Thermostable Xylanase Research Articles

Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0

A Bacillus spp. strain SPS-0, isolated from a hot spring in Portugal, produced an extracellular xylanase upon growth on wheat bran arabinoxylan. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange, gel filtration, and affinity chromatography. The optimum temperature and pH for activity was 75°C and 6.0. Xylanase was stable up to 70°C for 4 h at pH 6.0 in the presence of xylane. Xylanase was completely inhibited by the Hg 2+ ions. β-Mercaptoethanol, dithiothreitol, and Mn 2+ stimulated the xylanase activity. The products of birchwood xylan hydrolysis were xylose, xylobiose, xylotriose, and xylotetraose. Kinetic experiments at 60°C and pH 6.0 gave V max and K mvalues of 2420 nkat/mg and 0.7 mg/ml.

The Application of Xylanase on Hardwood Kraft Pulp Bleaching and its Operating Experience

Enzyme bleaching was introduced to the process (C-E/O-dnD) for bleaching of hardwood kraft pulp in Yonago mill in October, 1998. This is the first case in which an enzyme has been steadily applied for pulp bleaching in Japan. A thermostable xylanase from a microorganism that we isolated through screening was selected for use in this process. This microorganism, Bacillus sp. S-2113, produces two extracellular xylanases, XP1 and XP2. Expansion of the scale of enzyme production from the laboratory-scale to the mill-scale has been accomplished. It was found that hardwood kraft pulp is effective as a xylanase inducer in the case of this microorganism. The xylanase-containing culture can be applied to the bleaching process without removing the bacterial cells from the culture medium. After the enzyme bleaching process, the microorganism is killed by the bleaching chemicals used in the next step of the bleaching process. The pulp used in the culture medium is returned to the fiber line. Furthermore, because the quantity of enzyme used (the culture) is relatively low, there is no adverse influence on the quality of the pulp. One year has passed since the enzyme bleaching operation, with on-site enzyme production, was begun, and so far, this operation has been working well.The ECF bleaching sequence will start operation within the year 2000 in Yonago mill. The effects of enzyme bleaching in the ECF bleaching process are expected to be even greater then.

Production of a thermostable alkali-tolerant xylanase from Bacillus circulans AB 16 grown on wheat straw.

Bacillus circulans AB 16 was able to produce 50 IU/ml of xylanase, with negligible cellulase activity when grown on untreated wheat straw. The pH optimum of the crude enzyme was 6–7 with a temperature optimum of 80 ∘C. The enzyme showed high pH and thermal stability retaining 100% activity at 60 ∘C, pH 8 and 9 after 2.5 h of incubation. The residual activity at 70 ∘C after 2.5 h was 62% and 45% at pH 8 and 9, respectively. At 75 ∘C only 22.2% activity remained at pH 8 after 1 h incubation. Since Kraft pulp is alkaline this enzyme could be used for prebleaching of pulp at temperatures up to 70 ∘C without pH adjustment.

High resolution structure and sequence ofT. aurantiacus Xylanase I: Implications for the evolution of thermostability in family 10 xylanases and enzymes with ??-barrel architecture

Xylanase I is a thermostable xylanase from the fungus Thermoascus aurantiacus, which belongs to family 10 in the current classification of glycosyl hydrolases. We have determined the three-dimensional X-ray structure of this enzyme to near atomic resolution (1.14 A) by molecular replacement, and thereby corrected the chemically determined sequence previously published. Among the five members of family 10 enzymes for which the structure has been determined, Xylanase I from T. aurantiacus and Xylanase Z from C. thermocellum are from thermophilic organisms. A comparison with the three other available structures of the family 10 xylanases from mesophilic organisms suggests that thermostability is effected mainly by improvement of the hydrophobic packing, favorable interactions of charged side chains with the helix dipoles and introduction of prolines at the N-terminus of helices. In contrast to other classes of proteins, there is very little evidence for a contribution of salt bridges to thermostability in the family 10 xylanases from thermophiles. Further analysis of the structures of other proteins from thermophiles with eight-fold (beta)alpha-barrel architecture suggests that favorable interactions of charged side chains with the helix dipoles may be a common way in which thermophilic proteins with this fold are stabilized. As this is the most common type of protein architecture, this finding may provide a useful guide for site-directed mutagenesis aimed to improve the thermostability of (beta)alpha-barrel proteins. Proteins 1999;36:295-306.

