Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli

A genomic library consisting of 4- to 7-kb EcoRI DNA fragments from Fibrobacter succinogenes 135 was constructed using a phage vector, lambda gtWES lambda B, and Escherichia coli ED8654 as the host bacterium. Two positive plaques, designated lambda FSX101 and lambda FSX102, were identified. The inserts were 10.5 and 9.8 kb, respectively. A 2.3-kb EcoRI fragment that was subcloned from lambda FSX101 into pBR322 also showed xylanase activity. Southern blot analysis showed that the cloned EcoRI fragment containing the xylanase gene had originated from F. succinogenes 135. The cloned endo-(1,4)-beta-D-xylanase gene (pFSX02) was expressed constitutively in E. coli HB101 when grown on LB and on M9 medium containing either glucose or glycerol as the carbon source. Most of the beta-D-xylanase activity was located in the periplasmic space. Zymogram activity stains of nondenaturing polyacrylamide gels and isoelectric focusing gels showed that several xylanase isoenzymes were present in the periplasmic fraction of the E. coli clone FSX02 and they probably were due to posttranslational modification of a single gene product. Comparison of the FSX02 xylanase and the xylanase from the extracellular culture fluids of F. succinogenes 135 and S85 for their ability to degrade oat spelt xylan showed that, for equal units of beta-D-xylanase activity, hydrolysis by the cloned gene product was more complete. However, unlike the unfractionated mixture of xylanases from F. succinogenes 135 and S85, the enzyme from E. coli FSX02 was unable to release arabinose from oat spelt xylan.     

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 neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

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     

Molecular and biochemical characterization of a new alkaline active multidomain xylanase from alkaline wastewater sludge

A xylanase gene, xyn-b39, coding for a multidomain glycoside hydrolase (GH) family 10 protein was cloned from the genomic DNA of the alkaline wastewater sludge of a paper mill. Its deduced amino acid sequence of 1,481 residues included two carbohydrate-binding modules (CBM) of family CBM_4_9, one catalytic domain of GH 10, one family 9 CBM and three S-layer homology (SLH) domains. xyn-b39 was expressed heterologously in Escherichia coli, and the recombinant enzyme was purified and characterized. Xyn-b39 exhibited maximum activity at pH 7.0 and 60 °C, and remained highly active under alkaline conditions (more than 80 % activity at pH 9.0 and 40 % activity at pH 10.0). The enzyme was thermostable at 55 °C, retaining more than 90 % of the initial activity after 2 h pre-incubation. Xyn-b39 had wide substrate specificity and hydrolyzed soluble substrates (birchwood xylan, beechwood xylan, oat spelt xylan, wheat arabinoxylan) and insoluble substrates (oat spelt xylan and wheat arabinoxylan). Hydrolysis product analysis indicated that Xyn-b39 was an endo-type xylanase. The K _m and V _max values of Xyn-b39 for birchwood xylan were 1.01 mg/mL and 73.53 U/min/mg, respectively. At the charge of 10 U/g reed pulp for 1 h, Xyn-b39 significantly reduced the Kappa number (P < 0.05) with low consumption of chlorine dioxide alone.     

Characterization of function and activity of domains A, B and C of xylanase C from Fibrobacter succinogenes S85

