Partial purification of fibrinolytic and fibrinogenolytic protease from Gliricidia sepium seeds by aqueous two-phase system

Abstract Fibrinolytic proteases are enzymes that provide a promising alternative to drugs for thrombolytic therapy, since they degrade fibrin, the major component of blood clots. Aqueous two-phase systems (ATPS) using polyethylene glycol (PEG) 8000 and sodium phosphate were used to select optimal conditions for extracting a cysteine protease with fibrinolytic and fibrinogenolytic activity from the legume Gliricidia sepium PBSGS. For this purpose, extraction tests were conducted in accordance with a 24-factorial design, in which concentrations of PEG (CPEG), molar mass of PEG, concentrations of sodium phosphate (CPHO) and pH were used as independent variables, and partition coefficient, activity yield and purification factor as responses. The best results in terms of enzyme partial purification were obtained at CPEG of 12.5% (w/w), CPHO of 15% (w/w) and pH 8.0, which guaranteed a purification factor of 6.26, a partition coefficient of 1.32 and a yield of 141.57%. The results clearly showed preferential protease partitioning to the PEG-rich top phase. The enzyme, which was able to degrade human fibrinogen, was characterized as a cysteine protease (66 kDa) and exhibited optimum activity temperature of 60 °C and pH of 8.0; in addition, its activity was increased in the presence of potassium, iron, zinc and sodium ions and reduced in the presence of calcium ion. The results obtained suggest potential of ATPS-partially purified protease in thrombolytic treatment, which will be the object of future investigation.