Crystal structure at 1.8 Å resolution and proposed amino acid sequence of a thermostable xylanase from Thermoascus aurantiacus

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component in the biosphere. Crystals belonging to P2 1 space group with a = 41.7 Å, b = 68.1 Å, c = 51.4 Å and β = 113.6 °, Z = 2 were grown that could diffract to better than 1.8 Å resolution. The structure was solved by molecular replacement method using the Streptomyces lividans xylanase model. The amino acid sequence was determined from the electron density map aided by multiple alignment of related xylanase sequences. The sequence thus obtained provides a correction to the sequence reported earlier based on biochemical methods. The final refined protein model at 1.8 Å resolution with 301 amino acid residues and 266 water molecules has an R-factor of 16.0 % and free R of 21.1 % with good stereochemistry. The single polypeptide chain assumes (α/β) 8TIM-barrel fold and belongs to F/10 family of glycoside hydrolases. The active site consists of two glutamate residues located at the C terminus end of the β-barrel, conforming to the double displacement mechanism for the enzyme action. A disulphide bond and more than ten salt bridges have been identified. In particular, the salt bridge Arg124-Glu232 which is almost buried, bridges the β-strands β4 and β7 where the catalytic glutamate residues reside, and it may play a key role in the stability and activity at elevated temperature. To our knowledge, for the first time in the F/10 family xylanases, we observe a proline residue in the middle of the α-helix α6 which may be contributing to better packing. Earlier studies show that the enzyme retains its activity even at 70 °C. The refined protein model has allowed a detailed comparison with the other known structures in the F/10 family of enzymes. The possible causative factors for thermostability are discussed.

Efficient production of truncated thermostable xylanases from Rhodothermus marinus in Escherichia coli fed-batch cultures

A cultivation strategy for the production of two truncated thermostable recombinant xylanases (Xyn1ΔN and Xyn1ΔNC) was developed. Fed-batch cultivations of Escherichia coli strain BL21(DE3) with a controlled exponential glucose feed led to high specific production of the recombinant proteins. Addition of complex nutrients ( e.g. Tryptone Soya Broth (TSB)) to the media were shown to increase both the specific growth rate during the production phase and the production per cell. The final cellmass concentration depended on the time of induction in relation to both the feed-start and the expected time at which the cultivation had to be terminated due to oxygen transfer limitations or cell lysis. The gene used for the genetic constructions (encoding Xyn1ΔN and Xyn1ΔNC) was originally isolated from Rhodothermus marinus. Recombinant protein expression was controlled by the T7 lac-promoter and induced in the fed-batch phase at low glucose concentrations by the single addition of either lactose or isopropyl-thio-β- d-galactoside (IPTG). In lactose-induced cells, the production of recombinant xylanase was delayed for approximately 30 min in comparison with those induced with IPTG, but the specific product levels were comparable at 3 h after induction. At this time, approximately 35% of the intracellular protein content was constituted by recombinant xylanase. Under the cultivation conditions used, production of the shorter deletion derivative (Xyn1ΔNC) led to nonspecific leakage and cell lysis, starting 1.5 or 2 h after induction with IPTG or lactose, respectively. At 3 h after induction, 50% of the produced protein (Xyn1ΔNC) was found in the culture medium. This was not the case for the longer protein (Xyn1ΔN), where only 10% of the xylanase leaked into the medium.

