Thermostable and Alkalistable Endoxylanase of the Extremely Thermophilic Bacterium Geobacillus thermodenitrificans TSAA1: Cloning, Expression, Characteristics and Its Applicability in Generating Xylooligosaccharides and Fermentable Sugars

Xylanase encoding gene (1,224 bp) from Geobacillus thermodenitrificans was cloned in pET28a (+) vector and successfully expressed in Escherichia coli BL21 (DE3). The deduced amino acid sequence analysis revealed homology with that of glycosyl hydrolase (GH) 10 family with a high molecular mass (50 kDa). The purified recombinant xylanase is optimally active at pH 9.0 and 70 °C with T _1/2 of 10 min at 80 °C, and retains greater than 85 % activity after exposure to 70 °C for 180 min. The enzyme liberates xylose as well as xylooligosaccharides from birchwood xylan and agro-residues, and therefore, this is an endoxylanase. The xylan hydrolytic products (xylooligosaccharides, xylose, and xylobiose) find application as prebiotics and in the production of bioethanol. The xylanase being thermostable and alkalistable, it has released chromophores and phenolics from the residual lignin of pulps, suggesting its utility in mitigating chlorine requirement in pulp bleaching.     

Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K _m and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K _m of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Purification and Characterization of Novel Halo-Acid-Alkali-Thermo-stable Xylanase from Gracilibacillus sp. TSCPVG

An aerobic xylanolytic moderately halophilic and alkali-tolerant bacterium, Gracilibacillus sp. TSCPVG, produces multiple xylanases of unusual halo-acid-alkali-thermo-stable nature. The purification of a major xylanase from TSCPVG culture supernatant was achieved by hydrophobic and gel permeation chromatographic methods followed by electroelution from preparatory PAGE. The molecular mass of the purified xylanase was 42 kDa, as analyzed by SDS-PAGE, with a pI value of 6.1. It exhibited maximal activity in 3.5 % NaCl and retained over 75 % of its activity across the broad salinity range of 0–30 % NaCl, indicating a high halo-tolerance. It showed maximal activity at pH 7.5 and had retained 63 % of its activity at pH 5.0 and 73 % at pH 10.5, signifying the tolerance to broad acid to alkaline conditions. With birchwood xylan as a substrate, K _m and specific activity values were 21 mg/ml and 1,667 U/mg, respectively. It is an endoxylanase that degrades xylan to xylose and xylobiose and had no activity on p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl acetate, carboxymethylcellulose, and filter paper. Since it showed remarkable stability over different salinities, broad pH, and temperature ranges, it is promising for application in many industries.     

Screening and Xylanase Production by Streptomyces sp. Grown on Lignocellulosic Wastes

Xylanases have raised interest because of their potential applications in various industrial fields, including the pulp and paper industries, bioethanol production, and the feed industry. In bioethanol production from lignocellulosic compounds, xylanase can improve the hydrolysis of cellulose into fermentable sugars, since the xylan restricts the cellulases from acting efficiently. In this work, a new thermophilic Streptomyces sp. was selected for its ability to produce xylanase. Carbon source selection is an important factor in the production of hemicellulases. The highest activity was obtained when Streptomyces sp. I3 was grown in the presence of wheat bran. Xylanase activity was partially characterized concerning the effect of pH and temperature on activity and thermostability, and the effects of different metal ions were also tested. The pH and temperature profile showed optimal activity at pH 6.0/70 °C. Zymogram analysis showed multiple xylanases (39, 21, 18, and 17 kDa). Xylanases studied in this work are thermophilic, thermostable, and active in a wide pH range; they have potential to be used in the development of new processes of biotechnological interest.     

Use of corncob for endoxylanase production by thermophilic fungus Thermomyces lanuginosus IOC-4145

The production of cellulase-free end oxylanase by the thermophilic fungus Thermomyces lanuginosus was investigated insemisolid fermentation and liquid fermentation. Different process variables were investigated in semisolid fermentation, employing corncobas the carbon source. The best results were with the following conditions: grain size=4.5 mm, solid:liquid ratio=1:2, and inoculum size=20% (v/v). Corncob, xylan, and xylose were the best inducers for endoxylanase production. Additionally, organic nitrogen sources were necessary for the production of high endoxylanase activities. The crude enzyme had optimum activity at pH 6.0 and 75°C, displaying a high thermostability. The apparent K ₂₅ and V _max were 1.77 mg of xylan/mL and 21.5 U/mg of protein, respectively.     

Isolation,Purification and Characterisation of an Organic Solvent-Tolerant Ca2+-Dependent Protease from Bacillus megaterium AU02

A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl₂, and it retained 90 % activity at 50 °C for 3 h. The K _m and V _max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH₂) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.     

