2.1. Isolation of fibrinolytic enzyme from Paenibacillus sp. IND8 The optimized conditions of fibrinolytic enzyme production by Paenibacillus sp. IND8 were described previously [28]. This organism was cultured under solid-state fermentation for 72 h using wheat bran as the substrate. After 72 h, crude enzyme was extracted from the culture medium. The fibrinolytic enzyme was purified from the crude sample by various steps: ammonium sulfate precipitation, dialysis, ion exchange chromatography, and casein-agarose affinity chromatography. All purification steps were performed at 4°C until otherwise stated. The crude enzyme was precipitated at 70% saturation of ammonium sulfate, and the protein was collected by centrifugation (10,000×g for 10 min). It was suspended in minimal volume of double-distilled water. The precipitate was dialyzed and used for enzyme purification using column chromatography. The dialyzed sample was loaded on DEAE-cellulose column, which was equilibrated previously with the buffer A (50 mM sodium phosphate buffer, pH 7.4). The column was washed with the five bed volumes of buffer A, and the enzyme was eluted with buffer A …show more content…
The incubation mixture contained 2.5 ml of 1.2% (w/v) fibrin, 2.5 ml of 100 mM Tris–HCl buffer, 10 mM CaCl2 (pH 7.8), and 20 µg of enzyme. The incubation was carried out at 37°C for 30 min, and the reaction was stopped by adding 5 ml of 110 mM trichloroacetic acid containing 220 mM sodium acetate and 330 mM acetic acid. This reaction mixture was centrifuged at 3,000×g for 5 min, and the absorbance of the trichloroacetic acid (50 mM) soluble product was determined at 275 nm. One unit of fibrinolytic enzyme activity was defined as the amount of enzyme required to liberate 1 µg of L-tyrosine per minute at 37°C. The total protein determination was performed as described by Lowry et al.