Highly Thermostable Xylanase of the Thermophilic Fungus Talaromyces thermophilus: Purification and Characterization

A thermostable xylanase from a newly isolated thermophilic fungus Talaromyces thermophilus was purified and characterized. The enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl cellulose anion exchange chromatography, P-100 gel filtration, and Mono Q chromatography with a 23-fold increase in specific activity and 17.5% recovery. The molecular weight of the xylanase was estimated to be 25kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and gel filtration. The enzyme was highly active over a wide range of pH from 4.0 to 10.0. The relative activities at pH5.0, 9.0, and 10.0 were about 80%, 85.0%, and 60% of that at pH7.5, respectively. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 50°C (7days) and the half-life of the xylanase at 100°C was 60min. The enzyme was free from cellulase activity. K _m and V _max values at 50°C of the purified enzyme for birchwood xylan were 22.51mg/ml and 1.235μmol min⁻¹ mg⁻¹, respectively. The enzyme was activated by Ag⁺, Co²⁺, and Cu²⁺; on the other hand, Hg²⁺, Ba²⁺, and Mn²⁺ inhibited the enzyme. The present study is among the first works to examine and describe a secreted, cellulase-free, and highly thermostable xylanase from the T. thermophilus fungus whose application as a pre-bleaching aid is of apparent importance for pulp and paper industries.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Immobilization of cellulase in nanofibrous PVA membranes by electrospinning

Electrospinning is a nanofiber-forming process by which either polymer solution or melt is charged to high voltages. With high specific surface area and porous structure, electrospun fibrous membranes are excellent candidates for immobilization of enzymes. In this paper, immobilization of cellulase in nanofibrous poly(vinyl alcohol) (PVA) membranes was studied by electrospinning. PVA and cellulase were dissolved together in an acetic acid buffer (pH 4.6) and electrospun into nanofibers with diameter of around 200 nm. The nanofibrous membranes were crosslinked by glutaraldehyde vapor and examined catalytic efficiency for biotransformations. The activity of immobilized cellulase in PVA nanofibers was over 65% of that of the free enzyme. Nanofibers were superior to casting films from the same solution for immobilization of cellulase. The activity of immobilized cellulase descended with ascending in enzyme loading efficiency and crosslinking time, which retained 36% its initial activity after six cycles of reuse.     

Selection of Conditions for Cellulase and Xylanase Extraction from Switchgrass Colonized by <Emphasis Type="Italic">Acidothermus cellulolyticus</Emphasis>

Solid-state fermentation has been widely used for enzyme production. However, secreted enzymes often bind to the solid substrate preventing their detection and recovery. A series of screening studies was performed to examine the role of extraction buffer composition including NaCl, ethylene glycol, sodium acetate buffer, and Tween 80, on xylanase and cellulase recovery from switchgrass. Our results indicated that the selection of an extraction buffer is highly dependent on the nature and source of the enzyme being extracted. While a buffer containing 50 mM sodium acetate at pH 5 was found to have a positive effect on the recovery of commercial fungal-derived cellulase and xylanase amended to switchgrass, the same buffer had a significant negative effect on enzyme extraction from solid fermentation samples colonized by the bacterium Acidothermus cellulolyticus. Xylanase activity was more affected by components in the extraction buffers compared to cellulase. This study demonstrated that extraction followed by diafiltration is important for assessing enzyme recovery from solid fermentation samples. Reduction in activity due to compounds present in the switchgrass extracts is reversible when the compounds are removed via diafiltration.     

