Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food

Comp Biochem Physiol B Biochem Mol Biol. 2003 Jan;134(1):45-52. doi: 10.1016/s1096-4959(02)00183-5.


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.

MeSH terms

  • Amino Acid Sequence
  • Bacillus / enzymology*
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Fibrinogen / chemistry
  • Fibrinolytic Agents / pharmacology
  • Humans
  • Hydrogen-Ion Concentration
  • Isoelectric Focusing
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Serine Endopeptidases / pharmacology
  • Soybeans / enzymology*
  • Subtilisin / chemistry
  • Subtilisins / chemistry*
  • Subtilisins / metabolism
  • Temperature
  • Thrombin / chemistry
  • Urokinase-Type Plasminogen Activator / chemistry


  • Fibrinolytic Agents
  • Fibrinogen
  • Serine Endopeptidases
  • Subtilisins
  • Thrombin
  • Subtilisin
  • Urokinase-Type Plasminogen Activator
  • nattokinase