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.     

Specificity and mode of action of a thermostable xylanase from bacillus amyloliquefaciens on-line monitoring of hydrolysis products

A thermostable xylanase purified from a strain of Bacillus amyloliquefaciens MIR 32 was characterized with respect to its substrate specificity and mode of hydrolytic action. The enzyme was highly specific for xylans as substrate and displayed no activity toward other polysaccharides, including cellulose. The enzyme exhibited K_m and V_max of 4.5 mg/mL and 0.58 mmol/min/mg, respectively, with birchwood xylan as the substrate. Microdialysis sampling with anion exchange chromatography and integrated pulsed electrochemical detection were used for rapid on-line monitoring of products during hydrolysis of oat spelt and bagasse xylan, and xylooligosaccharides. Xylobiose and xylotriose were the main end products. Xylotetraose was the smallest oligosaccharide to be acted on by the xylanase. The product pattern confirmed that the enzyme was an endoxylanase.     

Purification and characterization of a cyclomaltodextrin glucanotransferase from Paenibacillus campinasensis strain H69-3

A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19) from a newly isolated alkalophilic and moderately thermophilic Paenibacillus campinasensis strain H69-3 was purified as a homogeneous protein from culture supernatant. Cyclomaltodextrin glucanotransferase was produced during submerged fermentation at 45 degrees C and purified by gel filtration on Sephadex G50 ion exchange using a Q-Sepharose column and ion exchange using a Mono-Q column. The molecular weight of the purified enzyme was 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the pI was 5.3. The optimum pH for enzyme activity was 6.5, and it was stable in the pH range 6.0-11.5. The optimum temperature was 65 degrees C at pH 6.5, and it was thermally stable up to 60 degrees C without substrate during 1 h in the presence of 10 mM CaCl(2). The enzyme activity increased in the presence of Co(2+), Ba(2+), and Mn(2+). Using maltodextrin as substrate, the K(m) and K(cat) were 1.65 mg/mL and 347.9 micromol/mg x min, respectively.     

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.     

An Extremely Alkaline Novel Xylanase from a Newly Isolated Streptomyces Strain Cultivated in Corncob Medium

Streptomyces sp. CS802, recently isolated from Korean soil, produced xylanase in corncob medium. An extracellular xylanase (Xyn802) was purified by a single-step gel filtration and biochemical properties were studied. It showed high activity in extremely alkaline condition with optimum pH at 12.0 and exhibited stability between pH?7.5 and 13.0. It produced xylobiose and xylotriose as the major products from xylan, suggesting its endoxylanase nature. N-terminal amino acid sequences of Xyn802 were ADRNANRD which are significantly different from the reported xylanase. The activity was enhanced by various detergents and a reducing agent and stable in various organic solvents. Xyn802 produced by utilizing corncob, an agro-waste material, might be a novel xylanase based on its peculiar biochemical characteristics, and it can be a suitable candidate for the production of xylooligosaccharides including other useful products.     

Thermostable Hemicellulases of a Bacterium,Geobacillus sp. DC3, Isolated from the Former Homestake Gold Mine in Lead,South Dakota

A thermophilic strain, Geobacillus sp. DC3, capable of producing hemicellulolytic enzymes was isolated from the 1.5-km depth of the former Homestake gold mine in Lead, South Dakota. The DC3 strain expressed a high level of extracellular endoxylanase at 39.5 U/mg protein with additional hemicellulases including β-xylosidase (0.209 U/mg) and arabinofuranosidase (0.230 U/mg), after the bacterium was grown in xylan for 24 h. Partially purified DC3 endoxylanase exhibited a molecular mass of approximately 43 kDa according to zymography with an optimal pH of 7 and optimal temperature of 70 °C. The kinetic constants, K _m and V _max, were 13.8 mg/mL and 77.5 μmol xylose/min·mg xylan, respectively. The endoxylanase was highly stable and maintained 70 % of its original activity after 16 h incubation at 70 °C. The thermostable properties and presence of three different hemicellulases of Geobacillus sp. DC3 strain support its potential application for industrial hydrolysis of renewable biomass such as lignocelluloses.     

Production and Characterization of Cellulase-Free Xylanase from <Emphasis Type="Italic">Trichoderma inhamatum</Emphasis>

The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25 degrees C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50 degrees C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T1/2 was 2.2 h at 40 degrees C and 2 min at 50 degrees C. The pH stability was high from 4.0 to 11.0. These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence.     

Production, Purification, Immobilization, and Characterization of a Thermostable β-Galactosidase from Aspergillus alliaceus

A fungal strain isolated from rotten banana and identified as Aspergillus alliaceus was found capable of producing thermostable extracellular ??-galactosidase enzyme. Optimum cultural conditions for ??-galactosidase production by A. alliaceus were as follows: pH?4.5; temperature, 30?°C; inoculum age, 25?h; and fermentation time, 144?h. Optimum temperature, time, and pH for enzyme substrate reaction were found to be 45?°C, 20?min, and 7.2, respectively, for crude and partially purified enzyme. For immobilized enzyme?Csubstrate reaction, these three variable, temperature, time, and pH were optimized at 50?°C, 40?min, and 7.2, respectively. Glucose was found to inhibit the enzyme activity. The K _m values of partially purified and immobilized enzymes were 170 and 210?mM, respectively. Immobilized enzyme retained 43?% of the ??-galactosidase activity of partially purified enzyme. There was no significant loss of activity on storage of immobilized beads at 4?°C for 28?days. Immobilized enzyme retained 90?% of the initial activity after being used four times.     

Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0–9.0 and between 30–40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (K_m = 26.06 mg·mL⁻¹ and V_max = 5.647 U·mg⁻¹). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.     

