Purification and characterization of a native lytic polysaccharide monooxygenase from Thermoascus aurantiacus.

Lytic polysaccharide monooxygenases (LPMOs) have emerged as key proteins for depolymerization of cellulose. These copper-containing enzymes oxidize C-1 and/or C-4 bonds in cellulose, promoting increased hydrolysis of the oxidized cellulose chains. The LPMO from Thermoascus aurantiacus, a thermophilic ascomycete fungus, has been extensively studied and has served as a model LPMO. A method was developed to purify the LPMO from culture filtrates of T. aurantiacus along with its native cellobiohydrolase and endoglucanase. The activity of the purified LPMO was measured with a colorimetric assay that established the Topt of the native LPMO at 60 °C. Purification of the components of the T. aurantiacus cellulase mixture also enabled quantification of the amounts of cellobiohydrolase, endoglucanase and LPMO present in the T. aurantiacus culture filtrate, establishing that the LPMO was the most abundant protein in the culture supernatants. The importance of the LPMO to activity of the mixture was demonstrated by saccharifications with Avicel and acid-pretreated corn stover.

Identification of a thermostable fungal lytic polysaccharide monooxygenase and evaluation of its effect on lignocellulosic degradation.

Auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) show significant synergism with cellulase in cellulose degradation. In recent years, there have been many reports on AA9 LPMOs; however, the identification of efficient and thermostable AA9 LPMOs with broad potential for industrial applications remains necessary. In this study, a new AA9 LPMO from Talaromyces cellulolyticus, named TcAA9A, was identified. An analysis of the oxidation products of phosphoric acid-swollen cellulose categorized TcAA9A as a type 3 AA9 LPMO, and TcAA9A exhibited a better synergistic effect with cellulase from Trichoderma reesei than what is seen with TaAA9A, a well-studied AA9 LPMO from Thermoascus aurantiacus. Two AA9 LPMOs were successfully expressed in T. reesei, and the transformants were named TrTcAA9A and TrTaAA9A. The activities and thermostabilities of the AA9 LPMOs in TrTcAA9A were higher than those of the AA9 LPMOs in TrTaAA9A or the parent. The enzyme solution of TrTcAA9A was more efficient than that of the parent or TrTaAA9A for the degradation of Avicel and delignified corncob residue. Thus, TcAA9A showed a better performance than TaAA9A in T. reesei cellulase cocktails. This study may offer an innovative solution for improving enzyme cocktail activity for lignocellulosic degradation.

Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs), found in family 9 (previously GH61), family 10 (previously CBM33), and the newly discovered family 11 of auxiliary activities (AA) in the carbohydrate-active enzyme classification system, are copper-dependent enzymes that oxidize sp(3)-carbons in recalcitrant polysaccharides such as chitin and cellulose in the presence of an external electron donor. In this study, we describe the activity of two AA10-type LPMOs whose activities have not been described before and we compare in total four different AA10-type LPMOs with the aim of finding possible correlations between their substrate specificities, sequences, and EPR signals. EPR spectra indicate that the electronic environment of the copper varies within the AA10 family even though amino acids directly interacting with the copper atom are identical in all four enzymes. This variation seems to be correlated to substrate specificity and is likely caused by sequence variation in areas that affect substrate binding geometry and/or by variation in a cluster of conserved aromatic residues likely involved in electron transfer. Interestingly, EPR signals for cellulose-active AA10 enzymes were similar to those previously observed for cellulose-active AA9 enzymes. Mutation of the conserved phenylalanine positioned in close proximity to the copper center in AA10-type LPMOs to Tyr (the corresponding residue in most AA9-type LPMOs) or Ala, led to complete or partial inactivation, respectively, while in both cases the ability to bind copper was maintained. Moreover, substrate binding affinity and degradation ability seemed hardly correlated, further emphasizing the crucial role of the active site configuration in determining LPMO functionality.

Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components.

The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

Purification, biochemical characterization, and biotechnological applications of a multifunctional enzyme from the Thermoascus aurantiacus PI3S3 strain.

