Thermostable Xylanase Research Articles

Purification and characterization of a cellulase-free, thermostable xylanase from Streptomyces rameus L2001 and its biobleaching effect on wheat straw pulp

A xylanase purified from Streptomyces rameus L2001 and the biobleaching effect on wheat straw pulp was investigated. The extracellular xylanase was purified 13.3-fold by precipitation with 40–60% (NH 4) 2SO 4, DEAE-52 and CM Sepharose Fast Flow ion exchange chromatography. It appeared as a monomeric protein on SDS-PAGE gel and had a molecular mass of approx. 21.1 kDa, with a specific activity of 3236.6 U/mg. The purified xylanase had an optimum pH of 5.3 and was stable over pH 4.3–6.7. The stable optimal temperature of the enzyme was 70 °C. The xylanase was activated by Co 2+ by up to 329% of baseline activity. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent K m values of the xylanase for birchwood and beechwood were 5.8 and 5.3 mg mL −1, respectively. The potential application of the xylanase was further evaluated in wheat straw pulp. The amount of reducing sugars released by the xylanase from wheat straw pulp was significantly greater with increasing time. Enzymatic treatment at a charge of 20 U/g dry pulp for 1 h before hypochlorite (3.8%) treatment revealed an increase in brightness index by 2.8% and increase in residual chlorine by 14.5%.

Improving the thermostability of Geobacillus stearothermophilus xylanase XT6 by directed evolution and site-directed mutagenesis

Protein engineering of the thermostable xylanase XT6 from Geobacillus stearothermophilus was performed to obtain enzymes with improved thermal tolerance. Mutants producing such enzymes were obtained after several rounds of directed evolution using error-prone PCR and sequence family shuffling, in combination with a consensus-based semi-rational approach. The most thermostable mutant enzyme contained 13 amino acid substitutions and its half-life of inactivation was 52-fold of that of the wild-type. Its reaction temperature for maximum activity increased from 77 degrees C to 87 degrees C, and catalytic efficiency (k(cat)/K(m)) increased by 90%. The mutant is of potential interest for industrial applications.

Purification and characterization of two thermostable xylanases from Malbranchea flava active under alkaline conditions

Two xylanases, MFX I and MFX II, from the thermophilic fungus Malbranchea flava MTCC 4889 with molecular masses of 25.2 and 30kDa and pIs of 4.5 and 3.7, respectively were purified to homogeneity. The xylanases were optimally active at pH 9.0 and at 60 degrees C, exhibited a half-life of 4h at 60 degrees C, and showed distinct mode of action and product profiles when applied to birchwood, oat spelt, and larchwood xylan, and to wheat and rye arabinoxylan. The xylanases were most active on larchwood xylan with K(m) values of 1.25 and 3.7mg/ml. K(cat)/K(m) values suggested that the xylanases preferentially hydrolyzed rye arabinoxylan. LC-MS/MS (liquid chromatography/mass spectrometry) analysis of tryptic digests of MFX I and MFX II revealed similarity with known fungal xylanases and suggests that that they belonged to the GH 11 and 10 glycosyl hydrolase super families, respectively. These xylanases can potentially be used in enzyme-assisted bleaching of the pulp derived from agro-residues, as well as production of xylooligosaccharides for pre-biotic functional food applications.

Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11

An open reading frame (XylX) with 1131 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in E. coli. It encodes a family 11 endoxylanase, designated as XylX, of 41 kDa. The homology of the amino acid sequence deduced from XylX is only 73% identical to the next closest sequence. XylX contains a family 11 catalytic domain of the glycoside hydrolase and a family 6 cellulose-binding module. The recombinant xylanase was fused to a His-tag for affinity purification. The XylX activity was 2392 IU/mg, with a K m of 6.78 mg/ml and a V max of 4953 mol/min/mg under optimal conditions (pH 7, 60 °C). At pH 11, 60 °C, the activity was still as high as 517 IU/mg. Xylanase activities at 60 °C under pH 5 to pH 9 remained at more than 69.4% of the initial activity level for 8 h. The addition of Hg 2+ at 5 mM almost completely inhibited xylanase activity, whereas the addition of tris-(2-carboxyethyl)-phosphine (TCEP) and 2-mercaptoethanol stimulated xylanase activity. No relative activities for Avicel, CMC and d-(+)-cellobiose were found. Xylotriose constitutes the majority of the hydrolyzed products from oat spelt and birchwood xylan. Broad pH and temperature stability shows its application potentials for biomass conversion, food and pulp/paper industries.

