Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.     

Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes

Soil metagenomes represent an unlimited resource for the discovery of novel biocatalysts from soil microorganisms. Three large-inserts metagenomic DNA libraries were constructed from different grassland soil samples and screened for genes conferring cellulase or xylanase activity. Function-driven screening identified a novel cellulase-encoding gene (cel01) and two xylanase-encoding genes (xyn01 and xyn02). From sequence and protein domain analyses, Cel01 (831 amino acids) belongs to glycoside hydrolase family 9 whereas Xyn01 (170 amino acids) and Xyn02 (255 amino acids) are members of glycoside hydrolase family 11. Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases. Both Xyn01 and Xyn02 were most active at 60°C with high activities from 4 to 10 and optimal at pH 7 (Xyn01) and pH 6 (Xyn02). The cellulase gene, cel01, was expressed in E. coli BL21 and the recombinant enzyme (91.9 kDa) was purified. Cel01 exhibited high activity with soluble cellulose substrates containing β-1,4-linkages. Activity with microcrystalline cellulose was not detected. These data, together with the analysis of the degradation profiles of carboxymethyl cellulose and barley glucan indicated that Cel01 is an endo 1,4-β-glucanase. Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.     

Homologous constitutive expression of Xyn III in Trichoderma reesei QM9414 and its characterization

Xylanase III (Xyn III), a specific endoxylanase that belongs to family 10 of the glycoside hydrolases, was overexpressed in Trichoderma reesei QM9414 using a constitutive strong promoter of the gene encoding pyruvate decarboxylase (pdc). The maximum recombinant xylanase activity achieved was 817.2?±?65.2 U/mL in the transformant fermentation liquid. The productivities of Xyn III accounted for approximately 53 % of the total protein secreted by the recombinant. The enzyme was optimally active at 60 °C and pH 6. The recombinant Xyn III was stable at pH 5–8. This is the first report on the homologous expression of xyn3 in T. reesei QM9414. The properties of Xyn III make it promising in a variety of industrial use.     

Production in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> of three xylanases from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>: a recombinant thermoxylanase for biobleaching of kraft pulp

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5–7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.     

Cloning, expression and characterization of a novel salt-tolerant xylanase from Bacillus sp. SN5

A xylanase gene (xyn10A) was cloned from Bacillus sp. SN5 and expressed in Escherichia coli. It encoded a 348-residue polypeptide of ~45?kDa. The deduced amino acid sequence had 68?% identity with the endo-1,4-beta-xylanase from Paenibacillus lactis 154 that belonged to family 10 of the glycoside hydrolases. Purified recombinant Xyn10A had maximum activity at 40?°C and pH 7.0, with the specific activity of 105?U/mg and a Km of 0.6?mg/ml for beechwood xylan. Xyn10A retained more than 80?% activity between 25 and 45?°C and 29?% activity at 5?°C. It exhibited the highest activity (134?%) in 0.5?M NaCl and still retained 90?% activity in 2.5?M NaCl. It retained about 87?% activity after incubation in 2?M NaCl for 24?h. The cold-active and halo-tolerant properties of Xyn10A make it promising for application in the food industry, especially in the processing of saline food and sea food.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

Thermostable xylanase10B from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC824

The Clostridium acetobutylicum xylanase gene xyn10B (CAP0116) was cloned from the type strain ATCC 824, whose genome was recently sequenced. The nucleotide sequence of C. acetobutylicum xyn10B encodes a 318-amino acid protein. Xyn10B consists of a single catalytic domain that belongs to family 10 of glycosyl hydrolases. The enzyme was purified from recombinant Escherichia coli. The Xyn10B enzyme was highly active toward birchwood xylan, oat-spelt xylan, and moderately active toward avicel, carboxymethyl cellulose, polygalacturonic acid, lichenan, laminarin, barley--glucan and various p-nitrophenyl monosaccharides. Xyn10B hydrolyzed xylan and xylooligosaccharides to produce xylobiose and xylotriose. The pH optimum of Xyn10B was 5.0, and the optimal temperature was 70°C. The enzyme was stable at 60°C at pH 5.0–6.5 for 1 h without substrate. This is one of a number of xylan-related activities encoded on the large plasmid in C. acetobutylicum ATCC 824.     

Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei

Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.     

Characterization of thermostable Xyn10A enzyme from mesophilic Clostridium acetobutylicum ATCC 824

A thermostable xylanase gene, xyn10A (CAP0053), was cloned from Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the C. acetobutylicum xyn10A gene encoded a 318-amino-acid, single-domain, family 10 xylanase, Xyn10A, with a molecular mass of 34 kDa. Xyn10A exhibited extremely high (92%) amino acid sequence identity with Xyn10B (CAP0116) of this strain and had 42% and 32% identity with the catalytic domains of Rhodothermus marinus xylanase I and Thermoascus aurantiacus xylanase I, respectively. Xyn10A enzyme was purified from recombinant Escherichia coli and was highly active toward oat-spelt and Birchwood xylan and slightly active toward carboxymethyl cellulose, arabinogalactouronic acid, and various p-nitrophenyl monosaccharides. Xyn10A hydrolyzed xylan and xylooligosaccharides larger than xylobiose to produce xylose. This enzyme was optimally active at 60°C and had an optimum pH of 5.0. This is one of a number of related activities encoded on the large plasmid in this strain.     

Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2

To improve the thermostability of Trichoderma reesei xylanase 2 (Xyn2), the thermostabilizing domain (A2) from Thermotoga maritima XynA were engineered into the N-terminal region of the Xyn2 protein. The xyn2 and hybrid genes were successfully expressed in Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from S. cerevisiae (α-factor). The transformants expressed the hybrid gene produced clearly increased both the thermostability and substrate-binding capacity compared to the corresponding strains expressed the native Xyn2 gene. The activity of the hybrid enzyme was highest at 65 °C that was 10 °C higher than the native Xyn2. The hybrid enzyme was stable at 60 °C and retained more than 85% of its activity after 30-min incubation at this temperature. The hybrid enzyme was highly specific toward xylan and analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with xylobiose and xylotriose as the main degradation products. These attributes should make it an attractive applicant for various applications. Our results also suggested that the N-terminal domain A2 is responsible for both the thermostability and substrate-binding capacity of T. maritima XynA.     

Molecular and Biochemical Characterization of Two Xylanase-Encoding Genes from Cellulomonas pachnodae

Two xylanase-encoding genes, named xyn11A and xyn10B, were isolated from a genomic library of Cellulomonas pachnodae by expression in Escherichia coli. The deduced polypeptide, Xyn11A, consists of 335 amino acids with a calculated molecular mass of 34,383 Da. Different domains could be identified in the Xyn11A protein on the basis of homology searches. Xyn11A contains a catalytic domain belonging to family 11 glycosyl hydrolases and a C-terminal xylan binding domain, which are separated from the catalytic domain by a typical linker sequence. Binding studies with native Xyn11A and a truncated derivative of Xyn11A, lacking the putative binding domain, confirmed the function of the two domains. The second xylanase, designated Xyn10B, consists of 1,183 amino acids with a calculated molecular mass of 124,136 Da. Xyn10B also appears to be a modular protein, but typical linker sequences that separate the different domains were not identified. It comprises a N-terminal signal peptide followed by a stretch of amino acids that shows homology to thermostabilizing domains. Downstream of the latter domain, a catalytic domain specific for family 10 glycosyl hydrolases was identified. A truncated derivative of Xyn10B bound tightly to Avicel, which was in accordance with the identified cellulose binding domain at the C terminus of Xyn10B on the basis of homology. C. pachnodae, a (hemi)cellulolytic bacterium that was isolated from the hindgut of herbivorous Pachnoda marginata larvae, secretes at least two xylanases in the culture fluid. Although both Xyn11A and Xyn10B had the highest homology to xylanases from Cellulomonas fimi, distinct differences in the molecular organizations of the xylanases from the two Cellulomonas species were identified.     