The filamentous Thermoascus aurantiacus fungus characterized by its thermophilic nature, is recognized as an exceptional producer of various enzymes with biotechnological applications. This study aimed to explore biotechnological applications using polygalacturonase (PG) derived from the Thermoascus aurantiacus PI3S3 strain. PG production was achieved through submerged fermentation and subsequent purification via ion-exchange chromatography and gel filtration methods. The crude extract exhibited a diverse spectrum of enzymatic activities including amylase, cellulase, invertase, pectinase, and xylanase. Notably, it demonstrated the ability to hydrolyze sugarcane bagasse biomass, corn residue, and animal feed. The purified PG had a molecular mass of 36 kDa, with optimal activity observed at pH 4.5 and 70 °C. The activation energy (Ea) was calculated as 0.513 kJ mol-1, highlighting activation in the presence of Ca2+. Additionally, it displayed apparent Km, Vmax, and Kcat values of at 0.19 mg mL-1, 273.10 U mL-1, and 168.52 s-1, respectively, for hydrolyzing polygalacturonic acid. This multifunctional PG exhibited activities such as denim biopolishing, apple juice clarification, and demonstrated both endo- and exo-polygalacturonase activities. Furthermore, it displayed versatility by hydrolyzing polygalacturonic acid, carboxymethylcellulose, and xylan. The T. aurantiacus PI3S3 multifunctional polygalacturonase showed heightened activity under acidic pH, elevated temperatures, and in the presence of calcium. Its multifunctional nature distinguished it from other PGs, significantly expanding its potential for diverse biotechnological applications.

Rationally selected single-site mutants of the Thermoascus aurantiacus endoglucanase increase hydrolytic activity on cellulosic substrates.

Variants of the Thermoascus aurantiacus Eg1 enzyme with higher catalytic efficiency than wild-type were obtained via site-directed mutagenesis. Using a rational mutagenesis approach based on structural bioinformatics and evolutionary analysis, two positions (F16S and Y95F) were identified as priority sites for mutagenesis. The mutant and parent enzymes were expressed and secreted from Pichia pastoris and the single site mutants F16S and Y95F showed 1.7- and 4.0-fold increases in k(cat) and 1.5- and 2.5-fold improvements in hydrolytic activity on cellulosic substrates, respectively, while maintaining thermostability. Similar to the parent enzyme, the two variants were active between pH 4.0 and 8.0 and showed optimal activity at temperature 70°C at pH 5.0. The purified enzymes were active at 50°C for over 12 h and retained at least 80% of initial activity for 2 h at 70°C. In contrast to the improved hydrolysis seen with the single mutation enzymes, no improvement was observed with a third variant carrying a combination of both mutations, which instead showed a 60% reduction in catalytic efficiency. This work further demonstrates that non-catalytic amino acid residues can be engineered to enhance catalytic efficiency in pretreatment enzymes of interest.

Fungal ß-glucosidase expression in Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae strains expressing ß-glucosidases from Thermoascus aurantiacus (Tabgl1) and Phanerochaete chrysosporium (PcbglB and Pccbgl1) were constructed and compared to S. cerevisiae Y294[SFI], previously identified as the best ß-glucosidase-producing strain. The PcbglB was also intracellularly expressed in combination with the lac12 lactose permease of Kluyveromyces lactis in S. cerevisiae Y294[PcbglB + Lac12]. The recombinant extracellular ß-glucosidases indicated maximum activity in the pH range 4-5 and temperature optima varying from 50 to 75 °C. The S. cerevisiae Y294[Pccbgl1] strain performed best under aerobic and anaerobic conditions, producing 2.6 times more ß-glucosidase activity than S. cerevisiae Y294[SFI] and an ethanol concentration of 4.8 g l(-1) after 24 h of cultivation on cellobiose as sole carbohydrate source. S. cerevisiae Y294[Tabgl1] was unable to grow on cellobiose (liquid medium), whereas S. cerevisiae Y294[PcbglB + Lac12] exhibited limited growth.

