Engineering a high-performance,metagenomic-derived novel xylanase with improved soluble protein yield and thermostability

The novel termite gut metagenomic-derived GH11 xylanase gene xyl7 was expressed in Escherichia coli BL21, and the purified XYL7 enzyme exhibited high specific activity (6340 U/mg) and broad pH active range of 5.5–10.0. Directed evolution was employed to enhance the thermostability of XYL7; two mutants (XYL7-TC and XYL7-TS) showed a 250-fold increase in half-life at 55 °C, with a 10 °C increase in optimal temperature compared to that of wild-type XYL7. A truncated enzyme (XYL7-Tr3) acquired by protein engineering showed similar catalytic properties as the wild-type, with a tenfold increase in soluble protein yield by the mutant. The reducing sugar produced by XYL7-TC was about fourfold greater than that produced by their parents when incubated with xylan at 60 °C for 4 h. The engineered novel xylanase exhibited superior enzymatic performance and showed promise as an excellent candidate for industrial application due to its high specific activity, stability and soluble protein yield.     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Biochemical characterization,cloning and molecular modeling of a detergent and organic solvent-stable family 11 xylanase from the newly isolated Aspergillus niger US368 strain

New β-1,4-d-xylan xylanohydrolase (XAn11) belonging to the xylanase 11 family was purified to homogeneity from a newly soil-isolated Aspergillus niger US368 strain. The pure xylanase is a glycosylated monomer having a molecular mass of about 26 kDa. The N-terminal sequence of the purified enzyme was determined and compared to some Aspergillus xylanases N-terminal ones. The gene encoding the XAn11 was cloned and sequenced.The maximal xylanase activity was obtained at pH 5.0 and 55 °C. The XAn11 was found to be stable in a wide range of pH (3–9) and in presence of some detergents and organic solvents. A specific activity of about 805.6 U/mg or 334 U/mg was measured using birchwood xylan or oatspelt xylan as substrate, respectively. A structural explanation of the difference between experimental and theoretical molecular mass as well as the stability of the enzyme against acidic pH was proposed by molecular modeling.     

Biochemical characterization of xylanase produced from Streptomyces sp. CS624 using an agro residue substrate

An extracellular and cellulase-free xylanase (EX624) was produced by Streptomyces sp. CS624 using an agricultural residue (wheat bran) as a growth substrate. EX624 was purified from culture supernatant using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The SDS-PAGE and the zymogram analysis of the purified xylanase indicated molecular mass of 40 kDa. Biochemical characterization of the purified EX624 revealed its highest activity at a temperature of 60 °C and pH 6.0. The xylanase was adequately stable in the pH range 4.5–10.0 and at temperatures ≤50 °C. EX624 displayed enhanced activity in the presence of several metal ions including Fe²⁺, Co²⁺ and Ca²⁺. HPLC results showed that EX624 was not only able to hydrolyze commercially available pure beechwood xylan to xylose, xylobiose and xylotriose, but also abundantly available lignocellulosic agricultural residues in nature such as wheat bran to xylooligosaccharides.     

Biochemical characterization of an endoxylanase from Pseudozyma brasiliensis sp. nov. strain GHG001 isolated from the intestinal tract of Chrysomelidae larvae associated to sugarcane roots

Endo-xylanases play a key role in the hydrolysis of xylan and recently they have attracted much attention due to their potential applications on the biofuel and paper industries. We isolated a Pseudozyma brasiliensis sp. nov. strain from the intestinal tract of Chrysomelidae larvae that parasitize sugarcane roots. This basidiomycetous yeast produces a xylanase designated PbXynA which was purified and characterized. The molecular weight of PbXynA is 24 kDa, it belongs to the GH11 family and its optimum pH and optimum temperature are 4.0 and 55 °C, respectively. PbXynA has as secondary structure predominantly β-sheets and sigmoidal kinetic behavior with elevated speed conversion from substrate-to-products (V_max = 2792.0 μmol product/min/mg protein). It is highly activated by bivalent cations such as Ca²⁺, however in the presence of Cu²⁺ xylanase activity was inhibited. It has a high specific activity and produces xylooligosaccharides that have a variety of industrial applications, indicating PbXynA has a great biotechnological potential.     

Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11

An open reading frame (XylX) with 1131 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in E. coli. It encodes a family 11 endoxylanase, designated as XylX, of 41 kDa. The homology of the amino acid sequence deduced from XylX is only 73% identical to the next closest sequence. XylX contains a family 11 catalytic domain of the glycoside hydrolase and a family 6 cellulose-binding module. The recombinant xylanase was fused to a His-tag for affinity purification. The XylX activity was 2392 IU/mg, with a K_m of 6.78 mg/ml and a V_max of 4953 mol/min/mg under optimal conditions (pH 7, 60 °C). At pH 11, 60 °C, the activity was still as high as 517 IU/mg. Xylanase activities at 60 °C under pH 5 to pH 9 remained at more than 69.4% of the initial activity level for 8 h. The addition of Hg²⁺ at 5 mM almost completely inhibited xylanase activity, whereas the addition of tris-(2-carboxyethyl)-phosphine (TCEP) and 2-mercaptoethanol stimulated xylanase activity. No relative activities for Avicel, CMC and d-(+)-cellobiose were found. Xylotriose constitutes the majority of the hydrolyzed products from oat spelt and birchwood xylan. Broad pH and temperature stability shows its application potentials for biomass conversion, food and pulp/paper industries.     

Purification and partial characterisation of a thermostable xylanase from salt-tolerant Thermobifida halotolerans YIM 90462T

ThxynA, an extracellular xylanase of T. halotolerans YIM 90462^T, was purified to homogeneity from a fermentation broth by ultra-filtration, ammonium sulphate precipitation, hydrophobic chromatography and ion exchange chromatography. The purified xylanase has a molecular mass of 24 kDa and is optimally active at 80 °C and pH 6.0. The enzyme is stable over a broad pH range (pH 6.0–10.0) and shows good thermal stability when incubated at 70 °C for 1 h. The K_m and V_max values of the enzyme are 11.6 mg/mL and 434 μmol mg^?1 min^?1, respectively, using oat spelt xylan as a substrate. Moreover, the enzyme seemingly has both xylanase activity and cellulase activity. These unique properties suggest that it may be useful for industrial applications.     

A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp. MEY-1

A complete gene, xyl10C, encoding a thermophilic endo-1,4-β-xylanase (XYL10C), was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. XYL10C shares highest nucleotide and amino acid sequence identities of 57.3 and 49.7%, respectively, with a putative xylanase from Aspergillus fumigatus Af293 of glycoside hydrolase family 10. A high expression level in P. pastoris (73,400 U ml⁻¹) was achieved in a 3.7–l fermenter. The purified recombinant XYL10C was thermophilic, exhibiting maximum activity at 85°C, which is higher than that reported from any fungal xylanase. The enzyme was also highly thermostable, exhibiting ~100% of the initial activity after incubation at 80°C for 60 min and >87% of activity at 90°C for 10 min. The half lives of XYL10C at 80 and 85°C were approximately 45 and 3 h, respectively. It had two activity peaks at pH 3.0 and 4.5–5.0 (maximum), respectively, and was very acid stable, retaining more than 80% activity after incubation at pH 1.5−6.0 for 1 h. The enzyme was resistant to Co²⁺, Mn²⁺, Cr³⁺ and Ag⁺. The specific activity of XYL10C for oat spelt xylan was 18,831 U mg⁻¹. It also had wide substrate specificity and produced simple products (65.1% xylose, 25.0% xylobiose and 9.9% xylan polymer) from oat spelt xylan.     