Xylanase recovery by ethanol and Na2SO4 precipitation

Xylans are the major components of the hemicellulosic fraction of lignocellulosic biomass and their hydrolysis can be obtained using xylanases fromPenicillium janthinellum. In this work, sugarcane bagasse hemicellulosic hydrolysate was used as the substrate for producing xylanase. The precipitation of these enzymes was studied using ethanol and Na₂SO₄ as precipitating agents. Ethanol precipitation experiments were performed batchwise in concentrations ranging from 10 to 80%, pH 4.0 to 7.0, at 4áC. The concentrations used in the precipitations with Na₂SO₄ were from 5 to 60% at pH 5.5 and 25áC. Solubility curves as a function of xylanase activity and total protein for both precipitating agents were made. According to the results, Na₂SO₄ is not appropriate for precipitating xylanases in this medium since at salt concentrations higher than 25%, the enzyme was denaturated and at this concentration less than 80% of the enzyme and total protein were precipitated. Because of differences in xylanase and total protein solubility, a fractionated precipitation using ethanol can be performed, since with 40% ethanol, 49% of the total protein was precipitated and more than 95% of the enzyme was kept in solution. On the other hand approx 100% of the xylanases were recovered by precipitation after adding 80% ethanol.     

Methods and Supports for Immobilization and Stabilization of Cyclomaltodextrin Glucanotransferase from Thermoanaerobacter

Thermoanaerobacter cyclomaltodextrin glucanotransferase (CGTase) was immobilized using different supports and immobilization methods to study the effect on activity recovery. The enzyme covalently attached into glyoxyl-silica showed low activity recovery of 1.5%. The hydrophobic adsorption of the enzyme on Octadecyl-Sepabeads yielded also low activity recovery, 3.83%, and the enzyme could easily leak from the support at low ionic strength, although the immobilization yield was satisfactory, approximately 76%. The CGTase encapsulated in a sol–gel matrix gave an activity recovery of 6.94% and maximum cyclization activity at 60 °C, at pH 6.0. The half-time life at 60 °C, pH 6.0, in the presence of substrate was 100 min, which was lower than that of the free enzyme. The best activity recovery in this work (6.94%) is approximately five times smaller than that obtained previously using glyoxyl-agarose as support and covalent immobilization. Thus, the best support and method we tested so far for immobilization of CGTase is covalent attachment on glyoxyl-agarose.     

Characterization of a Thermotolerant and Alkalotolerant Xylanase from a Bacillus sp.

In a recent screening for thermophilic bacteria from Azores hot springs, a Bacillus sp strain 3M, exhibiting cellulase-free extracellular xylanolitic activity, was isolated. Further enzyme characterization from liquid cultures grown on birchwood xylan revealed that the endo-l,4-βxylanase retains 100% of activity for at least 3 d at 55°C. At 80°C, it retains 47% of its maximal activity, and the enzyme is still active at 90°C. The optimum pH of the enzyme has a broad pH range, between 6.0 and 7.5, and it is remarkably active for the alkaline region, exhibiting 89% of relative activity at pH 9.O. The enzyme was partially inactivated by different divalent metal ions. Because of its tolerance for high temperature and pH conditions, and the absence of contaminating cellulase activity, the xylanase produced byBacillus sp 3M appears to be attractive for use in the pulp and paper industry. Indeed, the efficiency of the enzyme application to the kraftEucalyptus pulp was studied for bleaching pretreatment, resulting in a moderate increase of pulp bleachability.     

Cellulolytic enzymes produced by the edible mushroom,Pleurotus sajor-caju

Pleurotus sajor-caju grows efficiently and degrades all the components present in lignocellulosic residues. Production of cellulase and xylanase enzymes in submerged culture and during solid state cultivation has been studied. An initial pH of 5.0 was found to be optimal for the production of cellulase in shake flasks; this was attained in about 6–8 d in a medium containing either cellulose or rice straw as the sole source of carbon. On the cellulose medium, the maximum filter paper activity attained was 0.15 IU/mL in 7 d whereas the endoglycanase activity of 1.0 IU/mL, xylanase activity of 1.55 IU/mL, and Β-glucosidase activity of 0.57 IU/mL were acheived after 9 d fermentation. The reducing sugars were absent in the culture medium. The cellulases (filter paper activity and endoglucanases) were most active at pH 5.0 and 45‡C. Xylanase had maximum activity at pH 4.8 and 45‡C, and Β-glucosidase at pH 5.5 and 40‡C. In shake cultures,P. sajor-caju produced dispersed suspension of short mycelial threads and various sizes of pellets. The profile and extent of enzyme biosynthesis during submerged cultivation on rice straw was found to be of the same nature as obtained on cellulose. During solid state cultivation ofP. sajor-caju on rice straw beds for 36 d, the elaboration of enzyme activities did not appear to follow any definite pattern. However, filter paper activity, which is representative of cellulase action in hydrolyzing cellulose, remained more or less constant during the period of about the first 20 d of cultivation after the appearance of fruit bodies on the surface of rice straw beds. All the activities attained their minimum values after 23 d of cultivation, during which approximately 1 kg of fresh fruit bodies had been harvested. The total fruit bodies harvested till 36th days were approx. 1.1 kg. ThroughT. sajor-caju elaborates cellulase and xylanse extracellularly, the activity values were not as high as those of other cellulase producers such asTrichoderma reesei.     