Xylanase C from the ruminant bacterium Fibrobacter succinogenes is comprised of two catalytic domains, A and B, and a third domain, C, of unknown function. The DNA coding for domains A and B of xylanase C were separately cloned and expressed in Escherichia coli as fusion proteins with glutathione-S:-transferase. The fusion proteins were isolated by affinity chromatography on glutathione-Sepharose 4B, cleaved with thrombin and the released xylanase C catalytic domains A and B were purified to apparent homogeneity by anion-exchange chromatography on Mono Q. Electrospray mass spectrometry provided a molecular mass of 27 818 Da (expected, 27 820 Da) for domain B. The pH and temperature optima for activity of domain B on oat spelt xylan were 5.0 and 52 degrees C, respectively. A kinetic analysis of the activity of the catalytic domain A on oat spelt xylan, birch wood xylan and xylooligomers at pH 6.5 and 37 degrees C provided data significantly different to those obtained previously with a protease-derived form of the enzyme [Zhu et al. (1994) J. Bacteriol. 176, 3885-3894]. The isolated domain A was more active on barley-glucan than the protease-derived form and its affinity for birch wood xylan was enhanced resulting in greater overall catalytic efficiency as reflected by k(cat)/K:(M) values. Likewise, significant differences in the Michaelis-Menten parameters K:(M), k(cat) and k(cat)/K:(M) were obtained with domain B compared with values previously reported with this domain attached to domain C. In general, the presence of domain C appeared to decrease the overall efficiency of domain B 7- and 36-fold with birch wood xylan and xylopentaose as substrates, respectively, as reflected by values of k(cat)/K:(M). The removal of domain C also affected the mode of action of domain B such that it more closely resembled that of catalytic domain A. However, no change in either pH and temperature optima or stability were found with domain B compared with the combined domains B and C. The function of domain C remains unknown, but hydrophobic cluster analysis indicated that it may belong to a class of dockerin domains involved in the protein-protein interactions of cellulolytic and xylanolytic complexes.     

Expression of two xylanase genes from the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 cloned in pUC13

Two distinct xylanase genes (designated xynA and xynB) were subcloned in pUC13 from non-homologous restriction fragments of Ruminococcus flavefaciens 17 DNA originally isolated in lambda EMBL3. The products of the two genes showed similar pH optima for hydrolysis of oat spelt xylan (around 5.5) and had little or no activity against carboxymethylcellulose. Trace activities against p-nitrophenyl (pNP) cellobioside and pNP-xyloside were detected in clones containing xynA, but not in one harbouring xynB. The xylanase associated with clones carrying xynA produced mainly xylobiose and xylose from xylan and did not give hydrolysis of xylobiose, while that encoded by xynB produced mainly xylobiose and higher xylo-oligosaccharides from xylan. There was evidence of increased expression, at the RNA level, of these two genes, and of another cloned region encoding multiple activities including xylanase, in R. flavefaciens 17 grown with xylan, as compared with cellobiose, as energy source. Total cell-associated xylanase and beta-xylosidase activities, and supernatant xylanase activity, were shown to be similarly induced in xylan-grown R. flavefaciens, 17.     

Expression of Bacillus circulans Teri-42 xylanase gene in Bacillus subtilis

The xylanase gene of Bacillus circulans Teri-42 was cloned in both B. subtilis and Escherichia coli. The enzyme activity was almost 87% higher in B. subtilis (pBA7) than in E. coli (pAQ4). No cellulase activity was detected in the clones, B. subtilis (pBA7) and E. coli (pAQ4). Approximately 1120 U (80%) of the xylanase was secreted extracellularly by the clone B. subtilis (pBA7) as compared to 79 U (88%) excreted in E. coli (pAQ4). In B. subtilis (pBA7) the optimal xylanase activity was at pH 7.0 and 50 degrees C, which was the same as that of the parent B. circulans Teri-42. The recombinant xylanase in B. subtilis was more stable at higher temperatures than the parent B. circulans Teri-42. Purification of xylanase from the clone B. subtilis (pBA7) showed a 71 kDa polypeptide similar to that observed in B. circulans Teri-42.     

Xylanase IV, an Exoxylanase of Aeromonas caviae ME-1 Which Produces Xylotetraose as the Only Low-Molecular-Weight Oligosaccharide from Xylan

A novel xylanase (xylanase IV) which produces xylotetraose as the only low-molecular-weight oligosaccharide from oat spelt xylan was isolated from the culture medium of Aeromonas caviae ME-1. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the xylanase IV molecular weight was 41,000. Xylanase IV catalyzed the hydrolysis of oat spelt xylan, producing exclusively xylotetraose. The acid hydrolysate of the product gave d-xylose. The enzyme did not hydrolyze either p-nitrophenyl-(beta)-d-xyloside, small oligosaccharides (xylobiose and xylotetraose), or polysaccharides, such as starch, cellulose, carboxymethyl cellulose, laminarin, and (beta)-1,3-xylan.     