Thermostable xylanase production and partial purification by solid-state fermentation using agricultural waste wheat straw

A locally isolated strain of Trichoderma harzianum was studied for production of xylanase (EC 3.2.1.8) using lignocellulosic substrates for solid-state fermentation. Among the different substrates used, wheat straw produced the highest yields (146 IU/ml). The influence of temperature, pH, moistening agents, moisture level, carbon sources, nitrogen sources, pretreatments and metal ions were evaluated with respect to xylanase production. Highest xylanase production was obtained using wheat straw after 12 days of incubation. Different substrate combination ratios and the effect of particle size were also checked against xylanase production. Maximum xylanase production was observed in a wheat straw/rice straw ratio of 1:1, with a particle size of 0.45–0.5 mm. Media supplementation with xylose as a carbon source in a ratio 1:5 (carbon source/substrate) gave maximum activity (157 IU/ml). Nitrogen supplementations from yeast extract produced xylanase activity of 141 IU/ml. Optimum xylanase production was observed at 30°C and pH 5.0. Xylanase production was enhanced in the presence of Ca2+ and Zn2+ ions. Ammonium sulfate fractionation (20–80% saturation) of partially purified xylanase yielded 76.5% of the enzyme with a 3.53-fold purification. The molecular weight of xylanase was found to be ca. 29,000 Da by SDS–PAGE.

Purification and characterization studies of a thermostable β-xylanase from Aspergillus awamori

This study presents data on the production, purification, and properties of a thermostable β-xylanase produced by an Aspergillus awamori 2B.361 U2/1 submerged culture using wheat bran as carbon source. Fractionation of the culture filtrate by membrane ultrafiltration followed by Sephacryl S-200 and Q-Sepharose chromatography allowed for the isolation of a homogeneous xylanase (PXII-1), which was 32.87 kDa according to MS analysis. The enzyme-specific activity towards soluble oat spelt xylan, which was found to be 490 IU/mg under optimum reaction conditions (50°C and pH 5.0-5.5), was 17-fold higher than that measured in the culture supernatant. Xylan reaction products were identified as xylobiose, xylotriose, and xylotetraose. K (m) values (mg ml(-1)) for soluble oat spelt and birchwood xylan were 11.8 and 9.45, respectively. Although PXII-1 showed 85% activity retention upon incubation at 50 °C and pH 5.0 for 20 days, incubation at pH 7.0 resulted in 50% activity loss within 3 days. PXII-1 stability at pH 7.0 was improved in the presence of 20 mM cysteine, which allowed for 85% activity retention for 25 days. This study on the production in high yields of a remarkably thermostable xylanase is of significance due to the central role that this class of biocatalyst shares, along with cellulases, for the much needed enzymatic hydrolysis of biomass. Furthermore, stable xylanases are important for the manufacture of paper, animal feed, and xylooligosaccharides.

Improvement of xylanase production by thermophilic fungus Thermomyces lanuginosus SDYKY-1 using response surface methodology

Response surface methodology (RSM) was employed to optimize medium composition for the production of a thermostable xylanase from T. lanuginosus SDYKY-1 using economical carbon and nitrogen sources (soybean meal and corncobs, respectively). Plackett-Burman (P-B) design was applied to evaluate the effects of nine variables (powdered corncobs, soybean meal, Tween-80, CaCl(2), MgSO(4)·7H(2)O, FeSO(4), KH(2)PO(4), initial pH and inoculum culture volume). Corncobs, soybean meal and FeSO(4) were found to significantly influence on the xylanase production. The concentrations of these three factors were therefore optimized using central composite design and RSM. Adjusting the concentration of corncobs to 38.7g/L, soybean meal to 17.5g/L and FeSO(4) to 0.26g/L favored maximum xylanase production. Xylanase activity of 3078U/mL was obtained after optimization, which was a 144% increase that obtained before optimization (1264U/mL).