Codon-optimized expression and characterization of a pH stable fungal xylanase in Pichia pastoris

Novel xylanase (EC 3.2.1.8) is in great demand due to its industrial significance. In this study, we have developed and characterized a novel xylanase-producing yeast strain. This mature xylanase gene xyn11A consists of 870 base pairs and belongs to GH11 family. The gene sequence was optimized and synthesized, and was then cloned into yeast vector pGAPZαA under the control of the constitutive GAP promoter. SDS-PAGE analysis indicates that Xyn11A is extracellularly expressed as a glycosylated protein in P. pastoris. Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase retained more than 90% of its activity after incubation at 50 °C and 60 °C for up to 1 h. Xyn11A is also stable over a wide range of pH (2.0–11.0). Most metal ions tested such as copper (Cu²⁺) and lead (Pb²⁺) have little inhibitory effects on Xyn11A. It is also resistant to pepsin and proteinase K digestion, retaining 80% and 90% of its activity after digestion at 37 °C for 1 h, respectively. Those superior properties make Xyn11A a robust xylanase with great potential for industrial use. To the best of our knowledge, this is the first report of xylanase from the fungus Corynascus thermophilus.     

Multi-functional glycoside hydrolase: Blon_0625 from Bifidobacterium longum subsp. infantis ATCC 15697

We here describe a unique β-D-glucosidase (BGL; Blon_0625) derived from Bifidobacterium longum subsp. infantis ATCC 15697. The Blon_0625 gene was expressed by recombinant Escherichia coli. Purified recombinant Blon_0625 retains hydrolyzing activity against both p-nitrophenyl-β-D-glucopyranoside (pNPG; 17.3 ± 0.24 U mg⁻¹) and p-nitrophenyl-β-D-xylopyranoside (pNPX; 16.7 ± 0.32 U mg⁻¹) at pH 6.0, 30 °C. To best of our knowledge, no previously described BGL retains the same level of both pNPGase and pNPXase activity. Furthermore, Blon_0625 also retains the activity against 4-nitrophenyl-α-l-arabinofranoside (pNPAf; 5.6 ± 0.09 U mg⁻¹). In addition, the results of the degradation of phosphoric acid swollen cellulose (PASC) or xylan using endoglucanase from Thermobifida fusca YX (Tfu_0901) or xylanase from Kitasatospora setae KM-6054 (KSE_59480) show that Blon_0625 acts as a BGL and as a β-D-xylosidase (XYL) for hydrolyzing oligosaccharides. These results clearly indicate that Blon_0625 is a multi-functional glycoside hydrolase which retains the activity of BGL, XYL, and also α-l-arabinofuranosidase. Therefore, the utilization of multi-functional Blon_0625 may contribute to facilitating the efficient degradation of lignocellulosic materials and help enhance bioconversion processes.     

The cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 and the evidence for production of a large multi-enzyme complex

The cellulolytic and hemi-cellulolytic system of Bacillus licheniformis SVD1 was isolated and characterised in birchwood xylan cultures. The predominant activity in the crude culture was xylanase activity, but the crude culture also displayed Avicelase, carboxymethylcellulase (CMCase), mannanase, and pectinase activity. Most of the xylanase activity was found in the culture supernatant, but some activity was cell-associated. Using Sepharose 4B size exclusion chromatography, a 2000 kDa multi-enzyme complex (MEC) was purified. The MEC contained predominantly xylanase activity, as well as significant levels of mannanase and CMCase activity, but no Avicelase activity. SDS-PAGE revealed up to eight visible bands in the MEC while zymograms of the MEC displayed two xylanase active bands at 21 kDa and 45 kDa, and two CMCase active bands at 25 kDa and 30 kDa. More active bands were visible in the crude supernatant with an additional xylanase active band at 40 kDa and an additional CMCase active band at 55 kDa. Using thin layer chromatography (TLC), it was established that the crude fraction could release xylose from insoluble birchwood xylan, while the MEC was only able to produce xylobiose from this substrate. The MEC was further able to bind to insoluble xylan, but was unable to bind to crystalline cellulose. This MEC lacks many of the characteristic features of a cellulosome and is most likely a different type of complex. The presence of both high xylanase and mannanase activity makes this MEC unusual.     

