Cloning, expression, and characterization of a new xylanase with broad temperature adaptability from Streptomyces sp. S9

A new xylanase gene, xynAS9, was cloned from Streptomyces sp. S9, which was isolated from Turpan Basin, China. The full-length gene consists of 1,395 bp and encodes 465 amino acids including 38 residues of a putative signal peptide. The overall amino acid sequence shares the highest identity (50.8%) with a putative endo-1,4-beta-xylanase from Streptomyces avermitilis of the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to electrophoretic homogeneity and subsequently characterized. The optimal pH and temperature for the recombinant enzyme were 6.5 and 60 degrees C, respectively. The enzyme showed broad temperature adaptability, retaining more than 65% of the maximum activity when assayed at 50-80 degrees C. The enzyme also had good thermal and pH stability. The K (m) values for oat spelt xylan and birchwood xylan substrates were 2.85 and 2.43 mg ml(-1), with the V (max) values of 772.20 and 490.87 mumol min(-1) mg(-1), respectively. The hydrolysis products of xylan were mainly xylose and xylobiose. These favorable properties should make XynAS9 a good candidate in various industrial applications.     

Gene cloning, sequencing, and biochemical characterization of endoxylanase from Thermoanaerobacterium saccharolyticum B6A-RI.

The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli. A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA. An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa. xynA from T. saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases. The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively. The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan. The cloned xylanase was an endo-acting enzyme.     

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 new xylanase (APX-II) from the fungus Aureobasidium pullulans Y-2311-1.

Aureobasidium pullulans Y-2311-1 produced four major xylanases (EC 3.2.1.8) with pI values of 4.0, 7.3, 7.9, and 9.4 as revealed by isoelectric focusing and zymogram analysis when grown for 4 days on 1.0% oat spelt xylan. The enzyme with a pI of 9.4 was purified by ammonium sulfate precipitation, chromatography on a DEAE-Sephadex A-50 column, and gel filtration with a Sephadex G-75 column. The enzyme had a mass of about 25 kDa as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The purified enzyme had a Km of 7.6 mg . ml(-1) and Vmax of 2,650 micromol . min(-1) . mg(-1) for birchwood xylan at 28 degrees C and pH 4.5. It lacked activity towards carboxymethylcellulose, cellobiose, starch, mannan, p-nitrophenyl (pNP)-beta-D-xylopyranoside, pNP-beta-D-glucopyranoside, pNP-alpha-D-glucopyranoside, pNP-beta-D-cellobioside, pNP-beta-D-fucopyranoside, or pNP-alpha-D-galactopyranoside. The predominant end products of birchwood xylan or xylohexaose hydrolysis were xylobiose and xylose. The enzyme had the highest activity of pH 4.8 and 54 degrees C. Sixty percent of the activity remained after the enzyme had been incubated at 55 degrees C and pH 4.5 for 30 min. The sequence of the first 68 amino acid residues at the amino terminus showed homology to those of several other xylonases. Immunoblot analysis with antiserum raised against the purified xylanase revealed that two immunologically related polypeptides of 25 and 22 kDa were produced in A. pullulans cultures containing oat spelt xylan or xylose as carbon sources but not in cultures containing glycerol or glucose.     

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.     

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 Aeromonas caviae ME-1 Xylanase V, Which Produces Exclusively Xylobiose from Xylan

A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the xylanase were 46 kDa and 5.4, respectively. Xylanase V had a maximum activity at a pH of 6.8 and at a temperature between 30 and 37 degrees C. It was relatively stable at a pH between 5.0 and 8.6 and a temperature between 25 and 37 degrees C. When soluble birch xylan was used as the substrate, the enzyme had a K(m) and V(max) of 2 mg/ml and 182 mumol of xylose equivalent liberated . min . mg of protein, respectively. By the action of xylanase V on xylans (from oat spelt and birch), only one product corresponding to xylobiose was observed by thin-layer chromatography. The xylanase V putative product was confirmed to be xylobiose by acid and enzymatic hydrolyses. The xylanase had neither beta-xylosidase, alpha-l-arabinofuranosidase, cellulase, nor beta-1,3-xylanase activities. Xylotriose was the shortest substrate which the enzyme could attack. These findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism.     

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).     

An alkali-tolerant xylanase produced by the newly isolated alkaliphilic <Emphasis Type="Italic">Bacillus pumilus</Emphasis> from paper mill effluent

An alkaline active xylanase, XynBYG, was purified from an alkaliphilic Bacillus pumilus BYG, which was newly isolated from paper mill effluent. It had an optimum pH of 8.0–9.0, and showed good stability after incubated at pH 9.0 for 120 min. The optimum temperature for the activity was 50°C, and the enzyme retained below 55% of its original activity for 30 min at 55°C. The gene coding for XynBYG consists of 687 bp and encodes 229 amino acids. Similarity analysis indicated that XynBYG belong to family 11 glycosyl hydrolases. Site-directed mutagenesis was performed to replace five sites (Tyr/Ser) to Arg/Glu and the results demonstrated that the optimum temperature of the mutant Y7 (S39R-T146E) increased 5°C and the half-life of inactivation (T1/2) at 60 and 65°C was 1 h and 25 min, respectively. Thus, it provides a potential xylanase that can meet the harsh conditions in the industrial applications.     

