A thermo-halo-tolerant and proteinase-resistant endoxylanase from Bacillus sp. HJ14

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52 % amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55 % of the maximum activity when assayed at 40–75 °C, 23 % at 20 °C, 16 % at 85 °C, and even 8 % at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62 % xylanase activity and stability at the concentration of 3–30 % (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5–19.0, 15.3–19.0, 21.9–27.7, and 28.2–31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.     

A novel xylanase with tolerance to ethanol, salt, protease, SDS, heat, and alkali from actinomycete Lechevalieria sp. HJ3

A xylanase-coding gene (xynAHJ3, 1,104 bp) was cloned from Lechevalieria sp. HJ3 harbored in a saline soil sampled from Heijing town, aka the "town of salt", on the famous "Silk Route of the South". The gene encodes a 367-residue polypeptide (XynAHJ3) with the highest identity of 74.0 % with the endoxylanase from Streptomyces thermocarboxydus HY-15. The coding sequence of the mature protein (without the predicted signal peptide from M1 to S22) of xynAHJ3 was expressed in Escherichia coli BL21 (DE3). The activity of the purified recombinant XynAHJ3 (rXynAHJ3) was apparently optimal at 70 °C and pH 6.0, retained greater than 55 % xylanase activity at a concentration of 0.2-2.0 M Na(+) and 26 % at 4.0 M Na(+) (pH 7.5 20 °C), and showed 110.2 and 44.2 % xylanase activities in the presence of 100 mM SDS (pH 6.0 37 °C) and 10 % ethanol (pH 5.0 37 °C), respectively. rXynAHJ3 activity was stable at 50 °C and pH 4.0-11.0 for more than 60 min, in trypsin or proteinase K at 20 °C for 24 h (pH 7.5), in 10 % ethanol (v/v) (pH 5.0) at 30 or 37 °C for 72 h, in 80 % ethanol (v/v) for 1 h, and in 0.6 or 3 M NaCl (20 °C, pH 7.5) for 72 h. Compared with the majority of xylanases with tolerance to ethanol, salt, SDS, or protease (K (m) values of 1.42-15.1 mg ml(-1)), rXynAHJ3 showed a low K (m) value (0.8 mg ml(-1)) and showed only limited amino acid sequence identity with those other xylanases (less than 47 %).     

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.     

Heterologous expression,purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.     

Purification and characterization of a XIP-type endoxylanase inhibitor from rice (Oryza sativa)

A rice XIP-type inhibitor was purified by affinity chromatography with an immobilized Aspergillus aculeatus family 10 endoxylanase. Rice XIP is a monomeric protein, with a molecular mass of ca. 32 kDa and a pI of ca. 5.6. Its N-terminal amino acid sequence was identical to that of a rice chitinase homologue, demonstrating the difficulty when using sequence information to differentiate between endoxylanase inhibitors and (putative) chitinases in rice. Rice XIP inhibited different endoxylanases to a varying degree. In particular, it most strongly inhibited family 10 endoxylanases from A. niger and A. oryzae, while several family 11 enzymes from Bacillus subtilis, A. niger and Trichoderma sp. were not sensitive to inhibition. The above mentioned A. aculeatus endoxylanase was not inhibited either, although gel permeation chromatography revealed that it complexed rice XIP in a 1:1 molar stoichiometric ratio.     

GH115 α-glucuronidase and GH11 xylanase from Paenibacillus sp. JDR-2: potential roles in processing glucuronoxylans

