Characterization of a Fibrinolytic Enzyme Secreted by Bacillus velezensis BS2 Isolated from Sea Squirt Jeotgal

Bacillus sp. BS2 showing strong fibrinolytic activity was isolated from sea squirt (munggae) jeotgal, a traditional Korean fermented seafood. BS2 was identified as B. velezensis by molecular biological methods. B. velezensis BS2 grows well at 15% NaCl and at 10oC. When B. velezensis BS2 was cultivated in TSB broth for 96 h at 37°C, the culture showed the highest fibrinolytic activity (131.15 mU/µl) at 96 h. Three bands of 27, 35 and 60 kDa were observed from culture supernatant by SDS-PAGE, and fibrin zymography showed that the major fibrinolytic protein was the 27 kDa band. The gene ( aprEBS2 ) encoding the major fibrinolytic protein was cloned, and overexpressed in heterologous hosts, B. subtilis WB600 and E. coli BL21 (DE3). B. subtilis ransformant showed 1.5-fold higher fibrinolytic activity than B. velezensis BS2. Overproduced AprEBS2 in E. coli was purified by affinity chromatography. The optimum pH and temperature were pH 8.0 and 37°C, respectively. K m and V max were 0.15 mM and 39.68 µM/l/min, respectively, when N- succinyl -Ala-Ala-Pro-Phe-pNA was used as the substrate. AprEBS2 has strong α-fibrinogenase and moderate β-fibrinogenase activity. Considering its high fibrinolytic activity, significant salt tolerance, and ability to grow at 10°C, B. velezensis BS2 can be used as a starter for jeotgal.