Cloning, expression, and characterization of a xylanase 10 from Aspergillus terreus (BCC129) in Pichia pastoris

A full-length xylanase gene, encoding 326 amino acids belonging to the fungal glycosyl hydrolase family 10, from Aspergillus terreus BCC129 was cloned and sequenced. Sequence analysis suggested that the first 25 amino acids of this enzyme is the signal peptide. Therefore, only the mature xylanase gene of 906 bp was cloned into a yeast expression vector, pPICZalphaA, for heterologous expression in Pichia pastoris. A band of approximately, 33 kDa was observed on the SDS-PAGE gel after one day of methanol induction. The expressed enzyme was purified by gel filtration chromatography. The purified recombinant xylanase demonstrated optimal activity at 60 degrees C, pH 5.0 and a Km of 4.8 +/- 0.07 mg/ml and a Vmax of 757 +/- 14.54 micromol/min mg, using birchwood xylan as a substrate. Additionally, the purified enzyme demonstrated broad pH stability from 4 to 10 when incubated at 40 degrees C for 4 h. It also showed a moderate thermal stability since it retained 90% of its activity when incubated at 50 degrees C, 30 min, making this enzyme a potential use in the animal feed and paper and pulp industries.     

Production, purification, and characterization of xylanase from a hyperxylanolytic mutant of Aspergillus ochraceus

Enhancement of the productivity of xylanase and beta-xy-losidase of Aspergillus ochraceus was investigated by multistep mutagenesis. The spores of the wild strain were subjected to UV and N-methyl-N-nitro-N-nitro-soguanidine (NTG). The hyperxylanolytic mutant (NG-13), which showed good clearing on the surface of the xylan-agar plate, secretes xylanase and beta-xylosidase at high levels during growth on commercial xylan and on agricultural wastes. Both liquid and solid state cultures were employed in the study for enzyme production. The xylanase from NG-13 was purified to homogeneity by ammonium sulfate precipitation and gel filtration. This purified enzyme showed a pH optimum of 6.0 and was stable in the range of pH 5 to 10. Prolonged stability of the enzyme was observed at 45 degrees C though its activity was maximal at 50 degrees C. The molecular weight of the enzyme was estimated to be 4.3 x 10(4) by SDS-polyacrylamide gel electrophoresis and 5 x 10(4) by gel filtration on Sephadex G-75. The kinetic data showed that the K(m) and V(max) values for xylan were 1 x 10(-3)M and 19.6 mumol/ min/mg protein, respectively. The enzyme was both more active and thermostable in the presence of K(+)and was inactivated by thiol reagents such as Hg(2+), p-hydroxymercuribenzoate (PHMB), 3', 5'-dithiobis (2'-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM).     

Chemical modification of a xylanase from a thermotolerant Streptomyces. Evidence for essential tryptophan and cysteine residues at the active site.

Extracellular xylanase produced in submerged culture by a thermotolerant Streptomyces T7 growing at 37-50 degrees C was purified to homogeneity by chromatography on DEAE-cellulose and gel filtration on Sephadex G-50. The purified enzyme has an Mr of 20,463 and a pI of 7.8. The pH and temperature optima for the activity were 4.5-5.5 and 60 degrees C respectively. The enzyme retained 100% of its original activity on incubation at pH 5.0 for 6 days at 50 degrees C and for 11 days at 37 degrees C. The Km and Vmax. values, as determined with soluble larch-wood xylan, were 10 mg/ml and 7.6 x 10(3) mumol/min per mg of enzyme respectively. The xylanase was devoid of cellulase activity. It was completely inhibited by Hg2+ (2 x 10(-6) M). The enzyme degraded xylan, producing xylobiose, xylo-oligosaccharides and a small amount of xylose as end products, indicating that it is an endoxylanase. Chemical modification of xylanase with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and p-hydroxymercuribenzoate (PHMB) revealed that 1 mol each of tryptophan and cysteine per mol of enzyme were essential for the activity. Xylan completely protected the enzyme from inactivation by the above reagents, suggesting the presence of tryptophan and cysteine at the substrate-binding site. Inactivation of xylanase by PHMB could be restored by cysteine.     