Immobilization of <Emphasis Type="Italic">Aspergillus niger</Emphasis> Xylanase on Chitosan Using Dialdehyde Starch as a Coupling Agent

Dialdehyde starch (DAS) was used as a novel coupling agent to prepare chitosan carrier to immobilize the xylanase from Aspergillus niger A-25. Compared with glutaraldehyde-cross-linked chitosan (CS-GA) and pure chitosan beads, the DAS-cross-linked chitosan (CS-DAS) beads exhibited the highest xylanase activity recovery. The DAS adding amount and cross-linking time in CS-DAS preparation process were optimized with respect to activity recovery to the values of 1.0 g (6.7% w/v concentration) and 16 h, respectively. The optimum temperature of both the CS-DAS- and CS-GA-immobilized xylanase was observed to be 5 °C higher than that of free enzyme (50 °C). The CS-DAS-immobilized xylanase had the highest thermal and storage stability as compared to the CS-GA-immobilized and free xylanase. The apparent K _m and V _max values of the CS-DAS-immobilized xylanase were estimated to be 1.29 mg/ml and 300.7 μmol/min/mg protein, respectively. The CS-DAS-immobilized xylanase could produce from birchwood xylan high-quality xylo-oligosaccharides, mainly composed of xylotriose, as free xylanase did. The proposed CS-DAS carrier was more advantageous over the CS-GA or pure chitosan carrier for xylanase immobilization application.     

Catalytic Performance of Corn Stover Hydrolysis by a New Isolate <Emphasis Type="Italic">Penicillium</Emphasis> sp. ECU0913 Producing both Cellulase and Xylanase

A fungal strain, marked as ECU0913, producing high activities of both cellulase and xylanase was newly isolated from soil sample collected near decaying straw and identified as Penicillium sp. based on internal transcribed spacer sequence homology. The cultivation of this fungus produced both cellulase (2.40 FPU/ml) and xylanase (241 IU/ml) on a stepwisely optimized medium at 30 °C for 144 h. The cellulase and xylanase from Penicillium sp. ECU0913 was stable at an ambient temperature with half-lives of 28 and 12 days, respectively. Addition of 3 M sorbitol greatly improved the thermostability of the two enzymes, with half-lives increased by 2.3 and 188-folds, respectively. Catalytic performance of the Penicillium cellulase and xylanase was evaluated by the hydrolysis of corn stover pretreated by steam explosion. With an enzyme dosage of 50 FPU/g dry substrate, the conversions of cellulose and hemicellulose reached 77.2% and 47.5%, respectively, without adding any accessory enzyme.     

Production of cellulolytic and hemicellulolytic enzymes from Aureobasidium pulluans on solid state fermentation

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL beta-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and beta-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75 degrees C, respectively. These enzymes remained stable between a wide range of pH. The beta-glucosidase was the most thermostable enzyme, remaining 100% active when incubated at 75 degrees C for 1 h.     

