Synthesis of supermacroporous cryogel for bioreactors continuous starch hydrolysis.

A bioreactor was built by means of immobilizing alpha-amylase from Aspergillus oryzae by encapsulation, through cryopolymerization of acrylamide monomers for the continuous starch hydrolysis. The starch hydrolysis was evaluated regarding pH, the concentration of immobilized amylase on cryogel, the concentration of starch solution and temperature. The maximum value for starch hydrolysis was achieved at pH 5.0, concentration of immobilized enzyme 111.44 mg amylase /gcryogel , concentration of starch solution 45 g/L and temperature of 35°C. The immobilized enzyme showed a conversion ratio ranging from 68.2 to 97.37%, depending on the pH and temperature employed. Thus, our results suggest that the alpha-amylase from A. oryzae immobilized on cryogel monoliths represents a potential process for industrial production of maltose from starch hydrolysis.

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Arabinanases (ABNs, EC 3.2.1.99) are promising catalysts for environmentally friendly biomass conversion into energy and chemicals. These enzymes catalyze the hydrolysis of the α-1,5-linked L-arabinofuranoside backbone of plant cell wall arabinans releasing arabino-oligosaccharides and arabinose, the second most abundant pentose in nature. In this work, new findings about the molecular mechanisms governing activation, functional differentiation, and catalysis of GH43 ABNs are presented. Biophysical, mutational, and biochemical studies with the hyperthermostable two-domain endo-acting ABN from Thermotoga petrophila (TpABN) revealed how some GH43 ABNs are activated by calcium ions via hyperpolarization of the catalytically relevant histidine and the importance of the ancillary domain for catalysis and conformational stability. On the other hand, the two GH43 ABNs from rumen metagenome, ARN2 and ARN3, presented a calcium-independent mechanism in which sodium is the most likely substituent for calcium ions. The crystal structure of the two-domain endo-acting ARN2 showed that its ability to efficiently degrade branched substrates is due to a larger catalytic interface with higher accessibility than that observed in other ABNs with preference for linear arabinan. Moreover, crystallographic characterization of the single-domain exo-acting ARN3 indicated that its cleavage pattern producing arabinose is associated with the chemical recognition of the reducing end of the substrate imposed by steric impediments at the aglycone-binding site. By structure-guided rational design, ARN3 was converted into a classical endo enzyme, confirming the role of the extended Arg(203)-Ala(230) loop in determining its action mode. These results reveal novel molecular aspects concerning the functioning of GH43 ABNs and provide new strategies for arabinan degradation.

Oligomeric state and structural stability of two hyperthermophilic ß-glucosidases from Thermotoga petrophila.

The ß-glucosidases are enzymes essential for several industrial applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts. In a recent study, we have provided a biochemical characterization of two hyperthermostable ß-glucosidases from Thermotoga petrophila belonging to the families GH1 (TpBGL1) and GH3 (TpBGL3). Here, as part of a continuing investigation, the oligomeric state, the net charge, and the structural stability, at acidic pH, of the TpBGL1 and TpBGL3 were characterized and compared. Enzymatic activity is directly related to the balance between protonation and conformational changes. Interestingly, our results indicated that there were no significant changes in the secondary, tertiary and quaternary structures of the ß-glucosidases at temperatures below 80 °C. Furthermore, the results indicated that both the enzymes are stable homodimers in solution. Therefore, the observed changes in the enzymatic activities are due to variations in pH that modify protonation of the enzymes residues and the net charge, directly affecting the interactions with ligands. Finally, the results showed that the two ß-glucosidases displayed different pH dependence of thermostability at temperatures above 80 °C. TpBGL1 showed higher stability at pH 6 than at pH 4, while TpBGL3 showed similar stability at both pH values. This study provides a useful comparison of the structural stability, at acidic pH, of two different hyperthermostable ß-glucosidases and how it correlates with the activity of the enzymes. The information described here can be useful for biotechnological applications in the biofuel and food industries.

Assembling a xylanase-lichenase chimera through all-atom molecular dynamics simulations.

Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.

Boto, a class II transposon in Moniliophthora perniciosa, is the first representative of the PIF/Harbinger superfamily in a phytopathogenic fungus.

Boto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6-12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168.

Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.

Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-ß-D-mannanase.