Paenibacillus sp. JDR-2 (Pjdr2) has been studied as a model for development of bacterial biocatalysts for efficient processing of xylans, methylglucuronoxylan, and methylglucuronoarabinoxylan, the predominant hemicellulosic polysaccharides found in dicots and monocots, respectively. Pjdr2 produces a cell-associated GH10 endoxylanase (Xyn10A₁) that catalyzes depolymerization of xylans to xylobiose, xylotriose, and methylglucuronoxylotriose with methylglucuronate-linked α-1,2 to the nonreducing terminal xylose. A GH10/GH67 xylan utilization regulon includes genes encoding an extracellular cell-associated Xyn10A₁ endoxylanase and an intracellular GH67 α-glucuronidase active on methylglucuronoxylotriose generated by Xyn10A₁ but without activity on methylglucuronoxylotetraose generated by a GH11 endoxylanase. The sequenced genome of Pjdr2 contains three paralogous genes potentially encoding GH115 α-glucuronidases found in certain bacteria and fungi. One of these, Pjdr2_5977, shows enhanced expression during growth on xylans along with Pjdr2_4664 encoding a GH11 endoxylanase. Here, we show that Pjdr2_5977 encodes a GH115 α-glucuronidase, Agu115A, with maximal activity on the aldouronate methylglucuronoxylotetraose selectively generated by a GH11 endoxylanase Xyn11 encoded by Pjdr2_4664. Growth of Pjdr2 on this methylglucuronoxylotetraose supports a process for Xyn11-mediated extracellular depolymerization of methylglucuronoxylan and Agu115A-mediated intracellular deglycosylation as an alternative to the GH10/GH67 system previously defined in this bacterium. A recombinantly expressed enzyme encoded by the Pjdr2 agu115A gene catalyzes removal of 4-O-methylglucuronate residues α-1,2 linked to internal xylose residues in oligoxylosides generated by GH11 and GH30 xylanases and releases methylglucuronate from polymeric methylglucuronoxylan. The GH115 α-glucuronidase from Pjdr2 extends the discovery of this activity to members of the phylum Firmicutes and contributes to a novel system for bioprocessing hemicelluloses.

     

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli.

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

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.     

<Emphasis Type="Italic">Paenibacillus</Emphasis> sp. A59 GH10 and GH11 Extracellular Endoxylanases: Application in Biomass Bioconversion

The cost-efficient degradation of xylan to fermentable sugars is of particular interest in second generation bioethanol production, feed, food, and pulp and paper industries. Multiple potentially secreted enzymes involved in polysaccharide deconstruction are encoded in the genome of Paenibacillus sp. A59, a xylanolytic soil bacterium, such as three endoxylanases, seven GH43 β-xylosidases, and two GH30 glucuronoxylanases. In secretome analysis of xylan cultures, ten glycoside hydrolases were identified, including the three predicted endoxylanases, confirming their active role. The two uni-modular xylanases, a 32-KDa GH10 and a 20-KDa GH11, were recombinantly expressed and their activity on xylan was confirmed (106 and 85 IU/mg, respectively), with differences in their activity pattern. Both endoxylanases released mainly xylobiose (X2) and xylotriose (X3) from xylan and pre-treated biomasses (wheat straw, barley straw, and sweet corn cob), although only rGH10XynA released xylose (X1). rGH10XynA presented optimal conditions at pH 6, with thermal stability at 45–50 °C, while rGH11XynB showed activity in a wider range of pH, from 5 to 9, and was thermostable only at 45 °C. Moreover, GH11XynB presented sigmoidal kinetics on xylan, indicating possible cooperative binding, which was further supported by the structural model. This study provides a detailed analysis of the complete set of carbohydrate-active enzymes encoded in Paenibacillus sp. A59 genome and those effectively implicated in hemicellulose hydrolysis, contributing to understanding the mechanisms necessary for the bioconversion of this polysaccharide. Moreover, the two main free secreted xylanases, rGH10XynA and rGH11XynB, were fully characterized, supporting their potential application in industrial bioprocesses on lignocellulosic biomass.     

Purification and characterization of a XIP-type endoxylanase inhibitor from Rice (Oryza sativa)

A rice XIP-type inhibitor was purified by affinity chromatography with an immobilized Aspergillus aculeatus family 10 endoxylanase. Rice XIP is a monomeric protein, with a molecular mass of ca. 32?kDa and a pI of ca. 5.6. Its N-terminal amino acid sequence was identical to that of a rice chitinase homologue, demonstrating the difficulty when using sequence information to differentiate between endoxylanase inhibitors and (putative) chitinases in rice. Rice XIP inhibited different endoxylanases to a varying degree. In particular, it most strongly inhibited family 10 endoxylanases from A. niger and A. oryzae, while several family 11 enzymes from Bacillus subtilis, A. niger and Trichoderma sp. were not sensitive to inhibition. The above mentioned A. aculeatus endoxylanase was not inhibited either, although gel permeation chromatography revealed that it complexed rice XIP in a 1:1 molar stoichiometric ratio.     