Characterization of the biochemical properties of recombinant Xyn10C from a marine bacterium, <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> 2-40

Endo-1,4-β-xylanases are mostly classified into glycoside hydrolase (GH) family 10 or 11. In this study, we examined the catalytic functions of a recombinant endo-1,4-β-xylanase belonging to GH10 (Xyn10C) from a marine bacterium, Saccharophagus degradans 2-40. Optimal activity of this enzyme was evident at 30 °C and pH 7.0, but activity remained even at low temperatures, indicating its adaptation to cold. With respect to other xylanases known to be active in cold temperatures, Xyn10C is unique in that it showed maximal activity in the presence of 2 M of NaCl. The action patterns of recombinant Xyn10C on xylans from hardwood and softwood differed in part, but the enzyme hydrolyzed polysaccharidic substrates primarily to xylobiose and xylotriose through xylo-oligosaccharides, releasing a small amount of xylose. The K _m and V _max values on birchwood xylan were 10.4 mg mL^?1 and 253 µmol mg^?1 min^?1, respectively. The efficient catalytic function of Xyn10C on short-length xylo-oligosaccharide chains was similar to the typical function of other known GH10 xylanases.     

An acid and highly thermostable xylanase from <Emphasis Type="Italic">Phialophora</Emphasis> sp. G5

An endo-β-1,4-xylanase gene, designated xyn10G5, was cloned from Phialophora sp. G5 and expressed in Pichia pastoris. The 1,197-bp full-length gene encodes a polypeptide of 399 amino acids consisting of a putative signal peptide at residues 1–20, a family 10 glycoside hydrolase domain, a short Gly/Thr-rich linker and a family 1 carbohydrate-binding module (CBM). The deduced amino acid sequence of XYN10G5 shares the highest identity (53.4%) with a putative xylanase precursor from Aspergillus terreus NIH2624. The purified recombinant XYN10G5 exhibited the optimal activity at pH 4.0 and 70 °C, remained stable at pH 3.0–9.0 (>70% of the maximal activity), and was highly thermostable at 70 °C (retaining ~90% of the initial activity for 1 h). Substrate specificity studies have shown that XYN10G5 had the highest activity on soluble wheat arabinoxylan (350.6 U mg⁻¹), and moderate activity to various heteroxylans, and low activity on different types of cellulosic substrates. Under simulated gastric conditions, XYN10G5 was stable and released more reducing sugars from soluble wheat arabinoxylan; when combined with a glucanase (CelA4), the viscosity of barley–soybean feed was significantly reduced. These favorable enzymatic properties make XYN10G5 a good candidate for application in the animal feed industry.     

Direct cloning of genes encoding novel xylanases from the human gut

The aim of this study was to identify a novel 1,4-beta-xylanase gene from the mixed genome DNA of human fecal bacteria without bacterial cultivation. Total DNA was isolated from a population of bacteria extracted from fecal microbiota. Using PCR, the gene fragments encoding 5 different family 10 xylanases (xyn10A, xyn10B, xyn10C, xyn10D, and xyn10E) were found. Amino acid sequences deduced from these genes were highly homologous with those of xylanases from anaerobic intestinal bacteria such as Bacteroides spp. and Prevotella spp. Self-organizing map (SOM) analysis revealed that xynA10 was classified into Bacteroidetes. To confirm that one of these genes encodes an active enzyme, a full-length xyn10A gene was obtained using nested primers specific to the internal fragments and random primers. The xyn10A gene encoding the xylanase Xyn10A consists of 1146 bp and encodes a protein of 382 amino acids and a molecular weight of 43,552. Xyn10A was a single module novel xylanase. Xyn10A was purified from a recombinant Escherichia coli strain and characterized. This enzyme was optimally active at 40 degrees C and stable up to 50 degrees C at pH 6.5 and over the pH range 4.0-11.0 at 25 degrees C. In addition, 2 ORFs (ORF1 and ORF2) were identified upstream of xyn10A. These results suggested that many unidentified xylanolytic bacteria exist in the human gut and may contribute to the breakdown of xylan which contains dietary fiber.     