Utilization of Jatropha deoiled seed cake for production of cellulases under solid-state fermentation.

Toxic waste generated by Jatropha seed cake after utilization of biodiesel on one hand has stimulated the need to develop new technologies to treat the waste and on the other, forced us to reevaluate the efficient utilization of its nutritive potential for production of various high-value compounds and its conversion to non-toxic forms which could be used as animal feed stock. In this study, Jatropha seed cake was used for production of cellulases by new isolate of Thermoascus aurantiacus under solid-state fermentation. The interaction of nitrogen source concentration, moisture ratio, initial pH of the medium and inoculum size was investigated and modelled using response surface methodology (RSM) using Box-Behnken Design (BBD). Under optimized conditions endo-ß-1,4-glucanase, ß-glucosidase and filter paper activities were found to be 124.44, 28.86, 4.87 U/g of substrate, respectively. Characterization of endo-ß-1,4-glucanase, ß-glucosidase was done after partial purification by ammonium sulfate fractionation followed by desalting. The endo-ß-1,4-glucanase and ß-glucosidase showed maximum activity at 70 °C and pH 4. Saccharification studies performed with different lignocellulosic substrates showed that sugar cane bagasse was most susceptible to enzymatic hydrolysis. The study suggests that Jatropha seed cake can be used as a viable nutrient source for cellulase production without any pretreatment under solid-state fermentation by T. aurantiacus.

Application of a recombinant GH10 endoxylanase from Thermoascus aurantiacus for xylooligosaccharide production from sugarcane bagasse and probiotic bacterial growth.

Xylooligosaccharides (XOs) are a promising class of prebiotics capable of selectively stimulating the growth of the beneficial intestinal microbiota against intestinal pathogens. They can be obtained from xylan present in residual lignocellulosic material from agriculture. Thus, in this study we produced XOs by extracting xylan from sugarcane bagasse and hydrolyzing it using the GH10 xylanase from Thermoascus aurantiacus expressed by Pichia pastoris. An alkaline method to extract xylan is described, which resulted in 83.40% of xylan recovery and low amounts of cellulose and lignin. The enzymatic hydrolysate exhibited a mixture of XOs containing mainly xylobiose, xylotriose and xylotetraose. These oligosaccharides stimulated the growth of Lactobacillus casei, L. rhamnosus, L. fermentum and L. bulgaricus strains, which were able to produce organic acids, especially acetic acid. These findings demonstrate the possibility to redirect crop by-products to produce XOs and their use as a supplement to stimulate the growth of probiotic strains.

Inhibition of LPMOs by Fermented Persimmon Juice.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.

Heterologous secretory expression of ß-glucosidase from Thermoascus aurantiacus in industrial Saccharomyces cerevisiae strains.

The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer ß-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a ß-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.

Resonance assignments for the apo-form of the cellulose-active lytic polysaccharide monooxygenase TaLPMO9A.

The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts.

Butanol production employing fed-batch fermentation by Clostridium acetobutylicum GX01 using alkali-pretreated sugarcane bagasse hydrolysed by enzymes from Thermoascus aurantiacus QS 7-2-4.

Sugarcane bagasse (SB) is a potential feedstock for butanol production. However, biological production of butanol from SB is less economically viable. In this study, evaluation of eight pretreatments on SB showed that alkali pretreatment efficiently removed lignin from SB while retaining the intact native structure of the released microfibrils. In total, 99% of cellulose and 100% of hemicellulose in alkali-pretreated SB were hydrolysed by enzymes from Thermoascus aurantiacus. The hydrolysate was used to produce butanol in a fed-batch fermentation by Clostridium acetobutylicum. At 60h, 14.17 and 21.11gL(-1) of butanol and acetone-butanol-ethanol (ABE) were produced from 68.89gL(-1) of total sugars, respectively, yielding 0.22 and 0.33gg(-1) of sugars. The maximum yield of butanol and ABE reached 15.4g and 22.9g per 100g raw SB, respectively. This established process may have potential application for butanol production from SB.

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides.

