Characterization of Novel EGs Reconstructed from Bacillus subtilis Endoglucanase

Bacterial cellulases have taken on satisfactory application performance and economic value in detergent industry. Neutral endoglucanase (EG1) gene was cloned from Bacillus subtilis and expressed Pichia pastoris in our previous study. Redesigned endoglucanases enhanced cellulase domain, added and deleted carbohydrate-binding module (CBM), named EG2, EG3, and EG4, respectively, were constructed in this study. The redesigned EG genes were expressed in P. pastoris, and their characters were also discussed. The optimal temperature and pH value of the all EGs was 65 °C and 6.0, respectively, where their enzymatic activities in P. pastoris cultivation supernatant reached 867, 651, 966, and 881 U/mL. EG2 showed 24.9 % enzymatic activity loss compared to natural endoglucanase. EG4 showed specific activity 30 % loss and thermostability decrease compared to EG1, which suggested CBM played an important role in improving the catalytic power and heat stability of cellulase family which attached. The specific activity of EG2 and EG3 showed similar to EG1, which suggested neither enhancement of CD nor CBM to endoglucanase can improve its catalytic power, which might rest with its intact topologic structure.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

Production of Xylooligosaccharides by Immobilized His-tagged Recombinant Xylanase from Penicillium occitanis on Nickel-Chelate Eupergit C

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.     

Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production,Characterization, and Thermal Stability

A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g^?1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g^?1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K _M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.     

Stability and recovery ofPenicillium funiculosum cellulase under use conditions

The stability ofPenicillium funiculosum cellulase has been investigated under the conditions used for cellulose hydrolysis. Fifty five percent of filter paper activity (FPA) was inactivated on incubation at 50°C for 24 h, whereas there was no loss in endoglucanase and β-glucosidase activity. The addition of 2% polyethylene glycol (PEG) during incubation stabilized the FPA. The influence of pH during fermentation on the thermal stability of the enzyme is discussed. The recovery of enzymes after hydrolysis of bagasse at 50°C was between 8 and 14%. Under the optimal conditions of elution, the recovery of enzyme was 35% (1). Increasing the enzyme to the substrate ratio fivefold and presence of PEG during hydrolysis resulted in 80, 83, and 95% recovery of β-glucosidase, FPA, and endoglucanase activity, respectively. Index Entries: Stability; recovery of cellulase P.funiculosum.     

A Recombinant Highly Thermostable β-Mannanase (ReTMan26) from Thermophilic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (TBS2) Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and Its pH and Temperature Stability

A gene encoding a highly thermostable β-mannanase from a thermophilic Bacillus subtilis (TBS2) was successfully expressed in Pichia pastoris. The maximum activity of the recombinant thermostable β-mannanase (ReTMan26) was 5435 U/mL, which was obtained by high-density, fed-batch cultivation after 168-h induction with methanol in a 50-L bioreactor. The protein yield reached 3.29 mg/mL, and the protein had a molecular weight of ~42 kDa. After fermentation, ReTMan26 was purified using a 10-kDa cut-off membrane and Sephadex G-75 column. The pH and temperature optima of purified ReTMan26 were pH 6.0 and 60 °C, respectively, and the enzyme was stable at pH 2.0–8.0 and was active at 20–100 °C. HPLC analysis of the products of locust bean gum hydrolysis showed that the mannan-oligosaccharide content was 62.5%. ReTMan26 retained 58.6% of its maximum activity after treatment at 100 °C for 10 min, which was higher than any other β-mannanase reported up to now, suggesting its potential for industrial applications.     

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris αMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4–7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.     