Partial Characterization of the Streptomyces lividans xlnB Promoter and Its Use for Expression of a Thermostable Xylanase from Thermotoga maritima

Xylanase activity assays were used to screen a Streptomyces coelicolor genomic library in Escherichia coli, and a xylanase gene that is 99% identical to the xylanase B gene (xlnB) of S. lividans (GenBank accession no. M64552) was identified. The promoter region of this gene was identified by using a transcriptional fusion between the upstream region of the S. coelicolor xlnB gene and the xylE reporter gene. Transcription from the xlnB promoter was found to be induced by xylan and repressed by glucose. A single apparent transcription start site was identified by both primer extension analysis and in vitro run off transcription assays. Analysis of deletions of the promoter identified a region required for glucose repression. By using the transcriptional and protein localization signals of the Streptomyces xlnB gene, an in-frame translational fusion between the end of the xlnB signal sequence and the ATG of the Thermotoga maritima xynA gene was constructed. The xynA gene encodes a thermostable xylanase that has been demonstrated to be useful in the bleaching of Kraft pulp. The xlnB-xynA gene fusion was expressed in Streptomyces, and the activity of the protein produced was thermostable and was localized to the supernatant fraction of harvested cells.

Evidence for substrate binding of a recombinant thermostable xylanase originating from Rhodothermus marinus

The xyn1 encoded 5 domain xylanase from the thermophilic bacterium Rhodothermus marinus binds specifically to xylan, β-glucan and amorphous but not crystalline cellulose. Our results show that the binding is mediated by the full length xylanase, but not by the catalytic domain only. Based on similarities concerning both predicted secondary structure and binding specificity found with one cellulose binding domain of CenC from Cellulomonas fimi, we suggest that the binding is mediated by the two N-terminally repeated domains.

Thermophilic xylanase from Thermomyces lanuginosus: high-resolution X-ray structure and modeling studies.

The crystal structure of the thermostable xylanase from Thermomyces lanuginosus was determined by single-crystal X-ray diffraction. The protein crystallizes in space group P21, a = 40.96(4) A, b = 52. 57(5) A, c = 50.47 (5) A, beta = 100.43(5) degrees, Z = 2. Diffraction data were collected at room temperature for a resolution range of 25-1.55 A, and the structure was solved by molecular replacement with the coordinates of xylanase II from Trichoderma reesei as a search model and refined to a crystallographic R-factor of 0.155 for all observed reflections. The enzyme belongs to the family 11 of glycosyl hydrolases [Henrissat, B., and Bairoch, A. (1993) Biochem. J. 293, 781-788]. pKa calculations were performed to assess the protonation state of residues relevant for catalysis and enzyme stability, and a heptaxylan was fitted into the active-site groove by homology modeling, using the published crystal structure of a complex between the Bacillus circulans xylanase and a xylotetraose. Molecular dynamics indicated the central three sugar rings to be tightly bound, whereas the peripheral ones can assume different orientations and conformations, suggesting that the enzyme might also accept xylan chains which are branched at these positions. The reasons for the thermostability of the T. lanuginosus xylanase were analyzed by comparing its crystal structure with known structures of mesophilic family 11 xylanases. It appears that the thermostability is due to the presence of an extra disulfide bridge, as well as to an increase in the density of charged residues throughout the protein.

Properties of a thermostable xylanase produced by Melanocarpus albomyces IIS-68 in solid state fermentation

An extracellular xylanase was purified from the buffer extract obtained after the growth of a thermophilic fungus Melanocarpus albomyces IIS-68 in solid-state culture. A twenty five-fold purification employing chromatographic methods and a yield of 6·5% were achieved. Purified xylanase readily solubilized xylans isolated from wood and straws. The xylanase was stable at 70°C for more than 2 h and also in the pH range 5–10. The buffer extract also showed the presence of acetylesterase, a sidegroup-splitting enzyme.

Specificity and mode of action of a thermostable xylanase from bacillus amyloliquefaciens on-line monitoring of hydrolysis products

A thermostable xylanase purified from a strain of Bacillus amyloliquefaciens MIR 32 was characterized with respect to its substrate specificity and mode of hydrolytic action. The enzyme was highly specific for xylans as substrate and displayed no activity toward other polysaccharides, including cellulose. The enzyme exhibited Km and Vmax of 4.5 mg/mL and 0.58 mmol/min/mg, respectively, with birchwood xylan as the substrate. Microdialysis sampling with anion exchange chromatography and integrated pulsed electrochemical detection were used for rapid on-line monitoring of products during hydrolysis of oat spelt and bagasse xylan, and xylooligosaccharides. Xylobiose and xylotriose were the main end products. Xylotetraose was the smallest oligosaccharide to be acted on by the xylanase. The product pattern confirmed that the enzyme was an endoxylanase.