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and ß-glucosidase played an important role in mannan hydrolysis. Addition of 10mg/ml mannan reduced the glucose yield of Avicel (at 20g/l) from 40.1 to 24.3%. No inhibition of ß-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on ß-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.

Xylans inhibit enzymatic hydrolysis of lignocellulosic materials by cellulases.

Hemicelluloses have been found to be physical barriers in the hydrolysis of cellulose, and prevent the access of enzymes to cellulose surface. In addition, soluble hemicelluloses may strongly inhibit the cellulase activity. In this work, birchwood xylan clearly inhibited the enzymatic hydrolysis of wheat straw, Avicel and nanocellulose by cellulases. Hydrolysis efficiencies of cellobiohydrolase I (CBHI, from Thermoascus aurantiacus), cellobiohydrolase II (CBHII, from Trichoderma reesei) and endoglucanase II (from T. aurantiacus) were clearly inhibited by birchwood xylan, respectively. The strongest inhibitory effect of birchwood xylan was observed on the hydrolysis of Avicel by CBHI and CBHII, as a dramatically decreased formation of the main product, cellobiose. After additions of soluble and insoluble oat spelt xylan, cleaved cellobiose units by CBHI from cellulose chain decreased from 8 to 4 and 6, respectively. The results in this work demonstrated that xylans clearly inhibited the hydrolysis efficiencies of both endoglucanase and cellobiohydrolase.

Studies of enzymatic cleavage of cellulose using polysaccharide analysis by carbohydrate gel electrophoresis (PACE).

With the advent of fast genome analysis, many genes encoding novel putative cellulolytic enzymes are being identified in diverse bacterial and fungal genomes. The discovery of these genes calls for quick, robust, and reliable methods for qualitative and quantitative characterization of the enzymatic activities of the encoded proteins. Here, we describe the use of the polysaccharide analysis by carbohydrate gel electrophoresis (PACE) method, which was previously used, among other applications, to characterize various hemicellulose degrading enzymes; for structural elucidation of these carbohydrates; and for analysis of products resulting from enzymatic cleavage of cellulose. PACE relies on fluorescent labeling of mono-, oligo-, and polysaccharides at their reducing end and separation of the labeled carbohydrates by polyacrylamide gel electrophoresis. Labeling can be carried out before or after enzymatic digestion. PACE is very sensitive and allows analysis of both substrate specificities and kinetic properties of cellulolytic enzymes.

Cellulosic ethanol production by combination of cellulase-displaying yeast cells.

As an effort to find suitable endoglucanases to generate cellulolytic yeast strains, two fungal endoglucanases, Thermoascus aurantiacus EGI and Trichoderma reesei EGII, and two bacterial endoglucanases, Clostridium thermocellum CelA and CelD, were expressed on the yeast surface, and their surface expression levels, pH- and temperature-dependent enzyme activities, and substrate specificities were analyzed. T. aurantiacus EGI showed similar patterns of pH- and temperature-dependent activities to those of T. reesei EGII which has been widely used due to its high enzyme activity. Although EGII showed higher carboxymethyl cellulose (CMC) degradation activity than EGI, EGI showed better activity toward phosphoric acid swollen cellulose (PASC). For ethanol production from PASC, we combined three types of yeast cells, each displaying T. aurantiacus EGI, T. reesei CBHII (exoglucanase) and Aspergillus aculeatus BGLI (ß-glucosidase), instead of co-expressing these enzymes in a single cell. In this system, ethanol production can be easily optimized by adjusting the combination ratio of each cell type. A mixture of cells with the optimized EGI:CBHII:BGLI ratio of 6:2:1 produced 1.3 fold more ethanol (2.1g/l) than cells composed of an equal amount of each cell type, suggesting the usefulness of this system for cellulosic ethanol production.

Production of xylanase by a new strain of Thermoascus aurantiacus: obtainment of enzymatic extract with reduced cellulolytic activity for application in pulp and paper industries / Produção de xilanase por nova linhagem de Thermoascus aurantiacus: obtenção de extrato enzimático com reduzida atividade celulolítica para aplicação em indústria de papel e celulose

Xylanases are useful in several industrial segments, including pulp and paper bleaching, animal feed, and bread-making processes. However, the industrial use of these enzymes is closely related to its production cost and its catalytic properties. The process of solid state fermentation enables the use of agro-industrial residues as substrates for microbial cultivation and enzymes production, reducing costs. In the present study, different cultivation parameters were evaluated for the xylanase production by the thermophilic fungus Thermoascus aurantiacus, by solid state fermentation, using agro-industrial residues as substrates. High production of xylanase (1701.9 U g-1 of dry substrate) was obtained using wheat bran containing 65% of initial moisture, at 120 h of cultivation, and 45°C. The xylanase showed optimal activity at pH 5.0 and 75°C; its stability was maintained at pH 3.0­11.0. The enzyme retained its catalytic potential after 1 h, at 75°C. The enzymatic extract produced under optimized conditions showed reduced activities of endoglucanase and FPase. Our results, including the xylanase production by T. aurantiacus in low-cost cultivation medium, high structural stability of the enzyme, and reduced cellulolytic activity, encourage the application of this enzymatic extract in pulp and paper bleaching processes.

As xilanases apresentam aplicabilidade em diferentes segmentos industriais, como: branqueamento de papel e celulose, ração animal e panificação. No entanto, a utilização industrial dessas enzimas está intimamente relacionada com seu custo de produção e suas propriedades catalíticas. O processo de fermentação em estado sólido possibilita o uso de resíduos agroindustriais como substratos, para o cultivo microbiano e produção de enzimas, reduzindo o custo da produção enzimática. No presente trabalho, diferentes parâmetros de cultivo foram avaliados para produção de xilanase por cultivo em estado sólido do fungo termófilo Thermoascus aurantiacus, utilizando resíduos agroindustriais como substratos. A maior produção de xilanase, 1701,9 U g-1 de substrato seco, foi obtida no cultivo em farelo de trigo, contendo 65% de umidade inicial, em 120 horas de cultivo a 45°C. A xilanase produzida apresentou atividade ótima em pH 5,0 a 75°C, mantendo sua estabilidade em pH 3,0 a 11,0. A enzima manteve seu potencial catalítico após 1 h a 75°C. O extrato enzimático produzido nas condições otimizadas apresentou reduzida atividade de endoglucanase e FPase. Os resultados obtidos no presente trabalho (produção de xilanase pelo fungo em meios de cultivo de baixo custo, elevada estabilidade estrutural da enzima e reduzida atividade celulolítica) estimulam a aplicação desse complexo enzimático em processos de branqueamento de papel e celulose.

Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis.

Recombinant xylanase preparations from Nonomuraea flexuosa (Nf Xyn, GH11) and Thermoascus aurantiacus (Ta Xyn, GH10) were evaluated for their abilities to hydrolyze hydrothermally pretreated wheat straw. The GH family 10 enzyme Ta Xyn was clearly more efficient in solubilizing xylan from pretreated wheat straw. Improvement of the hydrolysis of hydrothermally pretreated wheat straw by addition of the thermostable xylanase preparations to thermostable cellulases was evaluated. Clear synergistic enhancement of hydrolysis of cellulose was observed when cellulases were supplemented even with a low amount of pure xylanases. Xylobiose was the main hydrolysis product from xylan. It was found that the hydrolysis of cellulose increased nearly linearly with xylan removal during the enzymatic hydrolysis. The results also showed that the xylanase preparation from T. aurantiacus, belonging to GH family 10 always showed better hydrolytic capacity of solubilizing xylan and acting synergistically with thermostable cellulases in the hydrolysis of hydrothermally pretreated wheat straw.

Xylooligosaccharides production from alkali-pretreated sugarcane bagasse using xylanases from Thermoascus aurantiacus.

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of L-arabinose, 4-O-methyl-D-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50 degrees C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5-3.5% (w/v), enzyme load 40-80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 2(2) factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.