Thermostable xylanase from Marasmius sp.: purification and characterization

We have screened 766 strains of fungi from the BIOTEC Culture Collection (BCC) for xylanases working in extreme pH and/or high temperature conditions, the so-called extreme xylanases. From a total number of 32 strains producing extreme xylanases, the strain BCC7928, identified by using the internal transcribed spacer (ITS) sequence of rRNA to be a Marasmius sp., was chosen for further characterization because of its high xylanolytic activity at temperature as high as 90 degrees C. The crude enzyme possessed high thermostability and pH stability. Purification of this xylanase was carried out using an anion exchanger followed by hydrophobic interaction chromatography, yielding the enzyme with >90% homogeneity. The molecular mass of the enzyme was approximately 40 kDa. The purified enzyme retained broad working pH range of 4-8 and optimal temperature of 90 degrees C. When using xylan from birchwood as substrate, it exhibits Km and Vmax values of 2.6 +/- 0.6 mg/ml and 428 +/- 26 U/mg, respectively. The enzyme rapidly hydrolysed xylans from birchwood, beechwood, and exhibited lower activity on xylan from wheatbran, or celluloses from carboxymethylcellulose and Avicel. The purified enzyme was highly stable at temperature ranges from 50 to 70 degrees C. It retained 84% of its maximal activity after incubation in standard buffer containing 1% xylan substrate at 70 degrees C for 3 h. This thermostable xylanase should therefore be useful for several industrial applications, such as agricultural, food and biofuel.     

Isolation, purification and characterization of xylanasefrom Staphylococcus sp. SG-13 and its application in biobleaching of kraft pulp

A haloalkalophilic Staphylococcus sp. SG-13 produced an alkalistable xylanase in wheat bran medium. A 12-fold purification was achieved by using standard purification techniques. The purified xylanase exhibited a dual pH optima of 7.5 and 9.2. The optimum temperature for enzyme activity was 50 degrees C. The enzyme was stable at 50 degrees C for more than 4 h. The xylanase exhibited Km and Vmax values of 4 mg ml-1, 90 micromol min-1 per mg for birchwood xylan and 7 mg ml-1, 55 micromol min-1 per mg for oatspelt xylan, respectively. The substrate binding affinity of xylanase was more for oatspelt xylan but birchwood xylan was hydrolysed more rapidly. The xylanase activity was stimulated by Fe2+, Ni2+, Cu2+ and dithiothreitol up to 60% and was strongly inhibited in the presence of Co2+, Hg2+, Pb2+, phenyl methane sulphonyl fluoride, ethylenediaminetetraacetic acid, and acetic anhydride up to 100%. The xylanase dose of 1.8 U g-1 moisture free pulp, exhibited bleach boosting of kraft pulps optimally at pH 9.5-10.0 and 50 degrees C after 4 h of reaction time. Pretreatment of pulp with xylanase and its subsequent treatment with 8% hypochlorite, reduced the kappa number by 30%, enhanced the brightness and viscosity by 11% and 1.8%, respectively, and improved the paper properties such as tensile strength and burst factor up to 10% and 17%, respectively.     

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 degrees C and 6.0. Xylanase was stable up to 70 degrees C for 4 h at pH 6.0 in the presence of xylane. Xylanase was completely inhibited by the Hg(2+) ions. beta-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 degrees C and pH 6.0 gave V(max) and K(m)values of 2420 nkat/mg and 0.7 mg/ml.     