Purification and Characterization of the Xylanase Produced by Jonesia denitrificans BN-13

Jonesia denitrificans BN-13 produces six xylanases: Xyl1, Xyl2, Xyl3, Xyl4, Xyl5, and Xyl6; the Xyl4 was purified and characterized after two consecutive purification steps using ultrafiltration and anion exchange chromatography. The xylanase-specific activity was found to be 77 unit (U)/mg. The molecular weight of the Xyl4 estimated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) revealed a monomeric isoenzyme of about 42 kDa. It showed an optimum pH value of 7.0 and a temperature of 50 °C. It was stable at 50 °C for 9.34 h. The enzyme showed to be activated by Mn⁺², β-mercaptoethanol, and dithiothreitol (DTT) with a high affinity towards birchwood xylan (with a K _m of 1 mg ml^?1) and hydrolysis of oat-spelt xylan with a K _m of 1.85 mg ml^?1. The ability of binding to cellulose and/or xylan was also investigated.     

Expression and Characterization of Hyperthermotolerant Xylanases,SyXyn11P and SyXyn11E,in Pichia pastoris and Escherichia coli

Both Syxyn11P and Syxyn11E, two codon-optimized genes encoding glycoside hydrolase (GH) family 11 hyperthermotolerant xylanases (designated SyXyn11P and SyXyn11E), were synthesized and inserted into pPIC9K^M and pET-28a(+) vectors, respectively. The resulting recombinant expression vectors, pPIC9K^M-Syxyn11P and pET-28a(+)-Syxyn11E, were transformed into Pichia pastoris GS115 and Escherichia coli BL21, respectively. The maximum activities of two recombinant xylanases (reSyXyn11P and reSyXyn11E) expressed in P. pastoris and E. coli reached 30.9 and 17.8 U/ml, respectively. The purified reSyXyn11P and reSyXyn11E displayed the same pH optimum at 6.5 and pH stability at a broad range of 4.5–9.0. The temperature optimum and stability of reSyXyn11P were 85 and 80 °C, higher than those of reSyXyn11E, respectively. Their activities were not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn²⁺ and Ag⁺. The K _m and V _max of reSyXyn11P toward birchwood xylan were 4.3 mg/ml and 694.6 U/mg, whose K _m was close to that (4.8 mg/ml), but whose V _max was much higher than that (205.6 U/mg) of reSyXyn11E. High-performance liquid chromatography analysis indicated that xylobiose and xylotriose as the major products were excised from insoluble corncob xylan by reSyXyn11P.     

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-β-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 °C and remained stable at weakly acidic to alkaline pH (pH 5.0–11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 °C for 1 h and more than 40% of the activity at 90 °C for 15 min. The K _m and V _max values for oat spelt xylan and birchwood xylan were 16.08 mg ml^?1 and 45.66 μmol min^?1 mg^?1 and 9.22 mg ml^?1 and 16.05 μmol min^?1 mg^?1, respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.     

Production and Accumulation of 4-<Emphasis Type="Italic">O</Emphasis>-Methyl-α-<Emphasis Type="SmallCaps">d</Emphasis>-Glucuronosyl-Xylotriose by Growing Culture of Thermophilic <Emphasis Type="Italic">Anoxybacillus</Emphasis> sp. Strain JT-12

A thermophilic Anoxybacillus sp. strain JT-12, isolated from soil, produced acidic xylotriose, 4-O-methyl-α-d-glucuronosyl-xylotriose (MeGlcAX₃), as a main product from birchwood xylan and accumulated them in the culture under optimum conditions at pH 7.0 and 55 °C using 0.75% (w/v) birchwood xylan as a carbon source for 42–72 h. The acidic xylotriose was purified by ethanol precipitation and high-performance liquid chromatography using NH₂ Lichosher® 100 column. The results of electrospray ionization mass spectrometry, mass to charge ratio (m/z) 603.23, confirmed that the purified sample was acidic xylotriose that had benefits and applications in many fields.     

Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from <Emphasis Type="Italic">Hypocrea orientalis</Emphasis>

A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7–22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19–29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K _m and V _max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.     

