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Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.

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Resumen La demanda de xilanasas fúngicas en los procesos biotecnológicos industriales muestra un claro aumento en todo el mundo, por lo que hay un interés en ajustar las condiciones de producción de xilanasas microbianas. En este estudio se optimizó la capacidad del hongo Fusarium solani para producir xilanasas extracelulares con escasa actividad celulolítica mediante el diseño de Box-Wilson. Se determinaron las mejores condiciones de cultivo para obtener una preparación enzimática cruda con una actividad xilanolítica significativa y poca actividad celulolítica. En la mayoría de los tratamientos, la actividad xilanolítica fue mayor que la actividad celulolítica. Se observó un efecto negativo sobre la producción de endoxilanasas, p-xilosidasasy endocelulasascon el aumento de la concentración dexilano. El aumento del tiempo de incubación afectó adversamente la producción de endocelulasas y p-xilosidasas. De acuerdo con el modelo matemático y las pruebas experimentales, es posible producir endoxilanasas con una actividad endocelulasa mínima aumentando el tiempo de incubación y la concentración de sulfato de amonio. Las condiciones de cultivo óptimas para producir una mayor cantidad de endoxilanasas (10,65 U/mg) y mínima cantidad de endocelulasas fueron 2,5% (p/v) de xilano y 5, 2 y 0,4 g/l de extracto de levadura, sulfato de amonio y urea, respectivamente, con 120 h de incubación.

Resumen La demanda de xilanasas fúngicas en los procesos biotecnológicos industriales muestra un claro aumento en todo el mundo, por lo que hay un interés en ajustar las condicionesde producción de xilanasas microbianas. En este estudio se optimizó la capacidad del hongo Fusarium solani para producir xilanasas extracelulares con escasa actividad celulolítica medi-ante el dise˜no de Box-Wilson. Se determinaron las mejores condiciones de cultivo para obteneruna preparación enzimática cruda con una actividad xilanolítica significativa y poca actividad celulolítica. En la mayoría de los tratamientos, la actividad xilanolítica fue mayor que laactividad celulolítica. Se observó un efecto negativo sobre la producción de endoxilanasas, xylanolytic activity and little cellulolytic activity. In most treatments, the xylanolytic activity was higher than the cellulolytic activity. A negative effect on the production of endoxylanases, p-xylosidases and endocellulases was observed with the increasing of xylan concentration. Increasing the incubation time adversely affected the production of endocellulases and p-xylosidases. According to the mathematical model and experimental tests, it is possible to produce endoxylanases with minimal endocellulase activity increasing incubation time and the concentration of ammonium sulfate. The optimal culture conditions to produce a greater amount of endoxylanases (10.65 U/mg) and low endocellulases from F. solani were: 2.5% (w/v) xylan, 5.0, 2.0 and 0.4g/l, of yeast extract, ammonium sulfate and urea, respectively, with 120 h of incubation.

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Biotechnology offers innovative alternatives for industrial bioprocesses mainly because it uses enzymes that biodegrade the hemicellulose releasing fermentable sugars. Caulobacter crescentus (C. crescentus) has seven genes responsible for xylanolytic cleavage, 5 to ß-xylosidases (EC 3.2.1.37) and 2 for endoxylanases, like xynA2 (CCNA_03137) that encodes Xylanase II (EC 3.2.1.8) of the glycohydrolases-GH10 group. The xynA2 gene was amplified by PCR, cloned into the pTrcHisA vector e efficiently overexpressed in E. coli providing a His-tag fusion protein. Recombinant xylanase (XynA2) was purified by affinity chromatography using a nickel sepharose column and exhibited a single 43 kDa band on SDS-PAGE gel. XynA2 showed an optimum alkaline pH (8) and stability at alkaline pH for 24 h. Although C. crescentus is mesophilic, XynA2 has optimum temperature of 60 °C and is thermo-resistance at 65 °C. XynA maintains 66% of the enzymatic activity at high temperatures (90 °C) without being denatured.The enzyme displayed a xylanolitic activity free of cellulase to xylan from beechwood and it was not inhibited in the presence of 50 µmol mL-1 of xylose. In addition, dithiothreitol (DTT) induced XynA2 activity, as it improved its kinetic parameters by lowering the KM (5.78 µmol mL-1) and increasing the KCat/KM ratio (1.63 U s-1). Finally, C. crescentus XynA2 efficiently hydrolyzed corn straw with high release of reducing sugars that can be applied in different branches of the industry.