Cloning, Expression, and Characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris

The β-mannanase gene (1,029 nucleotide) from Bacillus subtilis MAFIC-S11, encoding a polypeptide of 342 amino acids, was cloned and expressed in Pichia pastoris. To increase its expression, the β-mannanase gene was optimized for codon usage (mannS) and fused downstream to a sequence-encoding modified α-factor signal peptide. The expression level was improved by 2-fold. This recombinant enzyme (mannS) showed its highest activity of 24,600 U/mL after 144-h fermentation. The optimal temperature and pH of mannS were 50 °C and 6.0, respectively, and its specific activity was 3,706 U/mg. The kinetic parameters V _max and K _m were determined as 20,000 U/mg and 8 mg/mL, respectively, representing the highest ever expression level of β-mannanase reported in P. pastoris. In addition, the enzyme exhibited much higher binding activity to chitin, chitosan, Avicel, and mannan. The superior catalytic properties of mannS suggested great potential as an effective additive in animal feed industry.     

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.     

Production and Optimization of Physicochemical Parameters of Cellulase Using Untreated Orange Waste by Newly Isolated <Emphasis Type="Italic">Emericella variecolor</Emphasis> NS3

Cellulase enzymes have versatile industrial applications. This study was directed towards the isolation, production, and characterization of cellulase enzyme system. Among the five isolated fungal cultures, Emericella variecolor NS3 showed maximum cellulase production using untreated orange peel waste as substrate using solid-state fermentation (SSF). Maximum enzyme production of 31 IU/gds (per gram of dry substrate) was noticed at 6.0 g concentration of orange peel. Further, 50 °C was recorded as the optimum temperature for cellulase activity and the thermal stability for 240 min was observed at this temperature. In addition, the crude enzyme was stable at pH 5.0 and held its complete relative activity in presence of Mn²⁺ and Fe³⁺. This study explored the production of crude enzyme system using biological waste with future potential for research and industrial applications.     

Production and Characterization of a Novel Yeast Extracellular Invertase Activity Towards Improved Dibenzothiophene Biodesulfurization

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml^?1) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5–6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5–6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l^?1 sucrose and 250 μM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l^?1 sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its μ_max from 0.035 to 0.070 h^?1 and attained a 2-hydroxybiphenyl productivity of 5.80 μM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.     

Response Surface Methodology Based Optimization of β-Glucosidase Production from Pichia pastoris

The thermotolerant yeast Pichia etchellsii produces multiple cell bound β-glucosidases that can be used for synthesis of important alkyl- and aryl-glucosides. Present work focuses on enhancement of β-glucosidase I (BGLI) production in Pichia pastoris. In the first step, one-factor-at-a-time experimentation was used to investigate the effect of aeration, antifoam addition, casamino acid addition, medium pH, methanol concentration, and mixed feed components on BGLI production. Among these, initial medium pH, methanol concentration, and mixed feed in the induction phase were found to affect BGLI production. A 3.3-fold improvement in β-glucosidase expression was obtained at pH 7.5 as compared to pH 6.0 on induction with 1 % methanol. Addition of sorbitol, a non-repressing substrate, led to further enhancement in β-glucosidase production by 1.4-fold at pH 7.5. These factors were optimized with response surface methodology using Box–Behnken design. Empirical model obtained was used to define the optimum “operating space” for fermentation which was a pH of 7.5, methanol concentration of 1.29 %, and sorbitol concentration of 1.28 %. Interaction of pH and sorbitol had maximum effect leading to the production of 4,400 IU/L. The conditions were validated in a 3-L bioreactor with accumulation of 88 g/L biomass and 2,560 IU/L β-glucosidase activity.     

A Xylanase Gene Directly Cloned from the Genomic DNA of Alkaline Wastewater Sludge Showing Application Potential in the Paper Industry

A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.     

Production and Partial Characterization of Cellulases and Xylanases from Trichoderma atroviride 676 Using Lignocellulosic Residual Biomass

Trichoderma atroviride 676 was studied to evaluate its efficiency in the production of some lignocellulolytic enzymes, using lignocellulosic residual biomass. Best results were obtained when 3.0 % (w/v) untreated sugarcane bagasse was used (61.3 U mL^?1 for xylanase, 1.9 U mL^?1 for endoglucanase, 0.25 U mL^?1 for FPase, and 0.17 U mL^?1 for β-glucosidase) after 3–4 days fermentation. The maximal enzymatic activity for endoglucanase, FPase, and xylanase were observed at 50–60 °C and pH?4.0–5.0, whereas thermal stability at 50 °C (CMCase and FPase) or 40 °C (xylanase) was obtained after 8 h. Zymograms have shown two bands of 104 and 200 kDa for endoglucanases and three bands for xylanase (23, 36, and 55.7 kDa). The results obtained with T. atroviride strain 676 were comparable to those obtained with the cellulolytic strain Trichoderma reesei RUT-C30, indicating, in the studied conditions, its great potential for biotechnological application, especially lignocellulose biomass hydrolysis.     