A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization

A newly isolated bacterial strain, Bacillus sp. MX47, was actively producing extracellular xylanase only in xylan-containing medium. The xylanase was purified from the culture broth by two chromatographic steps. The xylanase had an apparent molecular weight of 26.4?kDa with an NH₂-terminal sequence (Gln-Gly-Gly-Asn-Phe) distinct from that of reported proteins, implying it is a novel enzyme. The optimum pH and temperature for xylanase activity were 8.0 and 40?°C, respectively. The enzyme activity was severely inhibited by many divalent metal ions and EDTA at 5?mM. The xylanase was highly specific to beechwood and oat spelt xylan, however, not active on carboxymethyl cellulose (CMC), avicel, pectin, and starch. Analysis of the xylan hydrolysis products by Bacillus sp. MX47 xylanase indicated that it is an endo-??-1,4-xylanase. It hydrolyzed xylan to xylobiose as the end product. The K _m and V _max values toward beechwood xylan were 3.24?mg?ml^?1 and 58.21???mol?min^?1?mg^?1 protein, respectively.     

Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant,Thermostable β-Xylosidase from a Tropical Strain of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> CBS 135684

From three cell-associated β-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60–70 °C) over a pH range of 5–9 with a specific activity of 163.3 units (U) mg^?1. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and 70 °C, respectively. Moreover, it was tolerant to xylose inhibition with a K _i value of 18 mM. The K _m and V _max values against p-nitrophenyl-β-d-xylopyranoside were 5.57 ± 0.27 mM and 137.0 ± 4.8 μmol min^?1 mg^?1 protein, respectively. When combining this β-xylosidase with xylanase from the same A. pullulans strain, the rate of black liquor xylan hydrolysis was significantly improved by up to 1.6-fold. The maximum xylose yield (0.812 ± 0.015 g g^?1 dry weight) was obtained from a reaction mixture containing 10% (w/v) black liquor xylan, 6 U g^?1 β-xylosidase and 16 U g^?1 xylanase after incubation for 4 h at 70 °C and pH 6.0.     

Improvement of Strain Penicillium sp. EZ-ZH190 for Tannase Production by Induced Mutation

In the search for an efficient producer of tannase, Penicillium sp. EZ-ZH190 was subjected to mutagenesis using heat treatment and strain EZ-ZH290 was isolated. The maximum tannase in this mutant strain was 4.32 U/mL with an incubation period of 84 h as compared to wild strain EZ-ZH190 where the incubation period was 96 h with a maximum enzyme activity of 4.33 U/mL. Also, the Penicillium sp. EZ-ZH290 tannase had a maximum activity at 40 °C and pH 5.5. Then, the spores of strain EZ-ZH290 were subjected to γ irradiation mutagenesis and strain EZ-ZH390 was isolated. Strain EZ-ZH390 exhibited higher tannase activity (7.66 U/mL) than the parent strain EZ-ZH290. It was also found that Penicillium sp. EZ-ZH390 tannase had an optimum activity at 35 °C and a broad pH profile with an optimum at pH 5.5. The tannase pH stability of Penicillium sp. EZ-ZH390 and its maximum production of tannase followed the same trend for five generations confirming the occurrence of stable mutant. This paper is shown that γ irradiation can mutate the Penicillium sp. leading to increase the tannase production.     

Purification and Properties of a Psychrotrophic <Emphasis Type="Italic">Trichoderma</Emphasis> sp. Xylanase and its Gene Sequence

A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5–9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K _m values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce xylobiose, but it hardly hydrolysed xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.     

Purification and properties of a SDS-resistant xylanase from halophilic Streptomonospora sp. YIM 90494

An extracellular xylanase from halophilic Streptomonospora sp. YIM 90494 was purified to homogeneity from a fermentation broth by ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 50 kDa. The xylanase had maximum activity at pH 7.5 and 55 °C. The enzyme was stable over a broad pH range (pH 4.0–10.0) and showed good thermal stability when being incubated at 60 °C for 2 h. Kinetic experiments indicated that the enzyme had K _m and V _max values of 19.24 mg/mL and 6.1 μmol/min/mg, respectively, using birch wood xylan as substrate. The inhibitory effects of various metal ions and chemical agents on the xylanase activity were investigated. It is greatly interesting to note that Ag⁺ ion and SDS, which strongly inhibited most xylanases reported previously increases the xylanase activity in this study. These characteristics suggest that the enzyme with new properties has considerable potential in industrial applications.     

