Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food.
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.
Peng
Yong
Y
College of Life Sciences, Sichuan University, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Chengdu 610064, PR China.
Huang
Qing
Q
Zhang
Ren-huai
RH
Zhang
Yi-zheng
YZ
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000737:chemistry; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000494:pharmacology; D013025:Glycine max / Q000201:enzymology*; D020860:Subtilisin / Q000737:chemistry; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D013917:Thrombin / Q000737:chemistry; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry
D005343:Fibrinolytic Agents; D005340:Fibrinogen; D012697:Serine Endopeptidases; D013381:Subtilisins; D013917:Thrombin; D020860:Subtilisin; D014568:Urokinase-Type Plasminogen Activator; C053771:nattokinase