Thermal stability of beta-xylanases produced by different Thermomyces lanuginosus strains

The thermostability of beta-xylanases produced by nine thermophilic Thermomyces lanuginosus strains in a coarse corn cob medium was assessed. The xylanase produced by T. lanuginosus strain SSBP retained 100% of its activity after 6 h at temperatures up to 65 degrees C. In comparison seven ATCC strains and the DSM 5826 strain of T. lanuginosus only retained 100% xylanase activity at temperatures up to 60 degrees C. Culture filtrates of T. lanuginosus strain SSBP grown on coarse corn cobs, oatspelts xylan, birchwood xylan, wheatbran, locust beangum, and sugar cane bagasse, retained 100% xylanase activity at temperatures up to 60 degrees C. The xylanase produced on corn cobs was the most thermostable and showed an increase of approximately 6% from 70 degrees C to 80 degrees C. The T(1/2) of all strains at 70 degrees C at pH 6.5 varied greatly from 63 min for strain ATCC 28083 to 340 min for strain SSBP. The xylanase of strain SSBP was much less thermostable at pH 5.0 and pH 12.0 with T(1/2) values of 11.5 min and 15 min, respectively at 70 degrees C. At 50 degrees C, the enzyme of T. lanuginosus strain SSBP produced on coarse corn cobs was stable within the pH range of 5.5-10.0. Furthermore, the enzyme retained total activity at 60 degrees C for over 14 days and at 65 degrees C for over 48 h. The xylanase of T. lanuginosus strain SSBP possesses thermo- and pH stability properties that may be attractive to industrial application.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

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.     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株BacilluspumilusWL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为2 6 0kD ,pI值9 5 ,以燕麦木聚糖为底物时的表观Km 值为16 6mg mL ,Vmax值为12 6 3μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7 2至8 0之间,是耐碱性木聚糖酶;最适作用温度为4 5℃～5 5℃,在37℃、4 5℃以下时该酶热稳定性均较好;5 0℃保温时,该酶活力的半衰期大约为2h ,在超过5 0℃的环境下,该酶的热稳定较差,5 5℃和6 0℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL_11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL_11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL_11主要合成内切型木聚     

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.     

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.     

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.     

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.     

Purification and properties of a xylanase from Streptomyces lividans.

An extracellular xylanase produced by a cellulase-negative mutant strain of Streptomyces lividans 1326 was purified to homogeneity. The purified enzyme has an apparent Mr of 43,000 and pI of 5.2. The pH and temperature optima for the activity were 6.0 and 60 degrees C respectively, and the Km and Vmax. values, determined with a soluble oat spelts xylan, were 0.78 mg/ml and 0.85 mmol/min per mg of enzyme. The xylanase showed no activity towards CM-cellulose and p-nitrophenyl beta-D-xyloside. The enzyme degraded xylan, producing mainly xylobiose, a mixture of xylo-oligosaccharides and a small amount of xylose as end products. Its pattern of action on beta-1,4-D-xylan indicates that it is a beta-1,4-endoxylanase (EC 3.2.1.8).     

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.     

Recombinant xylanase from Streptomyces coelicolor Ac-738: characterization and the effect on xylan-containing products

A xylanase gene was isolated from the genomic DNA of Streptomyces coelicolor Ac-738. The 723-bp full-length gene encoded a 241-amino acid peptide consisting of a 49-residue putative TAT signal peptide and a glycoside hydrolase family-11 domain. The mature enzyme called XSC738 was expressed in Escherichia coli M15[pREP4]. The electrophoretically homogeneous protein with a specific activity of 167 U/mg for beechwood xylan was purified. The pH optimum of XSC738 was at pH 6; a high activity was retained within a pH range of 4.5–8.5. The enzyme was thermostable at 50–60 °C and retained an activity at pH 3.0–7.0. Xylanase XSC738 was activated by Mn²⁺, Co²⁺ and largely inhibited by Cd²⁺, SDS and EDTA. The products of xylan hydrolysis were mainly xylobiose, xylotriose, xylopentaose and xylohexose. Xylotetraose appeared as a minor product. Processing of such agricultural xylan-containing products as wheat, oats, soy flour and wheat bran by xylanase resulted in an increased content of sugars.     

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.     

Xylanase from the extremely thermophilic bacterium "Caldocellum saccharolyticum": overexpression of the gene in Escherichia coli and characterization of the gene product

A xylanase encoded by the xynA gene of the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible lambda pR and pL promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42 degrees C, and the xylanase made up to 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E. coli. The temperature and pH optima were determined to be 70 degrees C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h if incubated at 60 degrees C, with half-lives of 8 to 9 h at 70 degrees C and 2 to 3 min at 80 degrees C. The enzyme had high activity on xylan and ortho-nitrophenyl beta-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl beta-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.     

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.     

Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Bacillus licheniformis xylanase A (BlxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. After 96-h 0.25% methanol induction, the activity of recombinant B. licheniformis xylanase A (reBlxA) in culture supernatant was 122.9 U/mg. Enzymatic properties assays showed that the optimum temperature and pH for reBlxA were 60 degrees C and pH 6.0, respectively. When treated at 70 degrees C, pH 6.0 for 2 min, the residual activities of the reBlxA were 76%. Over 80% of reBlxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1h at 25 degrees C. High performance liquid chromatography (HPLC) analysis revealed that xylotriose (X3) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reBlxA. The mode of action studies showed that reBlxA was an endo-acting xylanase and xylobiose (X2), xylotriose, xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by it. This is the first report on the expression of reBlxA in yeast and on determining and quantifying the hydrolysis products released from xylans by reBlxA.     

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.     

Expression and characterization of a thermostable β-xylosidase from the hyperthermophile, Thermotoga maritima

A thermostable beta-xylosidase from a hyperthermophilic bacterium, Thermotoga maritima, was over-expressed in Escherichia coli using the T7 polymerase expression system. The expressed beta-xylosidase was purified in two steps, heat treatment and immobilized metal affinity chromatography, and gave a single band on SDS-PAGE. The maximum activity on p-nitrophenyl beta-D-xylopyranoside was at 90 degrees C and pH 6.1. The purified enzyme had a half-life of over 22-min at 95 degrees C, and retained over 57% of its activity after holding a pH ranging from 5.4 to 8.5 for 1 h at 80 degrees C. Among all tested substrates, the purified enzyme had specific activities of 275, 50 and 29 U mg(-1) on pNPX, pNPAF, and pNPG, respectively. The apparent Michaelis constant of the beta-xylosidase was 0.13 mM for p NPX with a V (max) of 280 U mg(-1). When the purified beta-xylosidase was added to xylanase, corncob xylan was hydrolized completely to xylose.     

木聚糖酶XYNB分子中折叠股B1和B2间的疏水作用对酶热稳定性的影响

对来源于Streptomycesolivaceoviridis的高比活木聚糖酶XYNB进行同源建模,并结合嗜热木聚糖酶氮末端芳香族氨基酸疏水作用的结构分析,设计了XYNB的T11Y定点突变,观察XYNB分子中折叠股B1和B2的疏水作用对酶的热稳定性的影响。将突变酶XYNB′在毕赤酵母中表达,表达的XYNB′经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明,XYNB′的耐热性比XYNB有明显的提高,但最适温度与原酶一样为60℃。另外,XYNB′的最适pH、Km值及比活性均有一定的改变。实验证实了木聚糖酶XYNB的氮端芳香族氨基酸之间的疏水相互作用与其热稳定性相关,为进一步的结构与功能研究提供了优良的基因材料。     

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).