Cloning of a xylanase gene <Emphasis Type="Italic">xyn2A</Emphasis> from rumen fungus <Emphasis Type="Italic">Neocallimastix</Emphasis> sp. GMLF2 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its partial characterization

Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be 6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11.     

Engineering hyperthermostability into a mesophilic family 11 xylanase from Aspergillus oryzae by in silico design of N‐terminus substitution

A mesophilic xylanase from Aspergillus oryzae CICC40186 (abbreviated to AoXyn11A) belongs to glycoside hydrolase family 11. The thermostability of AoXyn11A was significantly improved by substituting its N‐terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11^TS. The suitable N‐terminus of AoXyn11A to be replaced was selected by the comparison of B‐factors between AoXyn11A and EvXyn11^TS, which were generated and calculated after a 15 ns molecular dynamic (MD) simulation process. Then, the predicted hybrid xylanase (designated AEx11A) was modeled, and subjected to a 2 ns MD simulation process for calculating its total energy value. The N‐terminus substitution was confirmed by comparing the total energy value of AEx11A with that of AoXyn11A. Based on the in silico design, the AEx11A was constructed and expressed in Pichia pastoris GS115. After 72 h of methanol induction, the recombinant AEx11A (reAEx11A) activity reached 82.2 U/mL. The apparent temperature optimum of reAEx11A was 80°C, much higher than that of reAoXyn11A. Its half‐life was 197‐fold longer than that of reAoXyn11A at 70°C. Compared with reAoXyn11A, the reAEx11A displayed a slight alteration in K_m but a decrease in V_max. Biotechnol. Bioeng. 2013; 110: 1028–1038. © 2012 Wiley Periodicals, Inc.     

Biochemical analysis of a highly specific, pH stable xylanase gene identified from a bovine rumen-derived metagenomic library

A metagenomic library was generated using microbial DNA extracted from the rumen contents of a grass hay-fed dairy cow using a bacterial artificial chromosome-based vector system. Functional screening of the library identified a gene encoding a potent glycoside hydrolase, xyn10N18, localised within a xylanolytic gene cluster consisting of four open-reading frames (ORFs). The ORF, xyn10N18, encodes an endo-β-1,4-xylanase with a glycosyl hydrolase family 10 (GH10) catalytic domain, adopts a canonical α8/ß8-fold and possesses conserved catalytic glutamate residues typical of GH10 xylanases. Xyn10N18 exhibits optimal catalytic activity at 35 °C and pH 6.5 and was highly stable to pH changes retaining at least 85 % relative catalytic activity over a broad pH range (4.0–12.0). It retained 25 % of its relative activity at both low (4 °C) and high (55 °C) temperatures, however the stability of the enzyme rapidly decreased at temperatures of >40 °C. The specific activity of Xyn10N18 is enhanced by the divalent cations Mn²⁺ and Co²⁺ and is dramatically reduced by Hg²⁺ and Cu²⁺. Interestingly, EDTA had little effect on specific activity indicating that divalent cations do not function mechanistically. The enzyme was highly specific for xylan containing substrates and showed no catalytic activity against cellulose. Analysis of the hydrolysis products indicated that Xyn10N18 was an endoxylanase. Through a combination of structural modelling and in vitro enzyme characterisation this study provides an understanding of the mechanism and the substrate specificity of this enzyme serving as a starting point for directed evolution of Xyn10N18 and subsequent downstream use in industry.     

Cloning,Sequencing, and Expression of the Gene Encoding a Cell-bound Multi-domain Xylanase from Clostridium josui,and Characterization of the Translated Product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, or carboxymethylcellulose.