Production of xylooligosaccharides from the steam explosion liquor of corncobs coupled with enzymatic hydrolysis using a thermostable xylanase

The production of xylooligosaccharides (XOs) from corncobs was studied using a two-stage process based on a steam explosion pretreatment followed by enzymatic hydrolysis. Corncobs with different chip sizes were subjected to steam explosion under different experimental conditions of temperature and time, namely 188–204 °C for 2.5–7.5 min. The results indicate that corncobs were optimally steam exploded at 196 °C for 5 min, resulting in hemicellulose recovery of 22.8%. Especially, corncobs with large chip sizes (∼100 mm) during steam explosion pretreatment were suitable to produce XOs. Furthermore, a thermostable xylanase from Paecilomyces themophila J18 was used to hydrolyze steam explosion liquor of corncobs (SELC) for the production of XOs. A maximum XOs yield of 28.6 g XOs/100 g xylan in corncobs was achieved and XOs syrup contained more than 90% of xylobiose and xylotriose when the hydrolysis was carried out under the optimized conditions (pH 7.0, 70 °C, 7.5 U mL −1 and 2.5 h). These results suggest that the process might be effective in production of XOs for industrial applications.

Highly thermostable purified xylanase from Rhizomucor miehei NRRL 3169

A thermostable xylanase was purified and characterized from the thermophilic fungus Rhizomucor miehei (Cooney & R.Emers.) Schipper. The enzyme was purified 29.1-fold to homogeneity by ammonium sulfate precipitation, sephadex G-100 gel filtration and diethylaminoethyl cellulose anion exchange chromatography. The enzyme was highly active with a range of pH from 5.0 to 6.5. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 70 and 75°C, and the half-life of the xylanase at 90°C was 30 min. Km and Vmax values at 50°C of the purified enzyme were 0.055 mg/ml and 113.5 μmol min−1 mg−1, respectively. The enzyme was activated by Ca2+, Cu2+, K+ and Na+ .On the other hand, Ag2+, Hg2+, Ba2+, and Zn2+ inhibited the enzyme. The molecular weight of the xylanase was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study is among the first work to examine and describe a secreted highly thermostable xylanase from the Rhizomucor miehei fungus. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for industrial and commercial application in pulp bleaching.

Improvement of Highly Thermostable Xylanases Production by Talaromyces thermophilus for the Agro-industrials Residue Hydrolysis

A newly isolated thermophilic fungal strain from Tunisian soil samples was identified as Talaromyces thermophilus and was selected for its ability to produce extracellular hemicellulases when grown on various lignocellulosic substrates. Following the optimization of carbon source, nitrogen source, and initial pH of the growth medium in submerged liquid cultures, yields as high as 10.00 +/- 0.15 and 0.21 +/- 0.02 U/ml were obtained for xylanase and beta-xylosidase, respectively. In fact, wheat bran was found to be a good inducer of hemicellulase enzymes, mainly beta-xylosidase. The optimal temperature and pH of the xylanase activity were 75 degrees C and 8.0, respectively. This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50 degrees C for 7 days at pH 7.0-8.0. The half-lives of the enzyme were 4 h at 80 degrees C, 2 h at 90 degrees C, and 1 h at 100 degrees C. T. thermophilus could therefore be considered as a satisfactory and promising producer of thermostable xylanases. Crude enzyme of T. thermophilus rich in xylanase and beta-xylosidase was established for the hydrolysis of lignocellulosic materials as wheat bran.

Biocatalytic approach for the utilization of hemicellulose for ethanol production from agricultural residue using thermostable xylanase and thermotolerant yeast

A hydrolysis of 62% and 50% for OSX (Oat spelt xylan) and WBH (Wheat bran hemicellulose) were obtained in 36 h and 48 h using Accellerase™ 1000 at 50 °C wherein thermostable xylanase from alkalothermophilic Thermomonospora sp. yielded 67% (OSX) in 3 h and 58% (WBH) in 24 h at 60 °C, favouring a reduction in process time and enzyme dosage. The rate of hydrolysis with thermostable xylanase was increased by 20% with the addition of nonionic surfactant tween 80 or biosurfactant sophorolipid. The simultaneous saccharification and fermentation (SSF) of OSX and WBH using thermostable xylanase and D. hansenii in batch cultures produced 9.1 g/L and 9.5 g/L of ethanol, respectively and had a shorter overall process time than the separate hydrolysis and fermentation (SHF). The immobilized yeast cells in Ca-alginate matrix produced ethanol with a yield of 0.46 g/g from hemicellulosic hydrolysates and were reused six times with 100% fermentation efficiency.