Aspergillus fumigatus Thermophilic and Acidophilic Endoglucanases

This study evaluated the production of cellulolytic enzymes by an Aspergillus fumigatus strain, isolated from sugar cane bagasse, according to its ability to grow on microcrystalline cellulose as the sole carbon source. The effect of the carbon source (brewer’s spent grain, sugarcane bagasse, and wheat bran) and of the nitrogen source (corn steep liquor and sodium nitrate) on cellulase production was studied using submerged and solid state cultivations at 30 °C. The highest levels of endoglucanase (CMCase) corresponded to 365 U L^-1 and was obtained using sugarcane bagasse (1%) and corn steep liquor (1.2%) in submerged fermentation within 6 days of cultivation. This supernatant was used to run a sodium dodecyl sulfate polyacrylamide gel electrophoresis that showed six bands with endoglucanase activity. CMCase activity was higher at 65 °C and pH 2.0, indicating that this microorganism produces a thermophilic and acid endoglucanase. Solid state cultivation favored FPase production, that reached 47 U g^-1 of dry substrate (wheat bran and sugarcane bagasse) within 3 days.     

Production of Extremely Alkaliphilic, Halotolerent, Detergent, and Thermostable Mannanase by the Free and Immobilized Cells of Bacillus halodurans PPKS-2. Purification and Characterization

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0–16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.     

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.     

Expression and Characterization of Recombinant GH11 Xylanase from Thermotolerant Streptomyces sp. SWU10

Xylans are major hemicellulose components of plant cell wall which can be hydrolyzed by xylanolytic enzymes. Three forms of endo-β-1,4-xylanases (XynSW1, XynSW2A, and XynSW2B) produced by thermotolerant Streptomyces sp. SWU10 have been reported. In the present study, we described the expression and characterization of the fourth xylanase enzyme from this bacteria, termed XynSW3. The gene containing 726 bp was cloned and expressed in Escherichia coli. The recombinant enzyme (rXynSW3) was purified from cell-free extract to homogeneity using Ni-affinity column chromatography. The apparent molecular mass of rXynSW3 was 48 kDa. Amino acid sequence analysis revealed that it belonged to a xylanase of glycoside hydrolase family 11. The optimum pH and temperature for enzyme activity were 5.5–6.5 and 50 °C, respectively. The enzyme was stable up to 40 °C and in wide pH ranges (pH 0.6–10.3). Xylan without arabinosyl side chain is the most preferable substrate for the enzyme. By using birch wood xylan as substrate, rXynSW3 produced several oligosaccharides in the initial stage of hydrolysis, and their levels increased with time, demonstrating that the enzyme is an endo-acting enzyme. The major products were xylobiose, triose, and tetraose. The rXynSW3 can be applied in several industries such as food, textile, and biofuel industries, and waste treatment.     

Purification, Sequencing, and Biochemical Characterization of a Novel Calcium-Independent α-Amylase AmyTVE from Thermoactinomyces vulgaris

α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS–PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl₂, and CaCl₂, and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl₂, suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.     

Molecular and Kinetic Characterization of Two Extracellular Xylanases Isolated from Leucoagaricus gongylophorus

In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 °C, optimum pH between 8.5 and 10.5, and Km?=?14.7?±?7.6 mg mL^?1. Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 °C, and Km?=?2.2?±?0.5 mg mL^?1. XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 °C) and pH (4.0), as well as the kinetic parameters (Km?=?7.4?±?2.0 mg mL^?1) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.