Production and characterization of a thermostable endo-type β-xylanase produced by a newly-isolated Streptomyces thermocarboxydus subspecies MW8 strain from Jeju Island

A xylanase-producing, Gram-positive, aerobic, and spore-forming bacterium was isolated from a soil sample collected from Jeju Island and was classified as a novel subspecies of Streptomyces thermocarboxydus on the basis of 16S rRNA gene sequence similarity, the results of DNA–DNA hybridization analysis, and phenotypic characteristics. The novel strain was named as S. thermocarboxydus subsp. MW8 (=KCTC29013 = DSM52054). This strain produced extracellular xylanase. Xylanase from the strain was purified to homogeneity and had an apparent molecular weight of 52 kDa. The NH₂-terminal sequence (Ala-Glu-Ile-Arg-Leu) was distinct from those of previously reported xylanases. The purified xylanase produced xylobiose as the end-product of birchwood xylan hydrolysis. The K_m and V_max values of the purified xylanase on birchwood xylan were 1.71 mg/ml and 357.14 U/mg, respectively. The optimum pH and temperature for the enzyme were found to be 7.0 and 50 °C, respectively, and the enzyme exhibited significant heat stability. In addition, the enzyme was active over broad pH ranges: 84% of the maximum activity at pH 5.0, 84–88% at pH 6.0, 88% at pH 8.0, and 75–81% (pH 9.0). These enzymatic properties may be very useful for use in bio-industrial applications.     

Fusarium graminearum xylanases show different functional stabilities,substrate specificities and inhibition sensitivities

When grown on arabinoxylan as the sole carbon source, the cereal phytopathogen Fusarium graminearum expresses four xylanases. Cloning and heterologous expression of the corresponding xylanase encoding genes and analysis of general biochemical properties, substrate specificities and inhibition sensitivities revealed some marked differences. XylA and XylB are glycoside hydrolase family (GH) 11 xylanases, while XylC and XylD belong to GH10. pH and temperature for optimal activity of the enzymes were between 6.0 and 7.0 and 40 °C, respectively. Interestingly, XylC displayed remarkable pH stability as it retained most of its activity even after pre-incubation at pH 1.0 and 13.0 for 120 min at room temperature. All xylanases hydrolysed xylotetraose, xylopentaose and xylohexaose, but to different extents, while only XylC and XylD hydrolysed xylotriose. The two GH10 xylanases released a higher percentage of smaller products from xylan and xylo-oligosaccharides than did their GH11 counterparts. Analysis of kinetic properties revealed that wheat arabinoxylan is the favoured XylC substrate while XylA and XylB prefer sparsely substituted oat spelt xylan. XylC and XylD were inhibited by xylanase inhibiting protein (XIP), while XylA and XylB were sensitive to Triticum aestivum xylanase inhibitor (TAXI). Because of its pH stability and preference for arabinoxylan, XylC is a valuable candidate for use in biotechnological applications.     

A novel fibrinolytic protease from Streptomyces sp. CS684

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. K_m, V_max and K_cat values for azocasein were 4.2 mg ml⁻¹, 305.8 μg min⁻¹ mg⁻¹ and 188.7 s⁻¹, respectively. The activity was suppressed by Co²⁺, Zn²⁺, Cu²⁺ and Fe²⁺, but slightly enhanced by Ca²⁺ and Mg⁺². Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.     