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.     

Fibrobacter succinogenes S85 has multiple xylanase genes

Four distinct DNA fragments encoding xylanase activities, pBX1.2, pXC30.2, pX14 and LX31, were cloned from plasmid and γ libraries constructed using genomic DNA from Fibrobacter succinogenes S85. pBX1.2 contained an insert which was homologous, and mapped similarly to that previously cloned in pBX1 while the three remaining clones pX14, pXC30 in plasmids, and LX31 in lambda, represented new xylanase activities. The X14 xylanase was a 73 kDa exo-type xylanase, which was exported to the periplasm of the Escherichia coli host, and produced large quantities of xylose and xylobiose from oat spelt xylan. The XC30 xylanase, also exported in E. coli, was a 77 kDa protein which exhibited both xylanase and endoglucanase activities, and a low cellobiosidase activity. The LX31 enzyme was a 58 kDa endoxylanase that produced a mixture of xylooligosaccharides. Zymograms of isoelectric focusing gels showed that the X14 xylanase had a neutral pI, XC30 contained acidic, neutral and basic enzymic components, while BX1 and LX31 were acidic. These results indicate that, in addition to the many other elements of its polysaccharide-degrading repertoire, F. succinogenes S85 possesses at least four distinct xylanases.     

Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product.

A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.     

Cloning and expression of an alpha-L-arabinofuranosidase gene (stxIV) from Streptomyces thermoviolaceus OPC-520, and characterization of the enzyme

The gene encoding alpha-L-arabinofuranosidase (STX-IV), located upstream of the previously reported stxI gene, was cloned and sequenced. The gene is divergently transcribed from the stxI gene, and the two genes are separated by 661 nucleotides. The stxIV gene consists of a 1,092-bp open reading frame encoding 363 amino acids. The deduced amino acid sequence of the gene showed that STX-IV was an enzyme consisting of only a catalytic domain, and that the enzyme had significant similarity with alpha-L-arabinofuranosidases belonging to family 62 of glycosyl hydrolases. The stxIV gene was expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. Arabinoxylan and oat spelt xylan were good substrates for STX-IV, however, the enzyme showed a low activity with p-nitrophenyl alpha-L-arabinofuranoside. The optimum pH and temperature were 5.0 and 60 degrees C, respectively.     

Hyperexpression of a Bacillus circulans xylanase gene in Escherichia coli and characterization of the gene product

A 4.0-kilobase (kb) fragment of Bacillus circulans genomic DNA inserted into pUC19 and encoding endoxylanase activity was subjected to a series of subclonings. A 1.0-kb HindIII-HincII subfragment was found to code for xylanase activity. Maximum expression levels were observed with a subclone that contained an additional 0.3-kb sequence upstream from the coding region. Enhancer sequences in the upstream region are thought to be responsible for these high expression levels. Southern hybridization analyses revealed that the cloned gene hybridized with genomic DNA from Bacillus subtilis and Bacillus polymyxa. Xylanase activity expressed by Escherichia coli harboring the cloned gene was located primarily in the intracellular fraction. Levels of up to 7 U/ml or 35 mg/liter were obtained. The protein product was purified by ion exchange and gel permeation chromatography. The xylanase had a molecular weight of 20,500 and an isoelectric point of 9.0.     

Xylanase encoded by genetically engineered Clostridium thermocellum gene in Bacillus subtilis

Summary Xylanase was produced with Bacillus subtilis(pJX18), constructed previously, which contains Clostridium thermocellum xylanase gene expressing with a strong Bacillus promoter. The enzyme hydrolyzed oat spelt xylan to mostly xylobiose and xylotriose which are preferred for industrial applications. The optimal temperature and pH for the activity of this enzyme were 60°C and 5.4, respectively, with moderate stability under these conditions.     

Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.     

