Produção, extração e purificação integrada de proteases com atividade fibrinolítica produzida por fungos do gênero Rhizopus e sua caracterização bioquímica

Abstract

Enzymes with fibrinolytic activity are obtained from different sources and can degrade fibrin, the main protein component of blood clots. The accumulation of fibrin in vessels can lead to thrombosis, a disease that occurs when clots form without bleeding. The work in question has as objectives to produce and purify proteases with fibrinolytic activity from filamentous fungi by submerged fermentation; carry out purification processes through an aqueous two-phase system (PEG/Citrate), in addition to biochemically characterize the fibrinolytic enzyme obtained from Rhizopus arrhizus UCP 1605. For production, a submerged fermentation was carried out with fungi of the genus Rhizophus using a complete 2³ factorial design to determine the best production conditions. The influence of the variables substrate type (TS), substrate concentration (CS) and glucose concentration (GC) were evaluated under the production of fibrinolytic proteases. The produced enzyme was extracted by an aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and sodium citrate, being carried out according to a 24 factorial design, to evaluate the influence of the variables: molar mass of PEG (MPEG), PEG concentration (CPEG), sodium citrate concentration (Ccit) and pH, on the partition coefficient (K), recovery (Y%) and purification factor (FP). Then the enzyme was characterized of biochemical and kinetic parameters. A 24 factorial design was also carried out to evaluate the influence of MPEG, CPEG, Ccit and pH on K and Y in extractive fermentation. The fibrinolytic protease from Rhizopus arrhizus UCP 1605 was produced under the conditions of 3% soybean and 0.5% glucose, under stirring at 120 rpm, at 30ºC for 96 hours of submerged fermentation, showing a protease activity of 10.37 U /mL and a fibrinolytic activity with a halo of 31 mm, corresponding to an activity of 850.60 U/mL. As for extraction in ATPS, the best results were obtained in the assay formed by 24.0% (m/m) of PEG 8000, 15% (m/m) of sodium citrate and pH 8. In this condition, the enzyme preferentially partitioned to the PEG-rich phase, with a K of 1.58, FP 4.07, Y of 97.0% and a fibrinolytic activity with a halo of 16 mm, corresponding to an activity of 44.39 U/ml. In the extractive fermentation process, the enzyme partitioned for both phases, with the best condition in the test composed of PEG 8000 g/mol, at a concentration of 20%, 15% citrate and pH 8.0, with a K of 2.4 and activity recovery (Y) 71.5% and 3.83 U/mL of activity protease. The enzyme present in both the enzymatic extract and the ATPS showed an optimal temperature of 50ºC and an optimal pH of 8. As for the kinetic parameters, the enzymatic extract presented a Michaelis Menten constant (Km) of 4.06 mg/mL with a maximum velocity of 45.05 U/mL. The enzyme obtained by extractive fermentation wasn't biochemically characterized. These results demonstrate the potential of the production of Rhizopus arrhizus UCP 1605 in submerged fermentation and of the fibrinolytic proteases extraction processes and their possibility to be explored in industrial applications as candidates for thrombolytic agents.