Cloning and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme

A novel exo-alpha-1,5-L-arabinanase gene (arn3) was isolated, cloned, and expressed in E. coli. The recombinant enzyme (ARN3) had a pH optimum of 6.0-7.0 and a pH 3.0-7.0 stability range. The temperature optimum was 50 degrees C with a stability less than or equal to 45 degrees C. The recombinant ARN3 cleaved carboxymethyl (CM)-arabinan, debranched arabinan, and linear arabinan at a decreasing rate and is inactive on sugar beet arabinan, wheat arabinoxylan, and p-nitrophenyl-alpha-L-arabinofuranoside. The enzyme hydrolyzed debranched arabinan and synthetic arabino-oligosaccharides entirely to arabinose. The apparent K(m) and V(max) values were determined to be 6.2+/-0.3 mg/ml and 0.86+/-0.01 mg ml(-1) min(-1), respectively (pH 7.0, 37 degrees C, CM-arabinan). Multiple sequence alignment and homology modeling revealed unique short sequences of amino acids extending the loop involved in partial blocking of one end of the substrate-binding site on the surface of the molecule.     

Xylanase from a newly isolated Fusarium verticillioides capable of utilizing corn fiber xylan

A fungus, Fusarium verticillioides (NRRL 26518), was isolated by screening soil samples using corn fiber xylan as carbon source. The extracellular xylanase from this fungal strain was purified to apparent homogeneity from the culture supernatant by ultrafiltration using a 30,000 cut-off membrane, octyl-Sepharose chromatography and Bio gel A-0.5 m gel filtration. The purified xylanase (specific activity 492 U/mg protein; MW 24,000; pI 8.6) displayed an optimum temperature at 50 degrees C and optimum pH at 5.5, a pH stability range from 4.0 to 9.5 and thermal stability up to 50 degrees C. It hydrolyzed a variety of xylan substrates mainly to xylobiose and higher short-chain xylooligosaccharides. No xylose was formed. The enzyme did not require metal ions for activity and stability.     

Molecular cloning, overexpression, and purification of a major xylanase from Aspergillus oryzae

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58 degrees C. It had a Km of 5.1 mg/ml and a Vmax of 123 micromol/min/mg when birch wood xylan was used as a substrate.     

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.     

Characterization of a thermostable D-stereospecific alanine amidase from Brevibacillus borstelensis BCS-1

A gene encoding a new thermostable D-stereospecific alanine amidase from the thermophile Brevibacillus borstelensis BCS-1 was cloned and sequenced. The molecular mass of the purified enzyme was estimated to be 199 kDa after gel filtration chromatography and about 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme could be composed of a hexamer with identical subunits. The purified enzyme exhibited strong amidase activity towards D-amino acid-containing aromatic, aliphatic, and branched amino acid amides yet exhibited no enzyme activity towards L-amino acid amides, D-amino acid-containing peptides, and NH(2)-terminally protected amino acid amides. The optimum temperature and pH for the enzyme activity were 85 degrees C and 9.0, respectively. The enzyme remained stable within a broad pH range from 7.0 to 10.0. The enzyme was inhibited by dithiothreitol, 2-mercaptoethanol, and EDTA yet was strongly activated by Co(2+) and Mn(2+). The k(cat)/K(m) for D-alaninamide was measured as 544.4 +/- 5.5 mM(-1) min(-1) at 50 degrees C with 1 mM Co(2+).     

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.     

Cloning of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> Xylanase Gene and Characterization of Recombinant Enzyme

Hemicellulose is a major component of lignocellulose biomass. Complete degradation of this substrate requires several different enzymatic activities, including xylanase. We isolated a strain of Bacillus licheniformis from a hot springs environment that exhibited xylanase activity. A gene encoding a 23-kDa xylanase enzyme, Xyn11, was cloned, and the recombinant protein was expressed in an Escherichia coli host and biochemically characterized. The optimum activity of the enzyme was at pH 5-7 and 40-50 degrees C. The enzyme was stable at temperatures up to 50 degrees C. Against birchwood xylan, the enzyme had an apparent K ( m ) of 6.7 mg/mL and V (max) of 379 mumol/min/mg.     

Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation

The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa. A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min. The amino acid composition of the purified enzyme was also determined.     

Purification and characterization of a thermostable xylanase from Saccharopolyspora pathumthaniensis S582 isolated from the gut of a termite

An extracellular thermostable xylanase produced by Saccharopolyspora pathumthaniensis S582 was purified 167-fold to homogeneity with a recovery yield of 12%. The purified xylanase appeared as a single protein band on SDS-PAGE, with a molecular mass of 36 kDa. The optimal temperature and pH of the xylanase were 70 °C and 6.5. The enzyme was stable within a pH range of 5.5-10.0. It retained its activity after incubation at 50 °C for 2 h. Its half lives at temperatures of 60 and 70 °C were 180 and 120 min respectively. Hydrolysis of beechwood xylan by the xylanase yielded xylobiose and xylose as major products. The enzyme acted specifically on xylan as an endo-type xylanase, and exhibited a K(m) value of 3.92 mg/mL and a V(max) value of 256 μmol/min/mg. Enzyme activity was completely inhibited by Hg(2+), and was stimulated by Rb(+) and Cs(+). The xylanase gene was cloned from genomic DNA of Saccharopolyspora pathumthaniensis S582 and sequenced. The ORF consisted of 1,107 bp and encoded 368 amino acid residues containing a putative signal peptide of 23 residues. This xylanase is a new member of family (GH) 10 that shows highest identity, of 63.4%, with a putative xylanase from Nocardiopsis dassonvillei subsp. dassonvillei.