Desenvolvimento de coquetéis enzimáticos a partir de diferentes fungos filamentosos e aplicação na hidrólise da biomassa lignocelulósica

Abstract

The use of biomass has emerged as one of the main strategies for the generation of renewable biofuels and chemicals. In addition, lignocellulosic biorefineries demand enzymatic cocktails of improved efficiency for generating fermentable sugars. In this scenario, lignocellulolytic fungi have enormous potential for the development of efficient enzymatic cocktails. In this work two fungal samples LMI01 and LMI03 isolated from the environment in’ the Amazon region and identified as belonging to the genera Penicillium and Aspergillus, had the fermentation medium optimized for a higher production of cellulases and xylanases. The complete formulation of the fermentation medium for the production of cellulases and xylanases by these isolates was optimized using experimental design and response surface techniques, where the amount of reagents was reduced at the same time as a significant increase in the enzymatic production was achieved. The electrophoretic profile of the enzymatic crude extracts of the Penicillium LMI01 fungus presents proteins with cellulolytic activity of approximate molecular mass ~100kDa to 50kDa e 35kDa, and the Aspergillus LMI03 fungus produces cellulases of ~80 a 52kDa, 37kDa e ~29kDa. The kinetic parameters of the apparent enzymatic activities of Penicillium LMI01 have: Km=17,95 g/L-1 Vmáx =39,84 g/L-1.min-1 (CMCase), Km= 1,67 mMol/L-1 Vmáx = 0,485 mMol/L-1.min-1 (β-glucosidase), Km= 12,54 g/L- 1 Vmáx = 270,02 g/L-1.min-1 (xylanase). Moreover, apparent activities CMCase, β-glucosidase and xylanase of Aspergillus LMI03 showed values of Km: 64, 50 g/L-1, 2, 17 mMol/L-1, 18 g/L- 1 and maximum velocities: 147, 06 g/L-1.min-1, 0, 95 mMol/L-1.min-1, 625 g/L-1.min-1, respectively. The optimized enzymatic cocktail for enzymatic hydrolysis of the pretreated pulp cellulose (cocktail UFAM1) is composed of 43% enzymatic extract of Aspergillus sp LMI03 and 57% of T. reesei QM9414, and the optimum cocktail for enzymatic hydrolysis of cassava husk (cocktail UFAM 2) consists of 50% enzymatic extract of Aspergillus sp LMI03, 25% P.citrinum and 25% T. reesei. The solid protein load is 52g / L of solid and 30mg of protein / g of substrate for cocktail UFAM1 with EH yield of 93,2%. For the cocktail UFAM2, the EH yield was 75,0 % for an optimized loading of 78g / L of solid and 19mg of protein / g of substrate for cocktail UFAM2. These results indicate that the enzymatic cocktails formulated with the proteic enzymatic extracts of P.citrinum LMI01, Aspergillus sp LMI03 and T. reesei may be an interesting alternative for hydrolysis of pretreated or untreated plant biomasses and for other processes that depend on biocatalysis.