Purification and characterization of a thermostable xylanase from Bacillus amyloliquefaciens

A Bacillus amyloliquefaciens strain isolated from soil produced xylanolytic enzymes in the extracellular medium when grown on xylan and nitrate. No cellulase activity was detected. Xylanase was purified from the culture supernatant in three steps which comprised anion-exchange adsorption, ammonium sulfate precipitation, and hydrophobic interaction chromatography. The pure enzyme appeared as two close protein bands on SDS-PAGE with molecular weights of 18.4 and 19.6 kD, respectively. The molecular weight obtained by sedimentation equilibrium under native conditions was about 18.5 kD. The isoelectric point was 10.1. The enzyme contains a relatively high content of aspartate, glycine, and threonine. Tryptophan seems to be essential for xylanase activity. The presence of carbohydrate was noted in the pure enzyme. Circular dichroism studies indicated that the protein contains almost equal proportions of α-helix, β-sheet, and β-turns. The optimum pH of activity was 6.8–7.0. The enzyme exhibited good stability even at pH 9.0. Excellent stability was observed at 50°C even though optimal activity was at 80°C. The activity was completely inhibited by Hg 2+ ions and was reduced drastically in the presence of Cu 2+ and Fe 3+ ions. Mn 2+ ions, EDTA, β-mercaptoethanol, and dithiothreitol up to 5 m m stimulated the enzyme activity.

Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp.

A new thermophilic strain of Bacillus SPS-0 which produces thermostable xylanases was isolated from a hot spring in Portugal. Xylanase production was 50 nkat/ml in the presence of wheat bran arabinoxylan. The temperature and pH for optimum activity were 75°C and 6–9, respectively. The hydrolysis patterns demonstrated that crude xylanases yield mainly xylose and xylobiose from xylan, whereas xylose and arabinose were produced from destarched wheat bran. An increase in xylose release was observed when SPS-0 xylanase was supplemented by a ferulic acid esterase. © Rapid Science Ltd. 1998

High level production of a cellulase-free xylanase in glucose-limited fed batch cultures of a thermophilic Bacillus strain

Bacillus sp. strain XE and its mutant derivative strain D3 produce a thermostable xylanase which is suitable for enzymatic bleaching of kraft pulp. Xylanase synthesis was shown to be induced by the soluble products of xylan hydrolysis (xylooligosaccharide) and equally, catabolically-repressed when these oligosaccharides accumulate in the medium. An optimal balance between these two antagonistic effects was obtained in a carbon-limited, fed-batch culture with continuous xylooligosaccharide feeding. To reduce substrate cost, the xylooligosaccharide could be 90% substituted by glucose without reduction of the xylanase production rate. Xylanase production ceased when the activity reached approximately 380 U ml −1 due to an amino acid shortage. A continuous supply of exogenous amino acids allowed the production to continue to more than 1000 U ml −1.

Crystallization and preliminary X-ray analysis of the thermostable alkaline-tolerant xylanase from Bacillus stearothermophilus T-6.

The extracellular thermostable xylanase (XT-6) produced by the thermophilic bacterium Bacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K, and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids with a calculated molecular weight of 43,808 and pI of 9.0. Crystallographic studies of XT-6 were initiated to study the mechanism of catalysis as well as to provide a structural basis for rational introduction of enhanced thermostability by site-specific mutagenesis. This report describes the crystallization and preliminary crystallographic characterization of the native XT-6 enzyme. The most suitable crystals were obtained by the vapor-diffusion method using ammonium sulfate and 2-methyl-2,4-pentanediol as an organic additive. The crystals belong to a primitive trigonal crystal system (space group P3(1) or P3(2)) with room-temperature cell dimensions of a = b = 114.9 and c = 122.6 A. At 103 K the volume of the unit cell decreased significantly with observed dimensions of a = b = 112.2 and c = 122.9 A. These crystals are mechanically strong and diffract X-rays to better than 2.2 A resolution. The crystals exhibit considerable radiation damage at room temperature even at relatively short exposures to X-rays. A full 2.3 A resolution diffraction data set (99.8% completeness) has recently been collected on flash-frozen crystals at 103 K using synchrotron radiation. Two derivatives of XT-6 were recently prepared. In the first derivative, a unique Cys residue replaced Glu265, the putative nucleophile in the active site. The second derivative was selenomethionyl xylanase which was produced biosynthetically. These derivatives have been crystallized and the resulting crystals were shown to be isomorphous to the native crystals and diffract X-rays to comparable resolutions.