A Highly Thermostable Alkaline Cellulase-Free Xylanase from Thermoalkalophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. JB 99 Suitable for Paper and Pulp Industry: Purification and Characterization

A highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99 using DEAE-Sepharose and Sephadex G-100 gel filtration with 25.7-fold increase in activity and 43.5% recovery. The molecular weight of the purified xylanase was found to be 20 kDA by SDS-PAGE and zymogram analysis. The enzyme was optimally active at 70 °C, pH 8.0 and stable over pH range of 6.0–10.0.The relative activity at 9.0 and 10.0 were 90% and 85% of that of pH 8.0, respectively. The enzyme showed high thermal stability at 60 °C with 95% of its activity after 5 h. The K _m and V _max of enzyme for oat spelt xylan were 4.8 mg/ml and 218.6 μM min⁻¹ mg⁻¹, respectively. Analysis of N-terminal amino acid sequence revealed that the xylanase belongs to glycosyl hydrolase family 11 from thermoalkalophilic Bacillus sp. with basic pI. Substrate specificity showed a high activity on xylan-containing substrate and cellulase-free nature. The hydrolyzed product pattern of oat spelt xylan on thin-layer chromatography suggested xylanase as an endoxylanase. Due to these properties, xylanase from Bacillus sp. JB 99 was found to be highly compatible for paper and pulp industry.     

Improvement of Highly Thermostable Xylanases Production by Talaromyces thermophilus for the Agro-industrials Residue Hydrolysis

A newly isolated thermophilic fungal strain from Tunisian soil samples was identified as Talaromyces thermophilus and was selected for its ability to produce extracellular hemicellulases when grown on various lignocellulosic substrates. Following the optimization of carbon source, nitrogen source, and initial pH of the growth medium in submerged liquid cultures, yields as high as 10.00?±?0.15 and 0.21?±?0.02 U/ml were obtained for xylanase and β-xylosidase, respectively. In fact, wheat bran was found to be a good inducer of hemicellulase enzymes, mainly β-xylosidase. The optimal temperature and pH of the xylanase activity were 75°C and 8.0, respectively. This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for 7 days at pH?7.0–8.0. The half-lives of the enzyme were 4 h at 80°C, 2 h at 90°C, and 1 h at 100°C. T. thermophilus could therefore be considered as a satisfactory and promising producer of thermostable xylanases. Crude enzyme of T. thermophilus rich in xylanase and β-xylosidase was established for the hydrolysis of lignocellulosic materials as wheat bran.     

Waste Biogas Residue from Cassava Dregs as Carbon Source to Produce Galactomyces sp. Cczu11-1 Cellulase and its Enzymatic Saccharification

In the conversion of cassava starch dregs to biogas by anaerobic fermentation, the biogas residue (BR) containing lignocellulosic materials still remained in the environment. In order to effectively utilize BR, the complexed 1-methyl-3-methylimidazolium dimethyl phosphate ([Mmim]DMP) media were used for pretreating cellulosic materials. After the optimization of pretreatment, the IL [Mmim]DMP-HCl-water (78.5:1.5:20, w/w/w) pretreament media were used for pretreating BR at 130 °C for 30 min. Furthermore, BR pretreated could be effectively saccharified by cellulase of Galactomyces sp. CCZU11-1. Moreover, BR could be used as a cheap carbon source for the production of Galactomyces sp. CCZU11-1 cellulase. After the culture optimization, the optimal culture conditions were obtained as follows: BR 5 g/L, (NH₄)₂SO₄ 5 g/L, K₂HPO₄ 2 g/L, MgSO₄ 0.2 g/L, NaCl 1 g/L, PEG6000 4 g/L, pH 5.5, and culture temperature 30 °C. After the fermentation for 6 days, the FPA and CMCase were 26.2 and 52.8 U/mL, respectively. In conclusion, waste BR could be chosen as a promising feedstock for biofuels.     

Expression of Aspergillus niger IA-001 Endo-β-1,4-Xylanase in Pichia pastoris and Analysis of the Enzymic Characterization

The xylanaseB (XynB) (JX560731.1) gene of Aspergillus niger IA-001 was optimized according to the codon usage of Pichia pastoris and expressed in P. pastoris GS115. The optimized XynB expression level was increased 2.8 times relative to that of the wild-type XynB, and the dual-copy XynB (optimized) expression level was increased 1.9 times relative to that of the single-copy XynB (optimized). The activity of the dual-copy XynB ((XynB-opt)₂) was maximized at 15,158.23?±?45.11 U/mL after 120 h of shaking. The optimal temperature and pH of (XynB-opt)₂ were 50 °C and 5.0, respectively. (XynB-opt)₂ showed a high specific activity of 6,853.00?±?20.08 U/mg. IC analysis of the standard xylooligosaccharides showed that (XynB-opt)₂ was an endo-xylanase with X2 as the main degradation product. (XynB-opt)₂ was highly specific towards different natural xylans. After 24 h of hydrolysis, more than 90 % of the total hydrolysis products of xylan were X2 and X1, almost no X4?~?X6. In addition, the enzyme exhibited resistance to many metal ions and low pH values. The superior catalytic properties of (XynB-opt)₂ suggested its great potential as an effective additive in animal feed industry.