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Demand for fungal xylanases in industrial biotechnological processes shows a clear increase worldwide, so there is an interest in adjusting the conditions of microbial xylanases production. In this study, the ability of the fungus Fusarium solani to produce extracellular xylanases with low cellulolytic activity was optimized by Box Wilson design. The best culture conditions were determined to obtain a crude enzyme preparation with significant xylanolytic activity and little cellulolytic activity. In most treatments, the xylanolytic activity was higher than the cellulolytic activity. A negative effect on the production of endoxylanases, ß-xylosidases and endocellulases was observed with the increasing of xylan concentration. Increasing the incubation time adversely affected the production of endocellulases and ß-xylosidases. According to the mathematical model and experimental tests, it is possible to produce endoxylanases with minimal endocellulase activity increasing incubation time and the concentration of ammonium sulfate. The optimal culture conditions to produce a greater amount of endoxylanases (10.65U/mg) and low endocellulases from F. solani were: 2.5% (w/v) xylan, 5.0, 2.0 and 0.4g/l, of yeast extract, ammonium sulfate and urea, respectively, with 120h of incubation.

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Abstract This study evaluated the production of endoxylanases by Streptomyces malaysiensis AMT-3 in submerged fermentation using by-products of the food industry at 28ºC. In shake-flasks experiments, the highest endoxylanase activity of 45.8 U.mL-1 was observed within 6 days in a medium containing (w/v) 2.5% wheat bran and 1.2% corn steep liquor. The same culture conditions were used to evaluate the enzyme production in a 2 L stirred tank reactor under different agitation (300, 450 and 600 rev.min-1) and aeration (30 and 60 L.h-1) conditions. The use of 450 rev.min-1 coupled to an aeration of 90 L.h-1 resulted on 81.3 U.mL-1 endoxylanase activity within 5 days. The effect of temperature and pH on endoxylanase activity and stability showed the highest activity at 60 ºC and pH 6.0. Zymography showed the presence of three xylanolytic bands with molecular masses of 690, 180 and 142 kDa. The results showed that the thermotolerant actinobacterial endoxylanase can be produced in high titers using by-product of the food industry.

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Agroforestry industries in the world generate lignocellulosic wastes that can be a huge problem of pollution, or the wastes can be used for different biotechonological applications such as substrates for microorganism growth and enzyme production. Fungi such as Aspergillus niger can grow in almost every substrate and produce hydrolytic enzymes such as endoxylanases, giving added value to agroforestry wastes generated by industries in the northeast of Argentina. In this context, the aim of this work was to use agroforestry wastes as substrates for the production of endoxylanases by Aspergillus niger and to optimize nitrogen sources and physical variables for the highest endoxylanase activity. A. niger LBM 055 and A. niger LBM 134 produced high endoxylanase levels when they were grown with sugarcane and cassava bagasses as carbon sources. A. niger LBM 134 reached the highest endoxylanase activity when nitrogen sources and physical variables were optimized. The fungus exhibited up to 110 U mL-1 of endoxylanase activity when it was grown with sugarcane bagasse and more than 160 U mL-1 with cassava bagasse. Therefore, endoxylanase production was optimized using agricultural bagasses and cost 20 times less than enzyme production using synthetic xylan.

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The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.