The breakdown of ß-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of ß-mannanases. In this work, a two-domain ß-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/ß)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-ß-1,3-glucanase from Thermotoga petrophila.

1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal ß-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

A novel mechanism of ß-glucosidase stimulation through a monosaccharide binding-induced conformational change.

It is urgent the transition from a fossil fuel-based economy to a sustainable bioeconomy based on bioconversion technologies using renewable plant biomass feedstocks to produce high chemicals, bioplastics, and biofuels. ß-Glucosidases are key enzymes responsible for degrading the plant cell wall polymers, as they cleave glucan-based oligo- and polysaccharides to generate glucose. Monosaccharide-tolerant or -stimulated ß-glucosidases have been reported in the past decade. Here, we describe a novel mechanism of ß-glucosidase stimulation by glucose and xylose. The glycoside hydrolase 1 family ß-glucosidase from Thermotoga petrophila (TpBgl1) displays a typical glucose stimulation mechanism based on an increased Vmax and decreased Km in response to glucose. Through molecular docking and dynamics analyses, we mapped putative monosaccharide binding regions (BRs) on the surface of TpBgl1. Our results indicate that after interaction with glucose or xylose at BR1 site, an adjacent loop region assumes an extended conformation, which increases the entrance to the TpBgl1 active site, improving product formation. Biochemical assays with TpBgl1 BR1 mutants, TpBgl1D49A/Y410A and TpBgl1D49K/Y410H, resulted in decreasing and abolishing monosaccharide stimulation, respectively. These mutations also impaired the BR1 looping extension responsible for monosaccharide stimulation. This study provides a molecular basis for the rational design of ß-glucosidases for biotechnological applications.

Diet Supplementation with Madagascar Cockroach Flour (Gromphadorhina portentosa) Improved Malnourished Mice Metabolism and Ameliorated Liver Inflammatory Markers.

BACKGROUND: Malnutrition and accessible high-quality protein food sources are two of the world's alimentary challenges. Edible insects are nowadays recognized as a possible functional food solution with lower environmental impacts and beneficial health effects. OBJECTIVE: In this context, the aim of the present study is to evaluate Madagascar cockroach (Gromphadorhina portentosa) flour supplementation effects on a malnourished mice model, considering its effects on metabolism, adiposity, and inflammatory liver profiles. METHOD: Male Swiss mice are divided into five groups and fed with experimental diets for eight weeks, including a standard diet (ST) ad libitum, AIN93 ad libitum (AIN), insect flour-enriched AIN93 (AIN+IM), AIN-40% feed restriction (AIN-FR), and insect flour-enriched AIN-40% of feed restriction (AIN-FR+IM). The metabolic profiles, adipose tissue, biochemical parameters, and liver IL-6 and IL-10 expression are evaluated. RESULTS: The main findings show a body weight and metabolism improvement followed by an increased recovery of the adipocyte area in the AIN-FR+IM group when compared to the AIN-FR malnourished group. Reduced hepatic IL-6 and increased IL-10 expression are also detected in the AIN-FR+IM group. CONCLUSION: The results show that insect flour supplementation enhances both body weight and adiposity gain/recovery. The results also show hepatic improvement of inflammatory markers.

Substrate cleavage pattern, biophysical characterization and low-resolution structure of a novel hyperthermostable arabinanase from Thermotoga petrophila.

Arabinan is a plant structural polysaccharide degraded by two enzymes; alpha-l-arabinofuranosidase and endo-1,5-alpha-l-arabinanase. These enzymes are highly diversified in nature, however, little is known about their biochemical and biophysical properties. We have characterized a novel arabinanase (AbnA) isolated from Thermotoga petrophila with unique thermostable properties such as the insignificant decrease of residual activity after incubation up to 90 degrees C. We determined the AbnA mode of operation through capillary zone electrophoresis, which accumulates arabinotriose and arabinobiose as end products after hydrolysis of arabinan-containing polysaccharides. Spectroscopic analyses by Far-UV circular dichroism and intrinsic tryptophan fluorescence emission demonstrated that AbnA is folded and formed mainly by beta-sheet structural elements. In silico molecular modeling showed that the AbnA structure encompasses a five-bladed beta-propeller catalytic core juxtaposed by distorted up-and-down beta-barrel domain. The low-resolution structure determined by small angle X-ray scattering indicated that AbnA is monomeric in solution and its molecular shape is in full agreement with the model.

The Angiotensin Converting Enzyme 2 (ACE2), Gut Microbiota, and Cardiovascular Health.

BACKGROUND: The renin-angiotensin system (RAS) is an important enzymatic system responsible for the regulation of biological functions, such as the arterial pressure, hydroelectrolytic control, vascular vasodilatation/vasoconstriction and more recently metabolic functions. OBJECTIVES: The aim of the present review is to discuss the associations between the gut microbiome and the renin-angiotensin system and the influence of their intimate relationship on the cardiovascular health. METHODS: A literature review of the main studies published regarding the relationship among the renin-angiotensin system, gut microbiota and cardiovascular health was performed. RESULTS: The association between the ACE2 and gut microbiota has been discussed. It is shown that the ACE2/Ang 1-7 axis modulates the immune response, influencing the microbiota composition, and thus being one of the causes for some diseases physiophatologies, such as diarrhea and intestinal inflammatory disease. CONCLUSION: The association between RAS and gut microbiota seems to have a strong influence on the genesis of cardiovascular diseases, through direct mechanisms, such as nerve stimulation, or indirectly on metabolic parameters, such as weight, adiposity and lipid profile.

Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries.

BACKGROUND: During the past few years, the first industrial-scale cellulosic ethanol plants have been inaugurated. Although the performance of the commercial cellulase enzymes used in this process has greatly improved over the past decade, cellulases still represent a very significant operational cost. Depending on the region, transport of cellulases from a central production facility to a biorefinery may significantly add to enzyme cost. The aim of the present study was to develop a simple, cost-efficient cellulase production process that could be employed locally at a Brazilian sugarcane biorefinery. RESULTS: Our work focused on two main topics: growth medium formulation and strain improvement. We evaluated several Brazilian low-cost industrial residues for their potential in cellulase production. Among the solid residues evaluated, soybean hulls were found to display clearly the most desirable characteristics. We engineered a Trichoderma reesei strain to secrete cellulase in the presence of repressing sugars, enabling the use of sugarcane molasses as an additional carbon source. In addition, we added a heterologous ß-glucosidase to improve the performance of the produced enzymes in hydrolysis. Finally, the addition of an invertase gene from Aspegillus niger into our strain allowed it to consume sucrose from sugarcane molasses directly. Preliminary cost analysis showed that the overall process can provide for very low-cost enzyme with good hydrolysis performance on industrially pre-treated sugarcane straw. CONCLUSIONS: In this study, we showed that with relatively few genetic modifications and the right growth medium it is possible to produce considerable amounts of well-performing cellulase at very low cost in Brazil using T. reesei. With further enhancements and optimization, such a system could provide a viable alternative to delivered commercial cellulases.

Xylan-specific carbohydrate-binding module belonging to family 6 enhances the catalytic performance of a GH11 endo-xylanase.

Xylanases catalyze the hydrolysis of ß-1,4-linked xylosyl moieties from xylan chains, one of the most abundant hemicellulosic polysaccharides found in plant cell walls. These enzymes can exist either as single catalytic domains or as modular proteins composed of one or more carbohydrate-binding modules (CBMs) appended to the catalytic core. However, the molecular mechanisms governing the synergistic effects between catalytic domains and their CBMs are not fully understood. Thus, the goal of this study was to evaluate the functional effects of the fusion of a CBM belonging to family 6, which exhibits high affinity to xylan, with the GH11 xylanase from Bacillus subtilis, which does not have a CBM in its wild-type form. The wild-type enzyme (BsXyl11) and the chimeric protein (BsXyl11-CBM6) were heterologously produced in Escherichia coli and purified to homogeneity for biochemical characterization. The molecular fusion did not alter the pH and temperature dependence, but kinetic data revealed an increase of 65% in the catalytic efficiency of the chimeric enzyme. Furthermore, the BsXyl11-CBM6 chimera was used to supplement the commercial cocktail Accellerase® 1500 and improved the reducing sugar release by 17% from pretreated sugarcane bagasse. These results indicate that CBM6 can be used as a molecular tool to enhance the catalytic performance of endo-xylanases (GH11) and provide a new strategy for the development of optimized biocatalysts for biotechnological applications.

Metformin Reduces Lipogenesis Markers in Obese Mice Fed a Low-Carbohydrate and High-Fat Diet.

Lipogenesis is the process by which fatty acids are synthesized. In metabolic syndrome, an insulin resistant state along with high plasma levels of free fatty acids (FFA) and hyperglycemia may contribute to the lipogenic process. The aim of the present study was to investigate the effects of oral administration of metformin on the expression of lipogenic genes and glycemic profile in mice fed with low-carbohydrate high-fat diet by evaluating their metabolic profile. SWISS male mice were divided into 4 groups (N = 7) that were fed with standard (ST), standard plus metformin (ST + MET), low-carbohydrate high-fat diet (LCHFD) and low-carbohydrate high-fat diet plus metformin (LCHFD + MET) (100 mg kg-1 diet) diets respectively. Food intake, body weight and blood parameters, such as glucose tolerance, insulin sensitivity, glucose, HDL-c, total cholesterol, triglycerides, ASL and ALT levels were assessed. Histological analyses were performed on hematoxylin and eosin-stained epididymal adipose tissue histological specimens. The expression levels of peroxisome proliferator-activated receptor (PPARγ), sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), were assessed by RT-PCR. This study showed that metformin decreased adipocyte area, body weight and food consumption in obese animals when compared to the standard group. Furthermore, the expression of lipogenic markers in adipose tissue were diminished in obese animals treated with metformin. This data showed that oral administration of metformin improved glucose and lipid metabolic parameters in white adipose tissue by reducing the expression of lipogenesis markers, suggesting an important clinical application of MET in treating obesity-related diseases in metabolic syndrome.

Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential.

Beta-glucosidases (BGLs) are enzymes of great potential for several industrial processes, since they catalyze the cleavage of glucosidic bonds in cellobiose and other short cellooligosaccharides. However, features such as good stability to temperature, pH, ions and chemicals are required characteristics for industrial applications. This work aimed to provide a comparative biochemical analysis of three thermostable BGLs from Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1 (GH1 from P. furiosus), TpBgl1 (GH1 from T. petrophila) and TpBgl3 (GH3 from T. petrophila) were cloned and proteins were expressed in Escherichia coli. The purified enzymes are hyperthermophilic, showing highest activity at temperatures above 80°C at acidic (TpBgl3 and PfBgl1) and neutral (TpBgl1) pHs. The BGLs showed greatest stability to temperature mainly at pH 6.0. Activities using a set of different substrates suggested that TpBgl3 (GH3) is more specific than GH1 family members. In addition, the influence of six monosaccharides on BGL catalysis was assayed. While PfBgl1 and TpBgl3 seemed to be weakly inhibited by monosaccharides, TpBgl1 was activated, with xylose showing the strongest activation. Under the conditions tested, TpBgl1 showed the highest inhibition constant (Ki=1100.00mM) when compared with several BGLs previously characterized. The BGLs studied have potential for industrial use, specifically the enzymes belonging to the GH1 family, due to its broad substrate specificity and weak inhibition by glucose and other saccharides.

Development of a chimeric hemicellulase to enhance the xylose production and thermotolerance.

Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-ß-xylanase fused to a GH43 ß-xylosidase, both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the ß-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis.

Effects of high pressure homogenization on the activity, stability, kinetics and three-dimensional conformation of a glucose oxidase produced by Aspergillus niger.

High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5-6.0 and a remarkable activity increase (30-300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.

Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and α-L-arabinofuranosidase.

Supplementation of cellulase cocktails with accessory enzymes can contribute to a higher hydrolytic capacity in releasing fermentable sugars from plant biomass. This study investigated which enzymes were complementary to the enzyme set of Trichoderma harzianum in the degradation of sugarcane bagasse. Specific activities of T. harzianum extract on different substrates were compared with the extracts of Penicillium echinulatum and Trichoderma reesei, and two commercial cellulase preparations. Complementary analysis of the secretome of T. harzianum was also used to identify which enzymes were produced during growth on pretreated sugarcane bagasse. These analyses enabled the selection of the enzymes pectinase and α-L-arabinofuranosidase (AF) to be further investigated as supplements to the T. harzianum extract. The effect of enzyme supplementation on the efficiency of sugarcane bagasse saccharification was evaluated using response surface methodology. The supplementation of T. harzianum enzymatic extract with pectinase and AF increased the efficiency of hydrolysis by up to 116%.