Purification and Characterization of a Major Fibrinolytic Enzyme from Bacillus amyloliquefaciens MJ5-41 Isolated from Meju

Abstract

Meju is a traditional Korean fermented soy product used as a key element for soy sauce and doenjang. Bacilli with antimicrobial activity were isolated from meju prepared by traditional methods at Sunchang county, Jeollabukdo, Korea. Six isolates were identified as Bacillus amylaliquefaciens by recA gene sequencing and RAPD PCR. One isolate, B. amylaliquefaciens MJ5-41, showed the strongest fibrinolytic activity. A 27 kDa active fibrinolytic enzyme, AprE5-41, was purified from the culture supernatant of MJ5-41 grown on LB by chromatographic methods. The optimum pH and temperature for purified AprE5-41 were 7.0 and 45 degrees C, respectively. AprE5-41 quickly degraded A alpha and B beta chains but not the gamma-chain of fibrinogen. AprE5-41 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide, a known substrate for alpha-chymotrypsin, cathepsin G, and subtilisin BPN'. The structural gene, aprE5-41, was cloned by PCR and successfully expressed in B. subtilis.