Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Abstract

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°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), b-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-PhepNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bb-chains and Aa-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.

Xylarinase: a novel clot busting enzyme from an endophytic fungusXylaria curta

“…Fibrinolytic enzymes from Plueurotous eryngii, Paecilomyces tenuipes, Ulva pertusa (ulvease), Bacillus subtilis DC 33 and TP-6 (TPAse) digested both Aa and Bb chain of fibrinogen. TPAse digested Aa and Bb chains of fibrinogen to generate D 0 and E 0 fragments which is similar to that generated by plasmin 10,32,[42][43][44] . Fibrinolytic enzymes from Streptomyces sp.…”

Characterization of Bacillus Subtilis Isolated from Asian Fermented Foods

“…Figure 2 shows that almost all bacteria, except B. subtilis SB4, FS-2 and CN-2, have high enzyme activities towards Suc-AAPF-pNA, a synthetic substrate specific for subtilisin and chymotrypsin (Peng et al, 2003). Moreover, these levels of enzyme activities were the same level as the reported enzyme activity of B. subtilis DC33, a highly proteolytic bacteria isolated from Chinese fermented soybean (Wang et al, 2006).Bz-FVR-pNA is a synthetic substrate specific for trypsin (Ahn et al, 2003). The bacterial enzyme activities varied among them, even with the bacteria belong to the same species, B. subtilis.…”

Partial Purification of Alkaline Protease as Thrombolytic Agent from Mutant Strain Bacillus licheniformis EMS250-O-1

“…Bacillus species have been recognized as source of protease enzymes having thrombolytic activities. 1,11,12 The nature of thrombolytic activity of microbial proteases in clot is similar to that of streptokinase. From out present study, it is evident that the thrombolytic proteases obtained from B. licheniformis strain EMS250-O-1 has significant clot lysis activity.…”