Produção, purificação e aplicação de proteases produzidas por fungos filamentosos isolados de solos da caatinga de Pernambuco

Abstract

This study aimed to evaluate the production, purification characterization of proteases produced by filamentous fungi isolated from Caatinga. 32 strains of filamentous fungi were previously selected from the best protease producer and the best culture conditions for the microorganisms were determined in shake flasks using a factorial design 23, the influence of the independent variables were evaluated: glucose and soy concentrations, and pH on proteinase activity. Based on the obtained results from the fermentation in shake flasks, a a factorial design 22 was performed in a bioreactor type stirred tank of 1.5L to verify the influence of the independent variables: agitation and aeration in the protease production. The resulting metabolic liquid from the fermentation was used in the following purification steps using aqueous two phases system (ATPS) and chromatographic methods. Another protease with fibrinolytic activity was studied and proprieties analyzed effect on coagulation, stability with solvents and the structure on different pH and fibrinogenolytic activity.The maximum protease production (69.77 U/ml) for shake flasks was obtained under the following conditions: 6.0 of pH, glucose 0.5% and soy 3%. On stirred-tank bioreactor maximum protease activity it was obtained 61.19 U/mL after 72 h of cultivation, under conditions of agitation and aeration at 450 rpm and 0.5 vvm. The protease characterization revealed that the optimum temperature of the enzyme was 50 °C and the activity was 75% when incubated for 3 hours at 20, 30 and 40 °C. The enzyme activity was slightly inhibited by metal ions (ZnSO4 and CuSO4) and phenylmethylsulfonyl fluoride (PMSF), indicate be a serine protease. The enzyme was parcial purified by 24% (w/w) PEG 400 and 20% (w/w) citrate system, at pH 6 the proteases preferably partitioned to the top phase, resulting in 146% recovery and a 1.6-fold increase in specific activity. The ATPS combined with varied chromatography methods reached 8 fold purified and a 12.5% recovery. The fibrinolytic protease on coagulation time was analyzed, a prolonged effect over time was observed in the activated partial thromboplastin time (APTT), indicating an inhibition of intrinsic pathway coagulation and the prothrombin time (PT) was less sensitive to time variation, even at high enzymatic concentration. The fibrinolytic protease was more stable in the presence of ethyl acetate solvents and less stable in the presence of ethanol. When fibrinogenolytic activity was evaluated, a preferential action of the fibrinolytic protease on the Aα and Bβ chains occurred. The fibrinolytic protease presented a structural disorder at pH scales with higher acidity and alkalinity. The two fungal enzymes studied present potential for application in the biotechnology industry.