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SIQUEIRA, A. C. R. Production of native peptidase and lipase from Fusarium oxysporum and obtainment of a recombinant chimera formed by peptidase and lipase expressed in Pichia pastoris. 2017. 116f. Doctoral Thesis. School of Pharmaceutical Sciences of Ribeirão Preto – University of São Paulo, Ribeirão Preto, 2017. Hydrolases, especially peptidases and lipases, are responsible for several industrial and biotechnological applications therefore high income in the world market. The main source of these proteins is wild-type microorganisms by bioprocesses or by heterologous expression. In this context, there is an incessant search for more stable enzymes with higher catalytic activity, and some techniques have been used to improve these parameters. Obtainment of peptidase and lipase by the fungus Fusarium oxysporum was performed by submerged bioprocess, generating peaks of 165 U / mL in 72 hours and 633 U / mL in 48 hours, respectively. Peptidase was purified using the Sephadex G-50 size exclusion resin and characterized using a peptide substrate with intramolecular fluorescence suppression. The enzyme class was determined as serine peptidase and demonstrated neutral and stable characteristics in a wide range of pH. The optimum temperature was 50 ° C and the thermal stability assays showed the maintenance of the proteolytic activity from 60 to 90% up to 60 ° C within one hour of exposure. As for catalytic efficiency, the S1, S2 and S'1 subsites have a certain specificity, since they do not demonstrate catalytic efficiency when the following amino acids are present in the respective positions of the substrates P1 (aspartic acid, proline and isoleucine), P2 (aspartic acid, Histidine, lysine, asparagine and tryptophan) and P'1 (aspartic acid, glutamic acid and proline), unlike subsites S3, S'2 and S'3, where all amino acids showed catalytic efficiency. The lipase was purified using ionic resin and presented basic characteristics and stability at neutral pH, its optimum temperature was 35 ° C and maintenance of the catalytic activity by 50% after 1 hour of exposure at 25 to 40 ° C. Production of the chimera was done by the search of a peptidase and a lipase from the fungus F. oxysporum in the database, they were then fused using a linker composed of five amino acids (GGAGG) in the C-terminal regions of the peptidase and NTerminal portion of the lipase. Both activities were detected in the chimera, making it functional. The biotechnological application of both wild and recombinant enzymes is an important step for innovation, and according to the characteristics presented each enzyme can be applied to several industrial processes.

Produção de peptidase e lipase nativas por Fusarium oxysporum e obtenção de uma quimera recombinante de peptidase e lipase expressa em Pichia pastoris