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0?±?0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe₃O₄@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

     

Covalent Immobilization of Cellulases onto a Water-Soluble–Insoluble Reversible Polymer

The covalent immobilization of a commercial preparation of cellulase on a reversibly soluble–insoluble enteric polymer Eudragit S-100 by carbodiimide coupling was carried out. The characteristics of covalent Eudragit cellulase were evaluated using Fourier transform infrared (FTIR) spectra, circular dichroism (CD) spectra, and fluorescence spectra. FTIR, CD, and fluorescence measurements also revealed that the cellulases were covalently bonded to the supports. Covalent Eudragit cellulase had binding efficiency of 81.08% which was higher than the noncovalent Eudragit cellulase 56.83%. The relative activity of the native cellulase and covalent Eudragit cellulase increased and reached the maximum (at pH 5.0, 50°C) and then decreased with further increases in pH and temperature. The covalent Eudragit cellulase shows higher stability especially at higher pH and temperature. The K _m value of covalent Eudragit cellulase (4.78 g·L⁻¹) was decreased compared to that of the native cellulase (2.89 g·L⁻¹). The affinity of the cellulase to its substrate was increased when it was immobilized on Eudragit S-100.     

Isolation and properties of a thermostable endoglucanase from a thermophilic mutant strain ofTielavia terrestris

A heat-stable enzyme was isolated from the cellulase complex of a thermophilic strain of the micromyceteThielavia terrestris. The purified enzyme exhibited both endoglucanase and xylanase activities and had a mol mass of 69,000 Daltons and an isoelectric point of 6.4. When the cells were grown at 48°C, the initial activity of the purified enzyme using carboxymethylcellulose as a substrate was 150 nkat/mg and the Michaelis constant was 6.6 g/L. The heat stability of the enzyme was high, losing only 20% of the initial activity after a 6-h incubation at 65 °C. When cultures were grown on microcrystalline cellulose and xylose was added after 48 h of growth, endoglucanase and xylanase activities were more than doubled. Similar increases in these activities were observed by growing the cultures on straw.     

Facile route to enzyme immobilization: core-shell nanoenzyme particles consisting of well-defined poly(methyl methacrylate) cores and cellulase shells

A one-step method for preparing cellulase-immobilized nanoparticles that consist of well-defined poly(methyl methacrylate) (PMMA) cores and cellulase shells has been developed. The core-shell nanoparticles are synthesized from a direct graft copolymerization of methyl methacrylate (MMA) from cellulase in an aqueous medium. Particle formation strongly depends on the surface nature of the cellulase (e.g., pH of reaction media) and MMA to cellulase weight ratio. Under optimized conditions, high MMA conversions (>90%) were achieved, and the PMMA-cellulase nanoparticles produced were very stable with narrow size distributions ( Dv/Dn < 1.20). Particle sizes in the range between 80 and 124 nm (volume average diameter) could be tailored by a variation of cellulase concentration. Transmission electron microscopy micrographs revealed that the nanoparticle had a well-defined PMMA core which was evenly coated with cellulase shell. Study of cellulase activity of the PMMA-cellulase nanoparticles indicated that even though activity of immobilized cellulase on the nanoparticles was 41% less than that of the native cellulase after the polymerization, the immobilized cellulase showed improved properties such as broader working pH range and better thermal stability. Other important advantages of this approach include that the PMMA-cellulase nanoparticles could be produced in high concentrations (up to 18% w/w solids content) and the nanoparticles have thick and evenly distributed enzyme shells. Thus, this method may provide a new commercially viable route to the immobilization of thermally stable enzyme to form nanoenzyme particles.     

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.     

Immobilization and Stabilization of Xylanase by Multipoint Covalent Attachment on Agarose and on Chitosan Supports