Surfactants from xylan: Production of n-octyl xylosides using a highly thermostable xylanase from Thermotoga neapolitana

A highly thermostable recombinant xylanase from Thermotoga neapolitana was used as a catalyst for single-step synthesis of n-octyl xylobioside and n-octyl xylotrioside from xylan and n-octanol. Effect of xylan concentration, enzyme dose, reaction water content, reaction temperature and initial pH on the yield of these surfactants has been studied. The optimal conditions for n-octyl xylobioside and n-octyl xylotrioside synthesis are found to be different. The maximum n-octyl xylobioside and n-octyl xylotrioside yield were 120 and 38 mg/g of xylan, respectively. The n-octyl xylobioside yield achieved in this study was better than the yield achieved in all other enzymatic synthesis studies reported so far except what is achieved with the use of supercritical fluoroform under high pressure. The n-octyl xylotrioside yield is the highest ever achieved through enzymatic synthesis. An integrated system of production and recovery of n-octyl xylosides has improved the yield of n-octyl xylobioside and n-octyl xylotrioside by a factor of 1.7 and 2.4, respectively.

Improvement of alkaliphily of thermostable GH family 10 xylanase from Thermotoga maritima by directed evolution

Xylanase B (XynTB) from hyperthermophilic bacterium Thermotoga maritima MSB8 is a thermostable xylanase classified into glycoside hydrolase family 10. XynTB is most active at pH 6.0, and shows lower activity at alkaline pHs. Improvement of alkaliphily of XynTB was attempted by directed evolution. One mutant enzyme that showed slightly higher activity under high temperature and alkaline pH conditions was acquired from a newly constructed random mutant library. Protein engineering study of this mutant revealed that the amino acid substitution N92D (Asn92 was substituted by Asp) could contribute to the improvement of alkaliphily.

Effect of Glycosylation and Additional Domains on the Thermostability of a Family 10 Xylanase Produced by Thermopolyspora flexuosa

The effects of different structural features on the thermostability of Thermopolyspora flexuosa xylanase XYN10A were investigated. A C-terminal carbohydrate binding module had only a slight effect, whereas a polyhistidine tag increased the thermostability of XYN10A xylanase. In contrast, glycosylation at Asn26, located in an exposed loop, decreased the thermostability of the xylanase. The presence of a substrate increased stability mainly at low pH.

Characterisation of a thermostable xylanase from Chaetomium sp. and its application in Chinese steamed bread

A novel thermophilic xylanase-producing fungus, Chaetomium sp. CQ31 produced 131 U ml −1 of xylanase when grown on a medium containing corncob (3.5%, w/v) at 37 °C for 6 days. A low molecular xylanase was purified 6.5-fold to homogeneity with a recovery yield of 17.5%. Its molecular mass was estimated to be 25.1 kDa by SDS–PAGE. The xylanase had an optimum pH of 7.5, and its optimal temperature was 65 °C. Apparent K m values of the xylanase for birchwood, beechwood and oat-spelt xylan were 1.3, 0.86 and 4.4 mg/ml, respectively. The influence of this xylanase on the quality of Chinese steamed bread (CSB) was further studied. Addition of xylanase in the range 2.5–5.0 ppm caused a 20–24.5% increase in specific volume over the control and remarkable decrease (8.9–24.2%) in firmness was also noticed. This is the first report on the purification, characterisation and application of a xylanase from Chaetomium sp. CQ31.

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-beta-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 degrees C and remained stable at weakly acidic to alkaline pH (pH 5.0-11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 degrees C for 1 h and more than 40% of the activity at 90 degrees C for 15 min. The K (m) and V (max) values for oat spelt xylan and birchwood xylan were 16.08 mg ml(-1) and 45.66 micromol min(-1) mg(-1) and 9.22 mg ml(-1) and 16.05 micromol min(-1) mg(-1), respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.

