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

• Published in: Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology Vol. 134; no. 1; pp. 45 - 52
• Main Authors: Peng, Yong, Huang, Qing, Zhang, Ren-huai, Zhang, Yi-zheng
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 2003
• Subjects: Amino Acid Sequence; Bacillus - enzymology; Bacillus amyloliquefaciens; Chinese soybean food; Cloning, Molecular; Douchi; Electrophoresis, Polyacrylamide Gel; Fibrinogen - chemistry; Fibrinolytic Agents - pharmacology; Fibrinolytic enzyme; Glycine max - enzymology; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Molecular Sequence Data; Nattokinase; Protein Structure, Tertiary; Purification; Serine Endopeptidases - pharmacology; Serine protease; Subtilisin - chemistry; Subtilisins - chemistry; Subtilisins - metabolism; Temperature; Thrombin - chemistry; Thrombolysis; Urokinase-Type Plasminogen Activator - chemistry; Purification; Fibrinolytic enzyme; Douchi; Serine protease; Bacillus amyloliquefaciens; Chinese soybean food; Nattokinase; Thrombolysis
• ISSN: 1096-4959; 1879-1107
• 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 p I of 8.0. The optimal reaction pH value and temperature were 9.0 and 48 °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.