Purification And Characterization Of A Fibrinolytic Enzyme Produced From Bacillus Subtilis

Bacillus subtilis LD-8547, which produced a strong fibrinolytic enzyme, was isolated from douchi, a traditional Chinese fermented food. After fermentation, fibrinolytic enzyme was purified with a series of suitable procedure including salt-out with (NH₄)₂SO₄, gel filtration with Sephadex G-100.Fibrinolytic enzyme purified from the supernatant of fermentation both displayed hydrophilicity and strong fibrinolytic activity. The optimal pH and temperature of this enzyme were 8.0 and 45℃, respectively. The metal ions Na⁺, K⁺, Ca²⁺, Mg²⁺, Cu²⁺, Fe³⁺ showed some inhibition, and Mn²⁺, Ba²⁺ showed stronger inhibition. The metal ions Al³⁺ and Zn²⁺ could increase the fibrinolytic activity. The effects of different organic solvents on the enzyme activity were investigated. The results indicated that methanol inhibited the enzyme activity, and the inhibition effect increased at the concentration of 10%-15% methanol. Alcohol increased enzyme activity a little at low concentration, but inhibited it at high concentration. Isopropyl alcohol had activation effect, which would make activity increased by about 70%.The PCR primer was designed and the fragment encoding the douchi fibrinolytic enzyme (DFE) was amplified from B. subtilis LD-8547 total DNA by PCR. The expression plasmid pET32a-DFE was transformed into E. coli BL21 (DE3). The target protein was highly expressed by IPTG induction. The protein was purified by Ni²⁺-NTA, and the function was also tested. The result showed that this fibrinolytic enzyme had some fibrinolytic activity.