Characterization of XynC from Bacillus subtilis subsp. subtilis strain 168 and analysis of its role in depolymerization of glucuronoxylan

Secretion of xylanase activities by Bacillus subtilis 168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAXn [where n represents the number of xylose residues]) in the hemicellulose component of lignocellulosics to biobased products. In addition to the characterized glycosyl hydrolase family 11 (GH 11) endoxylanase designated XynA, B. subtilis 168 secretes a second endoxylanase as the translated product of the ynfF gene. This sequence shows remarkable homology to the GH 5 endoxylanase secreted by strains of Erwinia chrysanthemi. To determine its properties and potential role in the depolymerization of MeGAXn, the ynfF gene was cloned and overexpressed to provide an endoxylanase, designated XynC, which was characterized with respect to substrate preference, kinetic properties, and product formation. With different sources of MeGAXn as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the beta-1,4-xylan chain. With MeGAXn from sweetgum as a preferred substrate, XynC exhibited a Vmax of 59.9 units/mg XynC, a Km of 1.63 mg MeGAXn/ml, and a k(cat) of 2,635/minute at pH 6.0 and 37 degrees C. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and 1H nuclear magnetic resonance data revealed that each hydrolysis product has a single glucuronosyl substitution penultimate to the reducing terminal xylose. This detailed analysis of XynC from B. subtilis 168 defines the unique depolymerization process catalyzed by the GH 5 endoxylanases. Based upon product analysis, B. subtilis 168 secretes both XynA and XynC. Expression of xynA was subject to MeGAXn induction; xynC expression was constitutive with growth on different substrates. Translation and secretion of both GH 11 and GH 5 endoxylanases by the fully sequenced and genetically malleable B. subtilis 168 recommends this bacterium for the introduction of genes required for the complete utilization of products of the enzyme-catalyzed depolymerization of MeGAXn. B. subtilis may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.     

Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat

Two genes encoding family 11 endo-beta-1,4-xylanases (XylA, XylB) from Fusarium graminearum were cloned and expressed in Escherichia coli. The amount of active endoxylanase in the cytoplasmic soluble fraction was considerably improved by varying different expression parameters, including host strain and temperature during induction. Both recombinant endoxylanases showed a temperature optimum around 35 degrees C and neutral pH optima (around pH 7 and 8 for XylB and XylA, respectively). For the first time this allowed one to test endoxylanases of a phytopathogenic organism for inhibition by proteinaceous endoxylanase inhibitors TAXI and XIP. Whereas XylA and XylB were inhibited by TAXI-I, no inhibition activity could be detected upon incubation with XIP-I. The insensitivity of both F. graminearum endoxylanases towards XIP is surprising, since the latter is typically active against endoxylanases produced by (aerobic) fungi. As F. graminearum is an important phytopathogen, these findings have implications for the role of endoxylanase inhibitors in plant defence.     

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.     

Characteristics of thermostable endoxylanase and β-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues

An extremely thermophilic bacterial isolate that produces a high titer of thermostable endoxylanase and β-xylosidase extracellularly in an inducible manner was identified as Geobacillus thermodenitrificans TSAA1. The distinctive features of this strain are alkalitolerance and halotolerance. The endoxylanase is active over a broad range of pH (5.0–10.0) and temperatures (30–100 °C) with optima at pH 7.5 and 70 °C, while β-xylosidase is optimally active at pH 7.0 and 60 °C. The T _1/2 values of the endoxylanase and β-xylosidase are 30 min at 80 °C, and 180 min at 70 °C, respectively. The endoxylanase activity is stimulated by dithiothreitol, but inhibited strongly by EDAC and Woodward’s reagent K. N-BS and DEPC strongly inhibited β-xylosidase. MALDI-ToF (MS/MS) analysis of tryptic digest of β-xylosidase revealed similarity with that of G. thermodenitrificans NG 80-2, and suggested that this belongs to the GH 52 glycosyl hydrolase super family. The action of endoxylanase on birch wood xylan and agro-residues such as wheat bran and wheat straw liberated xylooligosaccharides similar to endoxylanases of the family 10 glycoside hydrolases, while the enzyme preparation having both endoxylanase and β-xylosidase liberated xylose as main hydrolysis product.     