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Background: In this work, the xylanase production by Penicillium chrysogenum F-15 strain was investigated using agroindustrial biomass as substrate. The xylanase was purified, characterized and applied in hemicellulose hydrolysis. Results: The highest xylanase production was obtained when cultivation was carried out with sugar cane bagasse as carbon source, at pH 6.0 and 20°C, under static condition for 8 d. The enzyme was purified by a sequence of ion exchange and size exclusion chromatography, presenting final specific activity of 834.2 U·mg·prot-1. T he molecular mass of the purified enzyme estimated by SDS-PAGE was 22.1 kDa. The optimum activity was at pH 6.5 and 45°C. The enzyme was stable at 40°C with half-life of 35 min, and in the pH range from 4.5 to 10.0. The activity was increased in the presence of Mg+2 and Mn+2 and reducing agents such as DTT and ßmercaptoethanol, but it was reduced by Cu+2 and Pb+2 . The xylanase presented Km of 2.3 mM and Vmax of 731.8 U·mg·prot-1 with birchwood xylan as substrate. This xylanase presented differences in its properties when it was compared to the xylanases from other P. chrysogenum strains. Conclusion: The xylanase from P. chrysogenum F-15 showed lower enzymatic activity on commercial xylan than on hemicellulose from agroindustry biomass and its biochemistry characteristics, such as stability at 40°C and pH from 4.0 to 10.0, shows the potential of this enzyme for application in food, feed, pulp and paper industries and for bioethanol production.

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Background: Xylanase from bacteria finds use in prebleaching process and bioconversion of lignocelluloses into feedstocks. The xylanolytic enzyme brings about the hydrolysis of complex biomolecules into simple monomer units. This study aims to optimize the cellulase-free xylanase production and cell biomass of Bacillus tequilensis strain ARMATI using response surface methodology (RSM). Results: Statistical screening of medium constituents and the physical factors affecting xylanase and biomass yield of the isolate were optimized by RSM using central composite design at N = 30, namely 30 experimental runs with 4 independent variables. The central composite design showed 3.7 fold and 1.5 fold increased xylanase production and biomass yield of the isolate respectively compared to 'one factor at a time approach',inthe presence of the basal medium containing birchwood xylan (1.5% w/v) and yeast extract (1% w/v), incubated at 40°C for 24 h. Analysis of variance (ANOVA) revealed high coefficient of determination (R2)of0.9978 and 0.9906 for the respective responses at significant level (p < 0.05). The crude xylanase obtained from the isolate showed stability at high temperature (60°C) and alkaline condition (pH 9) up to 4 h of incubation. Conclusions: The cellulase-free xylanase showed an alkali-tolerant and thermo-stable property with potentially applicable nature at industrial scale. This statistical approach established a major contribution in enzyme production from the isolate by optimizing independent factors and represents a first reference on the enhanced production of thermo-alkali stable cellulase-free xylanase from B. tequilensis.

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Caulobacter crescentus (NA1000 strain) are aquatic bacteria that can live in environments of low nutritional quality and present numerous genes that encode enzymes involved in plant cell wall deconstruction, including five genes for ß-xylosidases (xynB1-xynB5) and three genes for xylanases (xynA1-xynA3). The overall activity of xylanases in the presence of different agro-industrial residues was evaluated, and it was found that the residues from the processing of corn were the most efficient in inducing bacterial xylanases. The xynA1 gene (CCNA_02894) encoding a predicted xylanase of group 10 of glyco-hydrolases (GH10) that was efficiently overexpressed in Escherichia coli LMG194 using 0.02 % arabinose, after cloning into the vector pJet1.2blunt and subcloning into the expression vector pBAD/gIII, provided a fusion protein that contained carboxy-terminal His-tags, named XynA1. The characterization of pure XynA1 showed an enzymatic activity of 18.26 U mL(-1) and a specific activity of 2.22 U mg-(1) in the presence of xylan from beechwood as a substrate. XynA1 activity was inhibited by EDTA and metal ions such as Cu(2+) and Mg(2+). By contrast, ß-mercaptoethanol, dithiothreitol (DTT), and Ca(2+) induced recombinant enzyme activity. Kinetic data for XynA1 revealed K M and V max values of 3.77 mg mL-(1) and 10.20 µM min-(1), respectively. Finally, the enzyme presented an optimum pH of 6 and an optimum temperature of 50 °C. In addition, 80 % of the activity of XynA1 was maintained at 50 °C for 4 h of incubation, suggesting a thermal stability for the biotechnological processes. This work is the first study concerning the cloning, overexpression, and enzymatic characterization of C. crescentus xylanase.

