Xylanases catalyze the random hydrolysis of xylan backbone from plant biomass and thus, they have application in the production of biofuels, Kraft pulps biobleaching and feed industry. Here, xylanases derived from Orpinomyces sp. PC-2 were engineered guided by molecular dynamics methods to obtain more thermostable enzymes. Based on these models, 27 amino acid residues from the N-terminal were predicted to reduce protein stability and the impact of this removal was validated to two enzyme constructs: small xylanase Wild-Type (SWT) obtained from Wild-Type xylanase (WT) and small xylanase Mutant (SM2) generated from M2 mutant xylanase (V135A, A226T). The tail removal promoted increase in specific activity of purified SWT and SM2, which achieved 5,801.7 and 5,106.8Umg of protein, respectively, while the WT activity was 444.1Umg of protein. WT, SWT and SM2 showed half-life values at 50°C of 0.8, 2.3 and 29.5h, respectively. Overall, in view of the results, we propose that the presence of non-structured amino acid in the N-terminal leads to destabilization of the xylanases and may promote less access of the substrate to the active site. Therefore, its removal may promote increased stability and enzymatic activity, interesting properties that make them suitable for biotechnological applications.

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Different lignocellulosic biomasses are found worldwide and each country has its own important industrial crop that can be converted into high-value products, such as ethanol. Therefore, evaluation of new biomasses to be used in biorefineries is important to decrease the dependence on non-renewable resources and to guarantee sustainable development. This work evaluated Brachiaria brizantha, a grass commonly used as animal forage, and the standard biomass for 2G-ethanol, sugarcane bagasse. The chemical compositions of both biomasses were determined and different times and temperature of acid pretreatment were tested. Morphological analysis via scanning electron microscopy showed more deconstructed fibers after harsher biomass pretreatments. Simultaneous saccharification and fermentation of pretreated Brachiaria brizantha presented higher efficiency than when using sugarcane bagasse as the carbon source. A biomass conversion of 46 % was achieved when Brachiaria brizantha grass was pretreated with 2% sulfuric acid for 60 minutes. Moreover, fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural. It was concluded that Brachiaria brizantha is a promising biomass for ethanol production.