Xylanases have important applications in industry. Immobilization and stabilization of enzymes may allow their reuse in many cycles of the reaction, decreasing the process costs. This work proposes the use of a rational approach to obtain immobilized commercial xylanase biocatalysts with optimized features. Xylanase NS50014 from Novozymes was characterized and immobilized on glyoxyl-agarose, agarose-glutaraldehyde, and agarose-amino-epoxy support and on differently activated chitosan supports: glutaraldehyde-chitosan, glyoxyl-chitosan, and epoxy-chitosan. Two different chitosan matrices were tested. The best chitosan derivative was epoxy-chitosan-xylanase, which presented 100% of immobilization yield and 64% of recovered activity. No significant increase on the thermal stability was observed for all the chitosan-enzyme derivatives. Immobilization on glyoxyl-agarose showed low yield immobilization and stabilization degrees of the obtained derivative. The low concentration of lysine groups in the enzyme molecule could explain these poor results. The protein was then chemically modified with ethylenediamine and immobilized on glyoxyl-agarose. The new enzyme derivatives were 40-fold more stable than the soluble, aminated, and dialyzed enzyme (70 °C, pH 7), with 100% of immobilization yield. Therefore, the increase of the number of amine groups in the enzyme surface was confirmed to be a good strategy to improve the properties of immobilized xylanase.     

Immobilization of Urease on (HEMA/IA) Hydrogel Prepared by Gamma Radiation

In the present study, the copolymeric hydrogels based on 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA) were synthesized by gamma radiation induced radical polymerization, in order to examine the potential use of these hydrogels in immobilization of Citrullus vulgaris urease. Gelation and Swelling properties of PHEMA and copolymeric P (HEMA/IA) hydrogels with different IA contents (96.5/3.5, 94.4/5.6 and 92.5/7.5 mol) were studied in a wide pH range. Initial studies of so-prepared hydrogels show interesting pH sensitivity in swelling and immobilization. C. vulgaris urease was immobilized on HEMA/IA (92.5/7.5) at 6 kGy with 41.3% retention of activity. The properties of free and immobilized urease were compared. Immobilized urease maintained a higher relative activity than free urease at both lower and higher pH levels, indicating that the immobilized urease was less sensitive to pH changes than the free urease. The K_m value of the immobilized urease was approximately 2 times higher than that of the free urease. Temperature stability was improved for immobilized enzyme. The free form exhibited a loss about 80% of activity upon incubation for 15 min at 80°C. The influence of various heavy metal ions at the concentration of l mM was improved after enzyme immobilization. The immobilization of C. vulgaris urease on HEMA/IA (92.5/7.5) at 6 kGy showed a residual activity of 47 % after 4 reuses.     

Xylanase and cellulase production byAspergillus fumigatus

When grown on a purified cellulose such as CF11 cellulose,Aspergillus fumigatus produces mainly exoglucanase (Avicelase) and endoglucanase (CMCase) with small amounts of Β-glucosidase and xylanase. In such cultures, the pH drops to 3–3.5 after 2 d incubation, which may account for the low levels of Β-glucosidase. The amounts of extracellular enzymes produced are larger when the organism is grown on hay or straw than when grown on CF11 cellulose. In particular, CMCase levels increase approximately seven times and xylanase levels increase 40–50 times. In such cultures the pH remains fairly constant at 6–7 over the 10-d incubation period used and so Β-glucosidase levels are also increased. Extraction of the hay or straw substrate with ethanol had little effect on enzyme production and so there appears to be no soluble material present that influences enzyme production. The organism produced elevated levels of CMCase and xylanase on barley straw, oat straw, and wheat straw, there being little difference between the varieties of each tested. However, grasses dried at elevated temperatures (260–500‡C) gave enzyme levels similar to CF11 cellulose. Similarly, chemical delignification of hay or straw gave enzyme levels similar to CF11 cellulose. Thus, both these treatments must lead to degradation of the hemicellulose present in the substrate. A. fumigatus was able to grow on a number of laboratory prepared and commercially available xylans (hay, barley straw, oat straw, and larch) as a pelletted mycelium. In all cases xylanase levels were increased 10–30 times over CF11 cellulose as substrate, but CMCase levels were similar to those with CF11 cellulose as substrate. Β-Glucosidase in most cases was not detectable, probably because the pH fell to 3–3.5 during incubation. Thus it appears that cellulase and xylanase can be independently induced in this organism. The optimum incubation time at 37‡C for xylanase production was 4–7 d and the optimum concentration of hay as substrate was 4–5%, even though this produces a very thick slurry that does not shake well.     

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.