Purification and characterization of a new xylanase from Acrophialophora nainiana

A new xylanase activity (XynII) was isolated from liquid state cultures of Acrophialophora nainiana containing birchwood xylan as carbon source. XynII was purified to apparent homogeneity by gel filtration and ion exchange chromatographies. The enzyme was optimally active at 55 degrees C and pH 7.0. XynII had molecular mass of 22630+/-3.0 and 22165 Da, as determined by mass spectrometry and SDS-PAGE, respectively. The purified enzyme was able to act only on xylan as substrate. The apparent K(m) values on soluble and insoluble birchwood xylans were 40.9 and 16.1 mg ml(-1), respectively. The enzyme showed good thermal stability with half lives of 44 h at 55 degrees C and ca. 1 h at 60 degrees C The N-terminal sequence of XynII showed homology with a xylanase grouped in family G/11. The enzyme did not show amino acid composition similarity with xylanases from some fungi and Bacillus amyloliquefaciens.     

Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose. The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.     

Purification and characterization of a family G/11 beta-xylanase from Streptomyces olivaceoviridis E-86

A beta-xylanase (GXYN) was purified from the culture filtrate of Streptomyces olivaceoviridis E-86 by successive chromatography on DE-52, CM-Sepharose and Superose 12. The molecular mass of the xylanase was estimated to be 23 kDa, indicating that the enzyme consists of a catalytic domain only. The enzyme displayed an optimum pH of 6, a temperature optimum of 60 degrees C, a pH stability range from 2 to 11 and thermal stability up to 40 degrees C. The N-terminal amino acid sequence of GXYN was A-T-V-I-T-T-N-Q-T-G-T-N-N-G-I-Y-Y-S-F-W-, and sharing a high degree of similarity with the N-terminal sequence of xylanases B and C from Streptomyces lividans, indicating GXYN belongs to family G/11 of glycoside hydrolases. GXYN was inferior to xylanase B from Streptomyces lividans in the hydrolysis of insoluble xylan because of its lack of a xylan binding domain.     

Purification and characterization of xylanases from<Emphasis Type="Italic">Aspergillus giganteus</Emphasis>

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 degrees C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50 degrees C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.     

Purification and characterization of an endoxylanase from the culture broth of Bacillus cereus BSA1

An extracellular xylanase from the fermented broth of Bacillus cereus BSA1 was purified and characterized. The enzyme was purified to 3.43 fold through ammonium sulphate precipitation, DEAE-cellulose chromatography and followed by gel filtration through Sephadex G-100 column. The molecular mass of the purified xylanse was about 33 kDa. The enzyme was an endoxylanase as it initially degraded xylan to xylooligomers. The purified enzyme showed optimum activity at 55 degrees C and at pH 7.0 and remained reasonably stable in a wide range ofpH (5.0-8.0) and temperature (40-65 degrees C). The Km and Vmax values were found to be 8.2 mg/ml and 181.8 micromol/(min mg), respectively. The enzyme had no apparent requirement ofcofactors, and its activity was strongly inhibited by Cu++, Hg++. It was also a salt tolerant enzyme and stable upto 2.5 M of NaCl and retained its 85% activity at 3.0 M. For stability and substrate binding, the enzyme needed hydrophobic interaction that revealed when most surfactants inhihited xylanase activity. Since the enzyme was active over wide range ofpH, temperature and remained active in higher salt concentration, it could find potential uses in biobleaching process in paper industries.     

Production, Purification, and Characterization of beta-(1-4)-Endoxylanase of Streptomyces roseiscleroticus

