Produção, purificação e caracterização bioquímica de Pleurotus pulmonarius (Pleurotaceae)

Abstract

Among cardiovascular diseases (CVD), thrombosis accounts for 30% of 70% of global deaths and is caused by the formation and/or development of blood clots in veins and arteries due to the accumulation of fibrin. Fibrinolytic proteases dissolve clots by activating plasmin. Among the most varied sources of obtaining these enzymes, edible mushrooms belonging to the genus Pleurotus sp. stand out as producers of enzymes with biotechnological potential. The objective of this study was initially to detect and quantify, among different culture media and methods, the production of fibrinolytic proteases, evaluate the level of cytotoxicity of the extracts obtained, purify and determine the molecular size and characterize biochemically the selected extract. For this purpose, four Amazonian fruit residues, three food products and three synthetic culture media were used to obtain the proteases. In all substrates evaluated, the production of proteases with fibrinolytic action was detected. The culture media with the highest fibrinolytic activity content were: coffee grounds residue - PPBB, 97.71 U/mL, oat bran - PPFA, 168.31 U/mL and the synthetic culture medium Malt Extract - PPEM, 173.20 U/mL. Then, all P. pulmonarius extracts were subjected to toxicity analysis using human fibroblast cell lines (MRC-5) for 72 hours. Among the 10 substrates analyzed, 2 substrates presented morphological alterations when compared to the negative control (DMSO). PPEM was then lyophilized and semipurified. Of the 21 fractions collected, fraction 1G11 presented the highest specific proteolytic and fibrinolytic activity. This fraction was then reinjected into the AKTA Avant system under the same initial conditions, obtaining a single active peak with 51% protease recovery and 5.94-fold purification (specific activity of 70.24 U/mL). Under the experimental conditions used, this fraction presented two distinct bands with molecular weights of 48 and 63 KDa, respectively, via SDS-PAGE, suggesting a homogeneous enzymatic fraction under non-reducing conditions, presenting optimal activity at pH 8.0, at 50 °C, being classified as metalloprotease, zinc and calcium dependent and partial inhibition of activity in the presence of methanol, isopropanol, acetone, butanol, acetonitrile, DMSO, Triton X 100 and CTAB. The action of these biocatalysts was inhibited by the presence of Fe+2. Thus, it was possible to conclude that the edible mushroom species P. pulmonarius produced proteases with fibrinolytic action in both cultivation methods evaluated, demonstrating the biotechnological potential of this mushroom species in the production of proteases with fibrinolytic action.

Keywords: Thrombosis, residues, culture media, proteases.