Purification and characterization of a thermostable glucoamylase from Chaetomium thermophilum

Thermostable amylolytic enzymes are currently being investigated to improve industrial processes of starch degradation. A thermostable extracellular glucoamylase (exo-1, 4-alpha-D-glucanohydrolase, E.C.3.2.1.3) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by using ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Phenyl-Sepharose Fast Flow chromatography. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 64 kDa. The glucoamylase exhibited optimum catalytic activity at pH 4.0 and 65 degrees C. It was thermostable at 50 degrees C and 60 degrees C, and retained 50% activity after 60 min at 65 degrees C. The half-life of the enzyme at 70 degrees C was 20 min. N-terminal amino acid sequencing (15 residues) was AVDSYIERETPIAWN. Different metal ions showed different effects on the glucoamylase activity. Ca2+, Mg2+, Na+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, and Hg2+ cause obvious inhibition. These properties make it applicable to other biotechnological purposes.     

Structure, function, and regulation of the aldouronate utilization gene cluster from Paenibacillus sp. strain JDR-2

Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX_n), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose. Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX_n and rapidly assimilates the principal products, β-1,4-xylobiose, β-1,4-xylotriose, and MeGAX₃, the aldotetrauronate 4-O-methylglucuronosyl-α-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 α-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 β-xylosidase/α-arabinofuranosidase). Expression of these genes, as well as xynA1 encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX_n and repressed by glucose. Sequences in the yesN, lplA, and xynA2 genes within the cluster and in the distal xynA1 gene show significant similarity to catabolite responsive element (cre) defined in Bacillus subtilis for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in Paenibacillus sp. strain JDR-2 operates as a regulon, coregulated with the expression of xynA1, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.     

假单胞菌碱性木聚糖酶的纯化及性质

假单胞菌(Pseudomonas)G62可产生两种胞外木聚糖酶,即XynA和XynB。经过硫酸铵沉淀、阴离子和阳离子交换层析、分子筛色谱,最终得到 两种电泳纯酶。XynA的分子量及等电点分别为42kD和91,XynB的分子量和等电点分别是 20kD和88。经薄层色谱分析证明,两酶以不同的方式水解木聚糖,但都不产生木糖,即 两酶都为内切酶,它们的最适作用温度均为50℃。XynA的最适作用pH为7.0～9.8,而XynB的为7.0～7.5。在65℃时的半寿期XynA为6 min,XynB为140 min。XynA的Km和Vmax分别是5.56 mg·ml-1和543μmol·min-1·mg-1,XynB的Km和Vmax分别是7.72 mg·ml-1和819μmol·min-1·mg-1。两酶受Cu2+、Fe3+、Pb2+、Zn2+和Hg2+强烈抑制。化学修饰的初步结果表明,两酶的活性位点氨基酸均含有色氨酸和羧基氨基酸。     

Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. Strain NG-27

Alkalophilic Bacillus sp. strain NG-27 produces a 42-kDa endoxylanase active at 70 degrees C and at a pH of 8.4. The gene for this endoxylanase was cloned and sequenced. The gene contained one open reading frame of 1,215 bases. An active site characteristic of the family 10 beta-glycanases was recognized between amino acids 303 and 313, with the active glutamate at position 310. Though highly thermostable, the enzyme contains no cysteine residue.     

Purification and characterization of a low molecular weight endoxylanase from solid-state cultures of alkali-tolerant Aspergillus fischeri

A low molecular weight, alkaline-stable endoxylanase (XylB) was purified to homogeneity from solid-state culture of Aspergillus fischeri Fxn1. XylB had a molecular mass of 13 kDa which is the lowest of reported xylanases. Optimal activity was at pH 6 and 55 degrees C. XylB was stable from pH 4.5 to 10 and up to 60 degrees C. It was non-glycosylated. The apparent K(m) and V(max) values of XylB on birch wood xylan were 0.53 mg ml(-1) and 0.2 mmol min-1 mg-1, respectively. The activity of XylB was not inhibited by Cd2+, Zn2+, Co2+, EDTA, iodoacetamide, beta-mercaptoethanol and acetic anhydride but strongly inhibited by 10 mm of N-bromosuccinimide, Hg2+, Pb2+ and p-hydroxymercuric benzoate. XylB is an endoxylanase since it hydrolysed xylan resulting the formation of xylo-oligomers but not of xylose residues.