Isolation and partial characterization of an antifungal protein produced by Bacillus licheniformis BS-3

An antifungal protein produced by Bacillus licheniformis strain BS-3 was purified to homogeneity by ammonium sulfate precipitation, DEAE-52 column chromatography and Sephadex G-75 column chromatography. The purified protein was designated as F2 protein, inhibited the growth of Aspergillus niger, Magnaporthe oryzae and Rhizoctonia solani. F2 protein was a monomer with approximately molecular weight of 31 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gave a single peak on High Performance Liquid Chromatography (HPLC). Using Rhizoctonia solani as the indicator strain, the EC50 of F2 protein was 35.82 μg/mL, displaying a higher antifungal activity in a range of pH 6.0 to pH 10.0, and at a temperature below 70 °C for 30 min. F2 protein was moderately resistant to hydrolysis by trypsin, proteinase K, after which its relative activities were 41.7% and 59.5%, respectively. F2 protein was assayed using various substrates to determine the enzymatic activities, the results showed the hydrolyzing activity on casein, however, no enzymatic activities on colloidal chitin, CM-cellulose, xylan, M. lysodeikticus, and p-nitrophenyl-N-acetylglucosaminide.     

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.     

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.     

Characterization of Xylanase and Cellulase Produced by a Newly Isolated <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> N2 and Its Efficient Saccharification of Barley Straw

Aspergillus fumigatus N2 was isolated from decaying wood. This strain produces extracellular xylanases and cellulases. The highest xylanase (91.9 U/mL) and CMCase (5.61 U/mL) activity was produced when 1% barley straw was used as the carbon source. The optimum pH and temperature for xylanase activity were 6.0 and 65 °C, respectively. CMCase revealed maximum activity at pH 4.0 and in the range of 65 °C. The FPase was optimally active at pH 5.0 and 60 °C. The zymograms produced by the SDS-PAGE resolution of the crude enzymes indicated that multiple enzymes were secreted into the fermentation supernatant. Five bands of proteins with xylanase activity and four bands of proteins with endoglucanase were observed in the zymogram gel. The crude enzymes were used in the barley straw saccharification; an additive effect was observed when the commercial cellulase was added as supplement.     

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, purification, and biochemical characterization of two beta-glucosidases from Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum produces beta-glucosidases in liquid culture with a variety of carbon sources, including cellulose (filter paper), xylan, barley straw, oat meal, and xylose. Analysis by native polyacrylamide gel electrophoresis (PAGE) followed by an activity staining with the specific chromogenic substrate, 5-bromo 4-chloro 3-indolyl beta-1,4 glucoside (X-glu) showed that two extracellular beta-glucosidases, designated as beta-glu1 and beta-glu2, were in the filter paper culture filtrate. Only one enzyme designated as beta-glu x was revealed by the same method in the xylose culture filtrate. Beta-glu1 and beta-glu2 were purified to homogeneity. The purification procedure consist of a common step of anion-exchange chromatography on DEAE-Sepharose CL6B, both high-performance liquid chromatography (HPLC) anion-exchange and gel filtration columns for beta-glu1 and only HPLC gel filtration for beta-glu2. Beta-glu1 has a molecular mass of 196 kDa and 96.5 kDa, as estimated by gel filtration and sodium dodecyl sulfate (SDS)-PAGE, respectively, suggesting that the native enzyme may consist of two identical subunits. The same analysis showed that beta-glu2 is a monomeric protein with an apparent molecular mass of about 76.5 kDa. Beta-glu1 and beta-glu2 hydrolyses PNPGlc and cellobiose, with apparent Km values respectively for PNPGlc and cellobiose of 0.1 and 1.9 mM for beta-glu1 and 2.8 and 8 mM for beta-glu2. Both enzymes exhibit the same temperature and pH optima for PNPGlc hydrolysis (60 degrees C and pH 5.0). beta-glu1 was stable over a pH range of 3-8 and kept 50% of its activity after 30 min of heating at 60 degrees C without substrate. It was further characterized by studying the effect of some cations and various reagents on its activity.     

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.     

Xylanase contribution to the efficiency of cellulose enzymatic hydrolysis of barley straw

In this study, different enzyme preparations available from Novozymes were assessed for their efficiency to hydrolyze lignocellulosic materials. The enzyme mixture was evaluated on a pretreated cellulose-rich material, and steam-exploded barley straw pretreated under different temperatures (190, 200, and 210 degrees C, respectively) in order to produce fermentable sugars. Results show that xylanase supplementation improves initial cellulose hydrolysis effectiveness of water-insoluble solid fraction from all steam-exploded barley straw samples, regardless of the xylan content of substrate. The mixture constituted by cellulase: beta-glucosidase: endoxylanase of the new kit for lignocellulose conversion at a ratio 10:1:5% ([v/w], enzyme [E]/substrate [S]) provides the highest increment of cellulose conversion in barley straw pretreated at 210 degrees C, for 10 min.     

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.     

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.     

Xylanolytic enzyme production by anAspergillus niger isolate

Production of xylanolytic enzymes by anAspergillus niger CCMI 850 isolate was investigated in batch cultures. The effect of the composition of a fermentation medium that did not include chemical inducers, on β-xylanase, β-xylosidase, α-l-arabinofuranosidase, and total cellulase activity was studied. With 4% xylan as the carbon source, about 65 U/mL of β-xylanase was obtained, whereas the total cellulase activity was undetectable, under the specified conditions. This β-xylanase activity represents the highest reported for a wild-type strain ofA. niger. The effect of pH and temperature on the activity of β-xylanase was studied. Partial characterization of the β-xylanase showed that with insoluble birchwood as substrate theK _m andV _max were 0.3 mM and 19 μmol/min, respectively. Aspects of using the crude β-xylanase preparation for applications in the pulp and paper industry were discussed.