 2.61- and 2.20-fold, respectively, clarifying its inducible nature. Specific induction or repression of the catR promoter provides the possibility for utilization of this promoter in heterologous protein production.

Cloning, Expression, and Characterization of a New Xylanase from Alkalophilic Paenibacillus sp. 12-11

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at 55 degrees C and was thermostable at 50 degrees C and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent Km and Vmax values were 1.18 mg/ml and 1,961 μmol/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.

High level expression of a recombinant xylanase by Pichia pastoris NC38 in a 5 L fermenter and its efficiency in biobleaching of bagasse pulp

A genetically modified XynA gene from Thermomyces lanuginosus was expressed in Pichia pastoris under the control of GAP promoter. P. pastoris expressed greater levels of xylanase (160IUml−1) on BMGY medium without zeocin after 56h. The xylanase production by recombinant P. pastoris was scaled up in a 5L fermenter containing 1% glycerol and the highest xylanase production of 139IUml−1 was observed after 72h. Further studies carried out in fermenter under controlled pH (5.5) yielded a maximum xylanase production of 177IUml−1 after 72h. The biobleaching efficacy of crude xylanase was also evaluated on bagasse pulp and a brightness of 47.4% was observed with 50IU of crude xylanase used per gram of pulp, which was 2.1 points higher in brightness than the untreated samples. Reducing sugars (24.8mgg−1) and UV absorbing lignin-derived compounds values were considerably higher with xylanase treated samples.

L -Arabitol Is the Actual Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)

The saprophytic fungus Hypocrea jecorina (anamorph, Trichoderma reesei) is an important native producer of hydrolytic enzymes, including xylanases. Regarding principles of sustainability, cheap and renewable raw materials, such as d-xylose (the backbone monomer of xylan), have been receiving increasing attention from industries. Recently, it was demonstrated that small (0.5 to 1 mM) amounts of d-xylose induce the highest expression of xylanase in H. jecorina. However, it was also reported that active metabolism of d-xylose is necessary for induction. In this report, we demonstrate that xylitol, the next intermediate in the pentose pathway after d-xylose, does not trigger transcription of xylanase-encoding genes in H. jecorina QM9414. The highest level of transcription of xylanolytic enzyme-encoding genes occurred in an xdh1 (encoding a xylitol dehydrogenase) deletion strain cultured in the presence of 0.5 mM d-xylose, suggesting that a metabolite upstream of xylitol is the inducer. The expression levels of xylanases in an xdh1-lad1 double-deletion strain were lower than that of an xdh1 deletion strain. This observation suggested that l-xylulose is not an inducer and led to the hypothesis that l-arabitol is the actual inducer of xylanase expression. A direct comparison of transcript levels following the incubation of the H. jecorina parental strain with various metabolites of the pentose pathway confirmed this hypothesis. In addition, we demonstrate that xyr1, the activator gene, is not induced in the presence of pentose sugars and polyols, regardless of the concentration used; instead, we observed low constitutive expression of xyr1.

Global and grain‐specific accumulation of glycoside hydrolase family 10 xylanases in transgenic maize (Zea mays)

In planta expression of cell wall degrading enzymes is a promising approach for developing optimized biomass feedstocks that enable low-cost cellulosic biofuels production. Transgenic plants could serve as either an enzyme source for the hydrolysis of pretreated biomass or as the primary biomass feedstock in an autohydrolysis process. In this study, two xylanase genes, Bacillus sp. NG-27 bsx and Clostridium stercorarium xynB, were expressed in maize (Zea mays) under the control of two different promoters. Severe phenotypic effects were associated with xylanase accumulation in maize, including stunted plants and sterile grains. Global expression of these xylanases from the rice ubiquitin 3 promoter (rubi3) resulted in enzyme accumulation of approximately 0.01 mg enzyme per gram dry weight, or approximately 0.1% of total soluble protein (TSP). Grain-specific expression of these enzymes from the rice glutelin 4 promoter (GluB-4) resulted in higher-level accumulation of active enzyme, with BSX and XynB accumulating up to 4.0% TSP and 16.4% TSP, respectively, in shriveled grains from selected T0 plants. These results demonstrate the potential utility of the GluB-4 promoter for biotechnological applications. The phenotypic effects of xylanase expression in maize presented here demonstrate the difficulties of hemicellulase expression in an important crop for cellulosic biofuels production. Potential alternate approaches to achieve xylanase accumulation in planta without the accompanying negative phenotypes are discussed.

Cloning and optimized expression of a GH-11 xylanase from Fusarium oxysporum in Pichia pastoris

The endo-1,4-β-xylanase gene xyn11a from Fusarium oxysporum, member of the fungal glycosyl hydrolase (GH) family 11, was cloned and expressed in Pichia pastoris. The mature xylanase gene, which generates after the excision of one intron and the secreting signal peptide, was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The final construction was integrated into the genome of the methylotrophic yeast P. pastoris X33 and the ability to produce xylanase activity was evaluated in flask cultures. Recombinant P. pastoris efficiently secreted xylanase into the medium and produced high level of enzymatic activity (110 U/ml) after 216 hours of growth, under methanol induction. To achieve higher enzyme production, the influence of initial pH, methanol concentration, agitation and flask design was evaluated. Under optimum culture conditions, production of the recombinant xylanase increased by 50%, reaching a final yield of 170 U/ml, underpinning aeration as the most important factor in improving enzyme production.