Resumo

Fibrinolytic enzymes have received attention owing to their medicinal potential for thrombolytic diseases. These are becoming a leading cause of morbidity and mortality worldwide yet most of current fibrinolytic agents available for clinic are barely satisfactory. Various natural active enzymes purified from animal, plant and microbial sources have been extensively studied. Aqueous two-phase partitioning can be used to separate and extract proteins from cell debris or to purify proteins from other proteins. Extraction of fibrinolytic protease produced by the fungal specie Mucor subtilissimus SIS 42 at submerse fermentation was carried using a PEG/ammonium sulphate aqueous two-phase system (ATPS). A 2³ full factorial design was used to investigate the influence of PEG molar mass, PEG concentration and salt concentration on the responses, namely partition coefficient (K), activity yield (Y) and purification factor (PF). The ATPS was composed of PEG (molar mass of 400, 3350 and 8000g/mol) concentrations 15.0, 17.5 and 20.0% (w/w) and ammonium sulphate concentrations 15, 20 and 25% (w/w). The results of the extraction of the fibrinolytic protease from the fermentation broth showed partition coefficient (K=1.5) and in the top phase rich in polymer was obtained (Y) of 79.2 % and (PF) of 12.2. The parameters of this assay were: PEG 8000, 15% and ammonium sulphate concentration 25%. The statistical analysis of the partition coefficient (K) showed that the independent variable ammonium sulphate concentration (%) significantly influenced this response exerted the greatest effect. It was found that increasing the concentration of salt and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the top phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. The practical application of ATPS has been demonstrated in a large number of cases including a number of industrial applications with excellent levels of purity and yield. Furthermore, higher values of purification and yield of this enzyme suggests suitable application of fibrinolytic protease from this strain in pharmaceutical industry as a thrombolytic agent. Although comprehensive studies on the optimization for fibrinolytic enzyme extraction and characterization have been in progress.