Produção, extração, purificação e caracterização de proteases fibrinolíticas produzidas por Streptomyces parvulus DPUA 1573

Abstract

Due to their fibrin degradation potential, fibrinolytic proteases are a promising alternative in the pharmaceutical industry for the treatment of cardiovascular diseases, especially thrombosis. There are several sources of fibrinolytic proteases, however, the microbial sources are the ones that stand out in terms of low cost and high production rates. From their production to application, enzymes need to go through several processes, which sounds negative, making the steps more costly and late. A method capable of overcoming these problems is the aqueous two-phase system (SDFA), a process capable of reducing downstream steps. The objective of this work was to produce, purify and biochemically characterize the fibrinolytic protease produced by Streptomyces parvulus DPUA 1573. The protease was produced by submerged fermentation using agro-industrial waste or co-products. The crude extract that showed the highest enzymatic activity (passion fruit peel flour) was subjected to extraction by SDFA consisting of polyethylene glycol (PEG) and phosphate salts (potassium and sodium), following a 24 plan. After extraction by SDFA, the protease was subjected to purification by gel filtration chromatography, and already purified had its biochemical characterization performed. The protease produced by S. parvulus DPUA 1573 showed fibrinolytic activity of 15.46 U/mL and was able to form a halo of 317.31 mm2 acting on fibrin degradation. In SDFA, the fibrinolytic protease partitioned preferentially to the PEG-rich phase. The best assay selected according to the combination of the highest specific activity index, purification factor and activity yield was 16, composed of PEG 8,000 gmol-1, 17.5 v/v PEG, pH 8.0 and 15 v/v of phosphate salts. The protease activity of the enzyme was highly stimulated in the presence of iron, reaching a 55% increase in activity, and drastically decreased in the presence of the protease inhibitor 2-mercaptoethanol (91%). The optimum temperature and pH for the enzymatic activity were 40ºC and pH 7.0, respectively, keeping the enzyme activity stable between 30ºC and 60ºC and in the pH range from 7.0 to 8.5. Based on the analyzed results, it was seen that S. parvulus DPUA 1573 proved to be a good producer of fibrinolytic proteases, and the PEG/Phosphate aqueous two-phase system proved to be a great alternative for the extraction and pre-purification of fibrinolytic proteases.