Fermentation Of Two Fibrinolytic Enzymes And Their Separation, Purification And Characterization

Cardiovascular diseases are the leading causes of death throughout the world, which are responsible for 29% of the total mortality rate in the world. The underlying pathophysiological process in myocardial infarction and stroke is the formation of a fibrin clot, which adheres to the unbroken wall of blood vessels. Accumulation of fibrin in the blood vessels usually increases thrombosis, leading to myocardial infarction and other cardiovascular diseases. A variety of fibrinolytic enzymes have been extensively investigated and used as thrombolytic agents. However, these enzymes are expensive, and patients may suffer from undesirable side effects. Therefore, the search for safer thrombolytic agents from other sources continues.Fermented shrimp paste, a traditional Asian seasoning, was shown to have a strong fibrinolytic activity. In this paper, Bacillus sp. nov. SK006, isolated from a fermented shrimp paste was physiological investigated and identified by 16S rDNA analysis.Fermentation process was optimized for production of fibrinolytic enzymes from Bacillus sp. nov. SK006. The results showed that enzyme activity was the highest (2.63 U/mL) in medium containing 2% of glucose and 3% tryptone, Na2HPO4·12H2O 1.5 %, NaH2PO4·2H2O 0.13%, MgSO4·7H2O 0.05%, CaCl2 0.01%, when Bacillus sp. nov. SK006 was cultured at 37°C for 24 h, and the initial pH was adjusted to 7.0.Fermentation parameters including temperature, pH and air flow rate, were further optimized in a Biostat B fermentor (5 L), while BP neural network model was constructed to simulate the expand production process of fibrinolytic enzymes.The acitivity staining by Native-PAGE revealed the presence of four isoenzymes of fibrinolytic activity in the culture broth. Two of them were purified to electrophoresis homogeneity by a combination of enthol precipitation and a serial of chromatographic stages. The molecular masses of the enzymes were determined to be 46.0 kDa and 12.3 kDa by SDS-PAGE and gel filtration chromatography.The activity of SPFE-1 was the highest at 30°C while it was inactivated rapidly at temperatures≥50°C and was inactivated totally at 65°C within 10 min. SPFE-1 was highly stable below 40°C. However, SPFE-2 was more sensitive to temperature compared to SPFE-1; it was inactivated rapidly at temperature≥40°C and was inactivated totally at 60°C within 10 min. It kept 60% of its activity following treatment at 40°C within 2 h.SPFE-1 remained active at pH values ranging from 5.0 to 11.0 while SPFE-2 from 4.0 to 8.0. The fibrinolytic activity of SPFE-1 was optimal at pH 6.0-8.0 while SPFE-2 at a lower pH value (5.0 to 6.0). However, both enzymes exhibit a significant loss of activities when the pH value was lower than 5.0. Only Zn2+ stimulated the enzyme activity of SPFE-1, whereas Cu2+, Ca2+, Fe3+ and Hg2+ caused its inhibition. It was predicted that the Zn2+ ion might be present in the active center of SPFE-1. This suggests strongly that the inhibition of enzyme activity caused by EDTA results from the chelation of a metal relevant for its activity, possibly Zn2+. Mn2+ and Fe2+ inhibited SPFE-1 up to approximately 30%. It was hypothesized that Mn2+ and Fe2+ intrude on the enzyme-catalyzed system by binding to carboxyl groups of the enzyme. In addition, SPFE-1 was strongly inhibited by PMSF, indicating it is a serine protease. It was also inhibited by Cu2+, Hg2+ and PCMB, which were supposed to react with the free SH-group of the critical cysteine, causing a strong reduction of the enzyme activity. As for SPFE-2, the highest activities were observed in the presence of Cu2+ and Ca2+, respectively. Zn2+ caused strong inhibition. 2-mercaptoethanol affects its activity significantly. EDTA was found to completely inactivate SPFE-2 even at a very low concentration. These results indicated that SPFE-2 is a metalloprotease with disulfide bonds in its active center. It was estimated that the combination of Zn2+ with some essential groups (disulfide bonds for example) in the active center lead to enzyme denature.During the degradation of fibrinogen by SPFE-1, Bβ-chains of fibrinogen were cleaved first, followed by slower release of theγ-chains. The Aα-subunit was resistant to the enzyme digestion. SPFE-2 was more effective with the Aα-subunit, but less sensitive towardsγ-chains.The enzymes are able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.After the activity of each enzyme was converted into caseinolytic activity for unification of the enzyme unit, the ratio of fibrinolytic activity to caseinolytic activity was calculated. Fibrinolytic activity of the enzymes was higher than other proteases. Hence, the enzymes have relatively high specificity for fibrin as a substrate.The two enzymes both had the highest affinity for N-Succ-Ala-Ala-Pro-Phe-pNA, which is a well-known substrate for subtilisin or chymotrypsin. The synthetic substrate were effectively hydrolyzed with amidolytic activity of 158.18 (SPFE-1) and 107.47 (SPFE-2) nmol/min/mL, Km of 0.60 (SPFE-1) and 0.51 (SPFE-2) mmol, as well as kcat/Km of 1.19×106 (SPFE-1) and 3.02×105 (SPFE-2) L·s-1 mol-1, respectively.The N-terminal amino acid sequence of SPFE-1 and SPFE-2 were found to be AQSVPYEQPHLSQ, which is different from that of other known fibrinolytic enzymes. CD of the enzymes showed that ratio ofα-Helix,β-sheet and turn structure were 0%, 64.6% and 5.6% (SPFE-1) to 47.8%, 0% and 0% (SPFE-2), respectively.