Isolation, Extraction, Purification, and Characterization of Fibrinolytic Enzyme from Pseudomonas aeruginosa and Estimation of the Molecular Weight of the Enzyme.

Abstract

Pseudomonas aeruginosa was isolated from injuries of patients' wounds and burns, and to ensure that the isolate was belonging to P. aeruginosa, several tests were performed, such as staining techniques, a biochemical test, morphological test, Vitek 2 system, and sensitivity test. The results of the gram stain test showed rod pink gram-negative bacteria, demonstrating that the isolate belonged to P. aeruginosa. Growth optimization of bacterial was performed by assessing different combinations of pH and temperatures. It is revealed that the best conditions for increasing the number of bacteria were achieved at 37°C with the bacterial number of 5.53×108 and pH 6 with the bacterial number of 5.87×108. Fibrinolytic enzyme is an agent that lysis fibrin clots. This fibrinolytic factor has prospective use to treat cardiovascular diseases, such as stroke and heart attack. Cardiovascular diseases have attracted worldwide attention for their elevation morbidity and mortality. Fibrinolytic enzyme was extracted by centrifugation at 10000 × g at 4°C for 10 min, the supernatant was kept and the pellet having bacterial cells was discarded. Purification of the fibrinolytic enzyme was achieved using salt precipitation, ion exchange, and gel filtration chromatographic techniques. The results showed that the gel filtration chromatography had optimal specific activity and purification fold at 562.6 U/ml, and the final specific activity of the purified enzyme increased 4.1 times. The molecular weight of the fibrinolytic enzyme was determined at26 kDa by gel filtration chromatography. The purified fibrinolytic enzyme had optimum activity atpH 7 and40°C.The pH stability for the enzyme activity was found in pH 6-7 and the range of 10-40°C.