Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi.
Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55 degrees C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS-PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), beta-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bbeta-chains and Aalpha-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.
Wang
Cheng Tao
CT
College of Food Science and Nutritional Engineering, China Agricultural University, East Campus, Beijing, China.
Ji
Bao Ping
BP
Li
Bo
B
Nout
Rob
R
Li
Ping Lan
PL
Ji
Hong
H
Chen
Long Fei
LF
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology*; D002681:China; D004795:Enzyme Stability; D005342:Fibrinolysis; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000378:metabolism; D008297:Male; D010958:Plasminogen / Q000378:metabolism; D051381:Rats; D013025:Glycine max / Q000382:microbiology*; D013379:Substrate Specificity; D013696:Temperature; D013917:Thrombin / Q000378:metabolism; D013927:Thrombosis / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D010958:Plasminogen; D007610:Kallikreins; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator