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.

Phenolic antioxidants have multiple benefits to human health. Polyphenols are responsible for the antioxidant activity in apples. Antioxidant activities were assessed using the ferric reducing/antioxidant power (FRAP) and 2,2-dipheny l-1-picrylhydrazyl (DPPH) radical scavenging assays in different cultivars and different parts of apples grown in China. The phenolic compounds and antioxidant activities differed significantly among the four apple cultivars. Guoguang had the highest phenolic concentration and antioxidant activity and Golden Delicious had the lowest. The peels had the highest total phenolic and flavonoid contents followed by the cores and flesh. Anthocyanins were detected only in red apple peels. Peel and core had greater antioxidant activities than apple flesh. FRAP values were inversely correlated with phenolic contents, whereas no clear relationship could be observed between DPPH values and phenolic contents. The higher levels of phenolic compounds and antioxidant activities of apple peels and cores than flesh may be of technological interest as a valuable source of antioxidants.