Twelve species of Streptomyces that formerly belonged to the genus Chainia were screened for the production of xylanase and cellulase. One species, Streptomyces roseiscleroticus (Chainia rosea) NRRL B-11019, produced up to 16.2 IU of xylanase per ml in 48 h. A xylanase from S. roseiscleroticus was purified and characterized. The enzyme was a debranching beta-(1-4)-endoxylanase showing high activity on xylan but essentially no activity against acid-swollen (Walseth) cellulose. It had a very low apparent molecular weight of 5,500 by native gel filtration, but its denatured molecular weight was 22,600 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had an isoelectric point of 9.5. The pH and temperature optima for hydrolysis of arabinoxylan were 6.5 to 7.0 and 60 degrees C, respectively, and more than 75% of the optimum enzyme activity was retained at pH 8.0. The xylanase had a K(m) of 7.9 mg/ml and an apparent V(max) of 305 mumol . min . mg of protein. The hydrolysis rate was linear for xylan concentrations of less than 4 mg/ml, but significant inhibition was observed at xylan concentrations of more than 10 mg/ml. The predominant products of arabinoxylan hydrolysis included arabinose, xylobiose, and xylotriose.     

Characterization of a cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp

A xylanase purified from the thermophilic fungus Thermomyces lanuginosus CBS 288.54 was characterized and its potential application in wheat straw pulp biobleaching was evaluated. Xylanase was purified 33.6-fold to homogeneity with a recovery yield of 21.5%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 26.2 kDa. The purified xylanase had a neutral optimum pH ranging from pH 7.0 to pH 7.5, and it was also stable over pH 6.5-10.0. The optimal temperature of the xylanase was 70-75 degrees C and it was stable up to 65 degrees C. The purified xylanase was found to be not glycosylated. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent Km values of the xylanase for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 4.0, 4.7, 2.0 and 23.4 mg ml-1, respectively. The potential application of the xylanase was further evaluated in biobleaching of wheat straw pulp. The brightness of bleached pulps from the xylanase pretreated wheat straw pulp was 1.8-7.79% ISO higher than that of the control, and showed slightly lower tensile index and breaking length than the control. Although chlorine consumption was reduced by 28.3% during bleaching, the xylanase pretreated pulp (15 U g-1 pulp) still maintained its brightness at the control level. Besides, pretreatment of pulp with the xylanase was also effective at an alkaline pH as high as pH 10.0.     

Synchrotron radiation small angle scattering studies of thermal stability of xylanase XYNII from Trichoderma longibrachiatum

Xylanase XYNII from Trichoderma longibrachiatum is a small protein of the molecular weight 21 kDa, belonging to the family 11 of glycosyl hydrolases, which catalyses hydrolysis of xylan. This article reports thermal stability study of xylanase XYN II conformation in the temperature range 15-65 degrees C by the small angle synchrotron radiation scattering. The study has been performed at different pH conditions: at pH 4.0 (below the physiological optimum of the enzyme activity) at pH 5.8 close to the optimum for enzymatic activity and at pH 8.0. The radius of gyration and the pair distance distribution function p(r) have been analyzed to characterize the changes of the enzyme conformation on heating. In the environment of the pH close to that of the optimum for the enzymatic activity, xylanase shows the greatest thermal stability and undergoes denaturation only above 55 degrees C. In the acidic and basic environments, the enzyme stability is much lower and denaturation begins at 45 degrees C. On the basis of the SAXS data, the shape of the xylanase molecule in solution in different temperatures has been reconstructed using ab initio method and program DAMMIN. The shape of the xylanase molecule at room temperature is similar to the right hand, which is typically observed for xylanase crystal structure. In higher temperatures (close to the enzyme activity optimum), the conformation of the right hand is loosened and half opened.     

Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32

Streptomyces cyaneus SN32 was used in this study to produce extracellular xylanase, an important industrial enzyme used in pulp and paper industry. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by anion exchange chromatography using DEAE-Sepharose column, with 43.0% yield. The enzyme was found to be a monomer of 20.5 kDa as determined by SDS gel electrophoresis and has a pI of 8.5. The optimum pH and temperature for purified xylanase activity was 6.0 and 60-65 degrees C, respectively. The half-lives of xylanase at 50 and 65 degrees C were approximately 200 and 50 min, respectively. The xylanase exhibited K(m) and V(max) values of 11.1 mg/ml and 45.45 micromol/min/mg. The 15 residue N-terminal sequence of the enzyme was found to be 87% identical up to that of endoxylanases from Steptomyces sp. Based on the zymogram analysis, sequence similarity and other characteristics, it is proposed that the purified enzyme from S. cyaneus SN32 is an endoxylanase and belongs to Group 1 xylanases (low molecular weight - basic proteins). The purified enzyme was stable for more than 20 week at 4 degrees C. Easy purification from the fermentation broth and its high stability will be highly useful for industrial application of this endoxylanase.     