Cloning,expression, and characterization of a thermostable β-xylosidase from thermoacidophilic Alicyclobacillus sp. A4

A β-xylosidase gene (xylA4) was identified in the genome sequence of thermoacidophilic Alicyclobacillus sp. A4. The deduced amino acid sequence was highly homologous with the β-xylosidases of family 52 of the glycoside hydrolases (GH). The full-length gene consisted of 2097 bp and encoded 698 amino acids without a signal peptide. The gene product was successfully expressed in Escherichia coli with an activity of 564.9 U/mL. Recombinant XylA4 was purified by Ni²⁺-NTA affinity chromatography with a molecular mass of 78.5 kDa. The enzyme showed optimal activity at pH 6.0 and 65 °C, and remained stable over the pH range of 5.0–9.0. The thermostability of XylA4 is noteworthy, retaining almost all of the activity after 1 h incubation at 65 °C. Using p-nitrophenyl-β-d-xylopyranoside (pNPX) as the substrate, XylA4 had the highest specific activity (261.1 U/mg) and catalytic efficiency (601.5/mM/s) known so far for GH52 xylosidases. The enzyme displayed high tolerance to xylose, with a K_i value of approximately 88.7 mM. It also had synergy with xylanase XynBE18 from Paenibacillus sp. E18 in xylan degradation, releasing more xylose (up to 1.43 folds) than XynBE18 alone. Therefore, this thermostable xylose-tolerant β-xylosidase may have a great application potential in many industrial fields.     

A novel thermostable cellulase free xylanase stable in broad range of pH from Streptomyces sp. CS428

A cellulase free thermostable xylanase from Streptomyces sp. CS428 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular xylanase was purified 26 fold with a 55% yield by CM Trisacryl cation exchange chromatography. The molecular mass of the enzyme (Xyn428) was approximately 37 kDa. Xyn428 was found to be stable over a broad pH range (4 to ～13.6) and to 50 °C and have an optimum temperature of 80 °C. Xyn428 had K_m and V_max values of 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, when beechwood xylan was used as substrate. N-terminal sequence of Xyn428 was INRTDHNENSYLEIHNNEAR. CS428 was grown on different agro waste xylan and produced 4197.1 U/mL of xylanase activity in 36 h of cultivation in wheat bran without supplements. Xyn428 activity was inhibited by Tris salt at concentrations above 20 mM, and produced xylose and xylobiose as major products. It was found to degrade agro waste materials by small unit of enzyme (20 U/g) as shown by electron microscopy. As being simple in purification, thermo tolerant, pH stability in broad range and ability to produce xylooligosaccharides show that Xyn428 has potential applications in industries as a biobleaching agent and for xylooligosaccharides production.     

Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution

This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38 U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55 °C and 50 °C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60 °C and apparent melting temperature of reBaxA50 were 9.74 min and 89.15 °C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.     

Purification and characterization of aminopeptidase B from goat brain

Aminopeptidase B was purified from goat brain with a purification fold of ～280 and a yield of 2.7%. The enzyme revealed a single band on both native acrylamide gel and SDS-PAGE thereby confirming apparent homogeneous preparation and its monomeric nature. The enzyme exhibited a molecular mass of 80.2 kDa and 79.7 kDa on Sephadex G-200 and SDS-PAGE respectively. The pH optimum was 7.4 and the enzyme was stable between pH 6.0 and 9.0. l-Arg-βNA was the most rapidly hydrolyzed substrate followed by Lys-βNA. The K_m value with Arg-βNA was found to be 0.1 mM. Metal chelating and –SH reactive agents strongly inhibited the enzyme activity. 1,10-Phenanthroline exhibited mixed type of inhibition with a K_i of 5 × 10^?5 M. The enzyme was highly sensitive to urea. Metal ions like Ni²⁺, Cd²⁺, Fe²⁺and Hg²⁺ inhibited the enzyme, whereas Co²⁺, Zn²⁺, Mn²⁺and Sn²⁺ slightly activated the enzyme.     

Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.     

Arginase from Bacillus thuringiensis SK 20.001: Purification,characteristics, and implications for l-ornithine biosynthesis

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn²⁺. The presence of Mn²⁺ and Ni²⁺ had strong effects on the enzyme activity, and Mn²⁺ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca²⁺, Fe²⁺ and Zn²⁺. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The K_m and V_max for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme.