Cellulases are a group of enzymes with several applications in biofuel production, and the paper, food, pharmaceutical, and chemical industries. Trichoderma harzianum P49P11 secrete all cellulases with high efficiency, representing an alternative to the current filamentous fungi in biotechnological industries. In this study, the cellulolytic mechanisms employed by the strain P49P11 to degrade crystalline cellulose in batch fermentation culture mode were elucidated by combining genome and secretome analysis. The strain P49P11 encodes nineteen cellulase genes from five different CAZyme families (GH5, GH6, GH7, GH12, and GH45), followed by several enzyme families for hemicellulose, pectin, and alpha-and beta-glucans degradation. The diverse CAZymes were also observed in the secretome, including cellulases, hemicellulases, and glucanases. In addition, β-glucosidases and xylanase activities detected during the fermentation process validated our secretome analysis. Taken together, our results revealed all enzymatic machinery used by the T. harzianum P49P11 to degrade cellulose in batch fermentation mode.Highlights ### Competing Interest StatementThe authors have declared no competing interest.

This work focused on mitigating carbon catabolic repression (CCR) and increasing cellulase production in Trichoderma harzianum based on the cre1 deletion. The CRE1 protein (encoded by cre1) has been described as a cellulase transcriptional repressor in various cellulotic fungi, but has not been investigated in T. harzianum. We constructed ∆cre1 T. harzianum by replacing the cre1 gene with the amdS gene from Aspergillus nidulans. Quantitative PCR analysis of some Cazymes genes showed that CRE1 acts positively on gh61, bgl1 and xyn2. The fed-batch strategy using hydrothermal sugarcane bagasse by the ∆cre1_Th15 produced a constant rate of FPase under glucose influence, suggesting that the knockout of the carbon catabolite regulator improved the glycoside hydrolases (FPase 1.96 ± 0.32 IU/mL; β-glucosidase 5.67 ± 0.28 IU/mL and xylanase 327. 26 ± 14.25 IU/mL), so that this strain can be used for biorefinery purposes.

Trichoderma harzianum has attracting attention for its potential alternative use in biofuel production, due to a recognized competence for high diversity glycoside hydrolases (GH) enzyme complex, including higher β-glucosidases and auxiliary proteins, using low-cost carbon sources. This strain constitutively overexpressed the global regulator putative methyltransferase - LAE1, in order to improve the GHs production. The recombinant strain achieved 79-fold increase in lae1 expression and high GHs productivity. The evaluation of the LAE1 impact to induce the GHs used soluble and lignocellulose inexpensive carbon sources in a stirred-tank bioreactor. Using sugarcane bagasse with sucrose, the overexpression of lae1 resulted in significantly increment of gh61b (31x), cel7a (25x), bgl1(20x) and xyn3 (20x) genes expression. Reducing sugar released from pretreated sugarcane bagasse, which hydrolyzed by recombinant crude enzyme cocktail, achieved 41% improvement. Therefore, lae1 overexpression effectively is a promising improving GHs target for biomass degradation by T. harzianum.

Production of biomass-degrading enzymes using inexpensive and readily available agricultural residues as substrates for solid-state fermentation (SSF) can contribute to a broader application of enzymes for the conversion of biomass into biofuels and chemicals. Among the operational parameters that affect SSF process efficiency, moisture content is one of the most important. This work evaluates the effect of initial moisture content on two Aspergillus strains (Aspergillus niger P47C3 and Aspergillus fumigatus P40M2), isolated from the Amazon rainforest and grown under SSF. Analyses were made of the biomass-degrading enzymes produced using different agro-industrial residues as carbon sources (wheat bran, sugar cane bagasse, soybean bran, and orange bagasse). The enzymatic complex produced by a selected strain of A. fumigatus was characterized in terms of optimum pH and temperature, and thermal stability. The most effective carbon sources for multienzyme production during Aspergillus cultivation were wheat and soybean bran, as well as a 1:1 mixture of sugar cane bagasse and wheat bran. Much higher activity values were achieved for β-glucosidase (105.8IU/g) and xylanase (1055.6IU/g) when wheat bran with 50% initial moisture content was used as substrate. Under this condition, endoglucanase and total cellulase activity values were 56.6IU/g and 5.0FPU/g, respectively. Characterization of the crude enzymatic complex showed that the A. fumigatus P40M2 enzymes were active in the acidic pH range, with maximal activities at the range of 50–65°C, demonstrating the potential of the organism for the production of acidophilic and thermophilic biomass-degrading enzymes.

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.

The successful strategy to produce cellulolytic enzymes includes both microorganism selection and improved fermentation process conditions. This work describes the isolation, screening and selection of biomass-degrading fungi species from the Amazon forest and analyzes the enzymatic complex produced by a selected strain of Aspergillus fumigatus cultivated using different agro-industrial residues (wheat bran, sugarcane bagasse, soybean bran, and orange peel) as substrate in solid state fermentation (SSF). The profile of endoglucanase (CMCase), FPase, beta-glucosidase and xylanase enzymatic activities obtained during 120 h of cultivation is presented. Enzyme activities up 160.1 IU g(-1) for CMCase, 5.0 FPU g (1) for FPAse, 105.82 IU g (1) for beta-glucosidase and 1055.62 IU g (1) for xylanase were achieved. The enzymatic extract with higher CMCase activity was used to run a zymogram analysis that showed 3 bands of endoglucanase activity. Characterization studies of this extract showed that the CMCase was most active at either 65 degrees C or pH 3-3.5, indicating that this microorganism produces a thermophilic and acid endoglucanase. These data demonstrate that the fungal isolates from the Amazon forest are a potential source of cellulases and xylanases, providing support to further studies related to the use of these microorganisms to obtain the enzymes needed for biomass conversion. (C) 2011 Elsevier Ltd. All rights reserved.