Orthophosphate anion enhances the stability and activity of endoxylanase from Bacillus sp

Endoxylanase, for which the optimum temperature is 60 degrees C (optimum pH 7), is labile to heat. Because the isoelectric point (pI) value of this xylanase is 10.6, the net charge of this enzyme is positive at pH 7. Thus, ions are likely to influence its enzyme structure and the thermal stability of endoxylanase may improve. Among the various ions tested, orthophosphate anion (HPO(4)(2-)) was found to significantly improve not only the stability but the activity of xylanase. When K(2)HPO(4) concentration was increased from 50 mM to 1.2 M, the T(m )value of xylanase was increased from 60.0 degrees C to 74.5 degrees C. The affinity of xylanase on xylan also increased along with K(2)HPO(4) concentration. Thus, the xylanase activity at 0.6 M K(2)HPO(4) was 2.3-fold higher than that at 50 mM K(2)HPO(4), and 120.2-fold higher than that in 40 mM MOPS buffer. This enhanced activity in the presence of K(2)HPO(4 )probably takes place because the orthophosphate anion affects the binding and catalytic residues of endoxylanase.     

A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization

AIMS: The enzymatic hydrolysis of xylan has potential economic and environment-friendly applications. Therefore, attention is focused here on the discovery of new extremophilic xylanase in order to meet the requirements of industry. METHODS AND RESULTS: An extracellular xylanase was purified from the culture filtrate of P. citrinum grown on wheat bran bed in solid substrate fermentation. Single step purification was achieved using hydrophobic interaction chromatography. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular weight of c. 25 kDa and pI of 3.6. Stimulation of the activity by beta mercaptoethanol, dithiotheritol (DTT) and cysteine was observed. Moderately thermostable xylanase showed optimum activity at 50 degrees C at pH 8.5. CONCLUSION: Xylanase purified from P. citrinum was alkaliphilic and moderately thermostable in nature. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work reports for the first time the purification and characterization of a novel endoglucanase free alkaliphilic xylanase from the alkali tolerant fungus Penicillium citrinum. The alkaliphilicity and moderate thermostability of this xylanase may have potential implications in paper and pulp industries.     

Purification and characterization of a thermostable xylanase from Fomitopsis pinicola

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular weight of F. pinicola xylanase was determined to be 58 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of 70 degreesC. The enzyme showed t(1/2) value of 33 h at 70 degrees C and catalytic efficiency (k(cat) = 77.4 s?1, k(cat)/K(m) = 22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.     

Production, characterization, and partial amino acid sequence of xylanase A from Schizophyllum commune.

Xylanase A, one of several extracellular xylanases produced by Schizophyllum commune strain Delmar when grown in submerged culture with spruce sawdust as carbon source, was purified 43-fold in 25% yield with respect to total xylanase activity. Although some polysaccharide was strongly bound to the purified enzyme, the complex could be dissociated by sodium dodecyl sulfate and appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the protein, calculated from the electrophoretic mobility, was 33,000. The molecular activity of the purified xylanase A, determined with soluble larch xylan as substrate, was 1.4 X 10(5) min-1, with xylobiose and xylose as the major products. The enzyme had a pH optimum of 5.0 and a temperature optimum of 55 degrees C in 10-min assays. The acid hydrolysate of xylanase A was rich in aspartic acid and aromatic amino acids. The sequence of 27 residues at the amino terminus showed no homology with known sequences of other proteins.