The construction and characterization of two xylan-degrading chimeric enzymes

Degradation of xylan requires several enzymes. Two chimeric enzymes, xyln-ara and xyln-xylo, were constructed by linking the catalytic portion of a xylanase (xyln) to either an arabinofuranosidase (ara) or a xylosidase (xylo) with a flexible peptide linker. The recombinant parental enzymes and chimeras were produced in E. coli at high levels and purified for characterization of their enzymatic and kinetic properties as well as activities on natural substrates. The chimeras closely resemble the parental enzymes or their mixtures with regard to protein properties. They share similar temperature profiles and have similar catalytic efficiencies as the parental enzymes when assayed using substrates 4-nitrophenyl-alpha-L-arabinofuranoside or 2-nitrophenyl- beta-D-xylopyranoside. The chimeras also show unique enzymatic characteristics. In xylanase activity assays using Remazol Brilliant Blue-xylan, while the parental xylanase has a pH optimum of pH 8, the chimeras showed shifted pH optima as a consequence of significantly increased activity at pH 6 (the optimal pH for ara and xylo). Both chimeras exhibited additive effects of the parental enzymes when assayed at wide ranges of pH and temperatures. The xyln-xylo chimera had the same activities as the xyln/xylo mixture in hydrolyzing the natural substrates oat spelt xylan and wheat arabinoxylan. Compared to the xyln/ara mixture, the xyln-ara chimera released the same amounts of xylose from oat spelt xylan and approximately 30% more from wheat arabinoxylan at pH 6. Our results demonstrate the feasibility and advantages of generating bifunctional enzymes for the improvement of xylan bioconversion.     

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.     

Purification and characterization of alpha-L-arabinofuranosidase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produced an alpha-L-arabinofuranosidase when grown in the presence of L-arabinose, sugar beet arabinan, or oat spelt xylan. At the end of a fermentation, about 40% of the activity was extracellular, and enzyme activity in the cell-free supernatant could reach 25 U/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 20000, and the enzyme was purified eightfold by anion-exchange and hydrophobic interaction chromatographies. The molecular weight of T-6 alpha-L-arabinofuranosidase was 256,000, and it consisted of four identical subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The native enzyme had a pI of 6.5 and was most active at 70 degrees C and at pH 5.5 to 6.0. Its thermostability at pH 7.0 was characterized by half-lives of 53, 15, and 1 h at 60, 65, and 70 degrees C, respectively. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax, a Km, and an activation energy of 749 U/mg, 0.42 mM, and 16.6 kcal/mol, (ca. 69.5 kJ/mol), respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+. T-6 alpha-L-arabinofuranosidase released L-arabinose from arabinan and had low activity on oat spelt xylan. The enzyme acted cooperatively with T-6 xylanase in hydrolyzing oat spelt xylan, and L-arabinose, xylose, and xylobiose were detected as the end reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a high-thermostable β-xylanase from newly isolated Thermomyces lanuginosus THKU-49

Highly thermostable β-xylanase produced by newly isolated Thermomyces lanuginosus THKU-49 strain was purified in a four-step procedure involving ammonium sulfate precipitation and subsequent separation on a DEAE-Sepharose fast flow column, hydroxylapatite column, and Sephadex G-100 column, respectively. The enzyme purified to homogeneity had a specific activity of 552 U/mg protein and a molecular weight of 24.9 kDa. The optimal temperature of the purified xylanase was 70°C, and it was stable at temperatures up to 60°C at pH 6.0; the optimal pH was 5.0–7.0, and it was stable in the pH range 3.5–8.0 at 4°C. Xylanase activity was inhibited by Mn²⁺, Sn²⁺, and ethylenediaminetetraacetic acid. The xylanase showed a high activity towards soluble oat spelt xylan, but it exhibited low activity towards insoluble oat spelt xylan; no activity was found to carboxymethylcellulose, avicel, filter paper, locust bean gum, cassava starch, and p-nitrophenyl β-d-xylopyranoside. The apparent K _m value of the xylanase on soluble oat spelt xylan and insoluble oat spelt xylan was 7.3 ± 0.236 and 60.2 ± 6.788 mg/ml, respectively. Thin-layer chromatography analysis showed that the xylanase hydrolyzed oat spelt xylan to yield mainly xylobiose and xylose as end products, but that it could not release xylose from the substrate xylobiose, suggesting that it is an endo-xylanase.