Bacillus amyloliquefaciens DC-4, which produces a strongly fibrinolytic enzyme, was isolated from douchi, a traditional Chinese soybean-fermented food. A fibrinolytic enzyme (subtilisin DFE) was purified from the supernatant of B. amyloliquefaciens DC-4 culture broth and displayed thermophilic, hydrophilic and strong fibrinolytic activity. Subtilisin DFE was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of 28000 Da and a pI of 8.0. The optimal reaction pH value and temperature were 9.0 and 48 degrees C, respectively. Subtilisin DFE not only hydrolyzed fibrin but also several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA, and phenylmethylsulfony fluoride can completely inhibit its fibrinolytic activity. These results indicated that subtilisin DFE is a subtilisin-family serine protease, similar to nattokinase from Bacillus natto. The first 24 amino acid residues of the N-terminal sequence of subtilisin DFE were AQSVPYGVSQIKAPALHSQGFTGS, which is identical to that of subtilisin K-54, and different from that of NK and CK. Results from subtilisin DFE gene sequence analysis showed that subtilisin DFE is a novel fibrinolytic enzyme.