Functional characterization of a recombinant thermostable xylanase from Pichia pastoris: A hybrid enzyme being suitable for xylooligosaccharides production

The hybrid xylanase gene Xyn2-A2 was previously constructed and expressed in Pichia pastoris. In this study, the hybrid recombinant enzyme (PTXC2) was prepared from a 2-L bioreactor and subsequently characterized. The produced xylanase was estimated by SDS-PAGE to be 26 kDa and practically free of cellulolytic activity. The optimal temperature and pH of PTXC2 was 65 °C and pH 6.0, respectively. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30 min incubation at this temperature. The specificity of PTXC2 towards different natural substrates was evaluated. PTXC2 was highly specific towards xylans tested but exhibited low activities towards cellulosic substrates, such as gellan gum (9.7%), Avicel (1.5%) and carboxymethylcellulose (CMC, 1.2%). The apparent K m values of oat-spelt xylan and birchwood xylan was 1.6 and 1.8 mg/mL, respectively. Analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as an endo-xylanase with xylotriose as the main hydrolysis products. These attributes should make the enzyme an attractive applicant for various applications, such as large-scale production of xylooligosaccharides.

Thermostable xylanase from Streptomyces thermocyaneoviolaceus for optimal production of xylooligosaccharides

A thermo stable xylanase was purified from Streptomyces thermocyaneoviolaceus M049 for the production of xylooligosaccharides from xylan. The enzyme showed thermostability by maintaining 65% of remaining enzyme activity after 1 h heat treatment at 70°C. The molecular weight of the purified protein was 35 kDa in SDS-PAGE, and the optimal pH and temperature for the enzymatic activity were pH 5.0 and 60°C, respectively. N-terminal amino acid sequences of the purified xylanase, DTITSNQTGTHNGYF, were similar to StxII from S. Thermoviolaceus and XlnB from S. lividans. Using those two genes, stxll and xlnB as probe DNA, a gene encoding xylanase, xynB, was cloned from genomic library of S. thermocyaneoviolaceus M049. The open reading frame of the xynB was composed of 1008 nucleotide sequences. Compared to N-terminal sequences from purified enzyme, it was proposed that the XynB contained a 40 amino acid long signal peptide to the N-terminus. For easy production and purification, a XynB overproduction strain was constructed using pET21a(+) and strain E. coli BLR(DE3). Consequently, the recombinant enzyme was tested for the production of xylooligosaccharides through TLC and HPLC analyses.

A comparative study of Thermomyces lanuginosus strains on thermostable xylanase production

Strains of Thermomyces lanuginosus could be differentiated into two groups based on their ability to produce xylanase using xylan or xylose as sole of carbon source. One group of these strains produced high xylanase activity either in the medium using xylan or xylose as a sole of carbon source. The xylanase production by T. lanuginosus ATCC 44008, THKU-11, and THKU-25, which were the representative members of this group, increased when xylose was added to the xylan medium. In contrast, there was another group that produced high xylanase activity only in the xylan medium. Addition of xylose to the xylan medium resulted decreasing of xylanase production in T. lanuginosus ATCC 46882, TISTR 3465 and THKU-85 that belonged to this group. The finding indicated that induction and repression mechanism of xylose on xylanase expression was different among the strains of T. lanuginosus. In addition, phylogenetic analysis obtained from random amplified polymorphic DNA (RAPD) pattern using primer UBC 241 pointed to greater diversity of high and low xylanase producing strains using xylose as a carbon source. Key words: Thermomyces lanuginosus, RAPD, thermostable xylanase

Optimized Expression of a Thermostable Xylanase 11 A Gene from Chaetomium thermophilum NIBGE 1 in Escherichia coli

The xylanase (Xyn11A) gene (883 bp) was amplified using C. thermophilum DNA as template and cloned into pET-32a (+) and expressed in E. coli BL21 under T(7) promoter. The recombinant xylanase on SDS-PAGE had a molecular mass of 30 kDa. Productivity profiles of xylanase in E. coli recombinant are more than 4-fold of that produced from T. reesei RUTC-30, 5-fold of that produced by the donor and significantly higher than the values reported on other E. coli, and Saccharomyces cerevisiae recombinants. Temperature stability, pH stability, and other kinetic parameters confirmed that the gene product was thermo-stable in alkaline buffer favoring its suitability to bio-bleaching of kraft pulp.