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The main limitation to the industrial scale hydrolysis of cellulose is the cost of cellulase production. This study evaluated cellulase and xylanase enzyme production by the cellulolytic mutant Penicillium echinulatum 9A02S1 using pretreated sugar cane bagasse as a carbon source. Most cultures grown with pretreated bagasse showed similar enzymatic activities to or higher enzymatic activities than cultures grown with cellulose or untreated sugar cane bagasse. Higher filter paper activity (1.253 ± 0.147 U · mL(-1)) was detected in the medium on the sixth day of cultivation when bagasse samples were pretreated with sodium hydroxide, hydrogen peroxide, and anthraquinone. Endoglucanase enzyme production was also enhanced by pretreatment of the bagasse. Nine cultures grown with bagasse possessed higher ß -glucosidase activities on the sixth day than the culture grown with cellulose. The highest xylanase activity was observed in cultures with cellulose and with untreated sugar cane bagasse. These results indicate that pretreated sugar cane bagasse may be able to serve as a partial or total replacement for cellulose in submerged fermentation for cellulase production using P. echinulatum, which could potentially reduce future production costs of enzymatic complexes capable of hydrolyzing lignocellulosic residues to form fermented syrups.

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Of the many reported applications for xylanase, its use as a food supplement has played an important role for monogastric animals, because it can improve the utilisation of nutrients. The aim of this work was to produce xylanase by extractive fermentation in an aqueous two-phase system using Aspergillus tamarii URM 4634, increasing the scale of production in a bioreactor, partially characterising the xylanase and evaluating its influence on monogastric digestion in vitro. Through extractive fermentation in a bioreactor, xylanase was obtained with an activity of 331.4 U mL(-1) and 72% yield. The xylanase was stable under variable pH and temperature conditions, and it was optimally active at pH 3.6 and 90 °C. Xylanase activity potentiated the simulation of complete monogastric digestion by 6%, and only Mg2+ inhibited its activity. This process provides a system for efficient xylanase production by A. tamarii URM 4634 that has great potential for industrial use.

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The effect of different carbon sources on morphology and cellulase and xylanase production of Penicillium echinulatum was evaluated in this work. Among the six carbon sources studied, cellulose and sugar cane bagasse were the most suitable for the production of filter paper activity, endoglucanases, xylanases, and ß-glucosidases. However, sucrose and glucose showed ß -glucosidase activities similar to those obtained with the insoluble sources. The polyacrylamide gels proved the enzymatic activity, since different standards bands were detected in the media mentioned above. Regarding morphology, it was observed that the mycelium in a dispersed form provided the greatest enzymatic activity, possibly due to greater interaction between the substrate and hyphae. These data are important in understanding the physiology of fungi and could contribute to obtaining enzyme with potential application in the technology of second generation ethanol.

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Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5-7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.

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The production of cellulases and xylanases by Penicillium echinulatum in an airlift bioreactor was evaluated. In batch production, we tested media with isolated or associated cellulose and sorbitol. In fed-batch production, we tested cellulose addition at two different times, 30 h and 48 h. Higher liquid circulation velocities in the downcomer were observed in sorbitol 10 g L(-1) medium. In batch production, higher FPA (filter paper activity) and endoglucanase activities were obtained with cellulose (7.5 g L(-1)) and sorbitol (2.5 g L(-1)), 1.0 U mL(-1) (120 h) and 6.4 U m L(-1) (100 h), respectively. For xylanases, the best production condition was cellulose 10 g L(-1), which achieved 5.5 U mL(-1) in 64 h. The fed-batch process was favorable for obtaining xylanases, but not for FPA and endoglucanases, suggesting that in the case of cellulases, the inducer must be added early in the process.