Purification and some properties of a thermostable xylanase from thermophilic fungus strain HG-1

An extracellular xylanase was purified to homogeneity from a wheat bran culture of the thermophilic fungus HG-1, an isolate from a compost heap. The enzyme had a molecular weight of 33,000 by SDS-PAGE and 31,000 by gel filtration; its isoelectric point was 6.8. The optimum temperature and pH for enzyme activity were 70°C and 4.5–5.0. The enzyme was stable in the pH range from 2 to 12 at 30°C. The K m values for birchwood xylan and oat-spelt xylan were 8.3 and 20 mg/ml, respectively. The enzyme produced xylobiose, xylotriose, and a trace of xylose as the endo products from birchwood xylan. The enzyme activity was strongly inhibited by SDS, and partially by Hg 2+, Mn 2+, Co 2+, Ca 2+, and iodoacetic acid. The enzyme hydrolyzed xylotriose to xylobiose and xylose and showed weak activity toward xylobiose.

Thermostable xylanases produced at 37°C and 45°C by a thermotolerant Aspergillus strain

Differences between xylanases produced by Aspergillus FP-470 growing under normal (37°C) and temperature stress conditions (45°C) were evaluated. Fungal growth at 45°C was seriously affected. However, the xylanase specific activity was 2.5 times higher than that produced at 37°C. Optimum pH and temperature were 6.5, 80°C and 4.3, 50°C for the xylanases produced at 37°C and 45°C, respectively. Electrophoresis showed that a more complex xylanolytic system was produced at 37°C. Stress induced by temperature produced a decrease in the number of extracellular xylanases released by fungi, but increased the production of some xylanases, possibly essential for growth under stress conditions.

Isolation and screening for alkaline thermostable xylanases

AbstractScreening of xylanase producing microorganisms from soil samples and hemicellulosic materials were carried out in three stages using wheat bran extract agar medium, xylan agar medium and liquid medium with xylan. One of the isolates, identified as Bacillus SSP‐34 produced 100 times more xylanase activity than the other isolates. Maximum enzyme production by this isolate was observed after 102 hours of growth. The optimum pH for the crude xylanase preparation range from 6–8 and optimum temperature was at 50 °C. The enzyme was stable up to 20 minutes at 50 °C and at pH range 4.5–9.0 for more than 2 hours.

Release of lignin from kraft pulp by a hyperthermophilic xylanase from Thermatoga maritima

The cloning, sequencing, and expression in E. coli of a gene from the hyperthermophile, Thermatoga maritima, encoding a thermostable xylanase is reported. The enzyme is active at 100°C for several hours and efficient in releasing lignin from kraft pulp. Comparison of the T. maritima recombinant enzyme with a commercially available xylanase, Pulpzyme, indicated that the hyperthermophilic enzyme has several advantages that make it an attractive biotechnological reagent. In studies of the release of reducing sugars and lignin from hardwood and softwood kraft pulp, the specific activity for the partially purified enzyme was 131 U mg −1. The enzyme released reducing sugars and aromatic materials from the pulp suspensions over a pH range from 3.5–10.

Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6

Xylanase T-6 is a thermostable alkaline-tolerant enzyme that is produced by Bacillus stearothermophilus T-6. Xylanase T-6 was found to bleach pulp effectively at pH 9 and 65 °C and was used successfully on an industrial-scale mill trial. To facilitate the future characterization of the protein via X-ray analysis and protein engineering, it was necessary to overexpress the enzyme in Escherichia coli. The xylanase gene was cloned into T-7 polymerase expression vectors and its expression was optimized. The enzyme was found to constitute over 70% of the cell protein and it was efficiently purified from the host proteins by a single heating step. Over 2 g of soluble and active enzyme per 1 culture were achieved.