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The development of more productive strains of microorganisms and processes that increase enzyme levels can contribute to the economically efficient production of second generation ethanol. To this end, cellulases and xylanases were produced with the S1M29 mutant strain of Penicillium echinulatum, using different concentrations of cellulose (20, 40, and 60 g L(-1)) in batch and fed-batch processes. The highest activities of FPase (8.3 U mL(-1)), endoglucanases (37.3 U mL(-1)), and xylanases (177 U mL(-1)) were obtained in fed-batch cultivation with 40 g L(-1) of cellulose. The P. echinulatum enzymatic broth and the commercial enzyme Cellic CTec2 were tested for hydrolysis of pretreated sugar cane bagasse. Maximum concentrations of glucose and xylose were achieved after 72 h of hydrolysis. Glucose yields of 28.0% and 27.0% were obtained using the P. echinulatum enzymatic extract and Cellic CTec2, respectively.

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In recent decades, xylanases have been used in many processing industries. This study describes the xylanase production by Penicillium glabrum using brewer's spent grain as substrate. Additionally, this is the first work that reports the purification and characterization of a xylanase using this agroindustrial waste. Optimal production was obtained when P. glabrum was grown in liquid medium in pH 5.5, at 25 °C, under stationary condition for six days. The xylanase from P. glabrum was purified to homogeneity by a rapid and inexpensive procedure, using ammonium sulfate fractionation and molecular exclusion chromatography. SDS-PAGE analysis revealed one band with estimated molecular mass of 18.36 kDa. The optimum activity was observed at 60 °C, in pH 3.0. The enzyme was very stable at 50 °C, and high pH stability was verified from pH 2.5 to 5.0. The ion Mn(2+) and the reducing agents ß -mercaptoethanol and DTT enhanced xylanase activity, while the ions Hg(2+), Zn(2+), and Cu(2+) as well as the detergent SDS were strong inhibitors of the enzyme. The use of brewer's spent grain as substrate for xylanase production cannot only add value and decrease the amount of this waste but also reduce the xylanase production cost.

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Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of ß-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 ± 411.2 U g(-1), while ß-glucosidase production was increased about 2.6-fold, reaching 20.7 ± 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis ß-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for ß-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The ß-glucosidase maintained about 95 % of its activity after 26 h in water at 55 °C, with half-lives of 15.7 h at 60 °C and 5.1 h at 65 °C. The presence of xylose during heat treatment at 65 °C protected ß-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 °C. Xylose stimulated ß-glucosidase activity up to 1.7-fold, at 200 mmol L(-1). The notable features of both xylanase and ß-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

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Solid-state fermentation (SSF) is defined as the growth of microbes without a free-flowing aqueous phase. The feasibility of using a citrus peel for producing pectinase and xylanase via the SSF process by Aspergillus niger F3 was evaluated in a 2 kg bioreactor. Different aeration conditions were tested to optimize the pectinase and xylanase production. The best air flow intensity was 1 V kg M (volumetric air flow per kilogram of medium), which allowed a sufficient amount of O2 for the microorganism growth producing 265 U/g and 65 U/g pectinases and xylanases, respectively. A mathematical model was applied to determine the different kinetic parameters related to SSF. The specific growth rate and biomass oxygen yield decreased during fermentation, whereas an increase in the maintenance coefficient for the different employed carbon sources was concurrently observed.

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Fibrolytic enzyme production by Aspergillus japonicus C03 was optimized in a medium containing agro-industrial wastes, supplemented with peptone and yeast extract. A 2(3) full factorial composite and response surface methodology were used to design the experiments and analysis of results. Tropical forages were hydrolyzed by A. japonicus C03 enzymatic extract in different levels, and they were also tested as enzymatic substrate. Optimal production to xylanase was obtained with soybean bran added to crushed corncob (1:3), 0.01% peptone, and 0.2% yeast extract, initial pH 5.0, at 30 °C under static conditions for 5 days of incubation. Optimal endoglucanase production was obtained with wheat bran added to sugarcane bagasse (3:1), 0.01% peptone, and 0.2% yeast extract, initial pH 4.0, at 30 °C, for 6 days, under static conditions. Addition of nitrogen sources as ammonium salts either inhibited or did not influence xylanase production. This enzymatic extract had a good result on tropical forage hydrolyzes and showed better performance in the Brachiaria genera, due to their low cell wall lignin quantity. These results represent a step forward toward the use of low-cost agricultural residues for the production of valuable enzymes with potential application in animal feed, using fermentation conditions.

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