Studies On Preparation, Enzymic Characteristics And Functional Properties Of Dochi Fibrinolytic Enzyme

Dochi Fibrinolytic Enzyme (DCFE, E.C.3.4.21.7) is a kind of serine protease and acts as an important fibrinolytic and antithrombotic enzyme. Due to its potent fibrinolytic effect, it has the potential to become a new fibrinolytic drug.In this study, the main content was as follows:(1) To screen and identify Bacillus bacteria that can secret fibrinolytic enzyme(2) To optimize the cultivation process aiming at high DCFE production(3) To purify and characterize DCFE from the bacterium(4) To study the function of DCFEDC-S6 was screened by medium-fibrin plate and the fermented broth showed strong fibrinolytic activity in fibrin plate. The enzymatic activity of DCFE is 1,200 IU/mL. Choosing fibrin as screen, UV, DES and ⁶⁰Co as mutagen, UγD8, one of stable DCFE high-producing mutant with the enzymatic activity of 2900 IU/mL ,1.4 times as high as DC-S6,was isolated after consecutive mutagenesis After examining the colony, cell morphology and metabolism characteristic, the strain DC-S6 was found to be a Bacillus sp.. The fermented liquid usng UγD8 was safe when it was orally administered by test rats.Optimal conditions of the mutant UγD8's shaked flask culture were studied. The propagation of UγD8 was prohibited as the pH of medium decreased significantly. The growth of the UγD8 and DCFE production was prohibited when ammonium sulfate or ammonium chloride were used as nitrogen source. UγD8 favors organic nitrogen than inorganic nitrogen during DCFE production. The L(9)34 orthogonal experiments were carried out to inspect the optimal medium and the most suitable fermentation conditions. The results showed that the concentration of soy serum was the most significant factor influencing DCFE production by UγD8. The optimized medium for DCFE production by UγD8 was determined and the results are as follows (g/L): sugar 20, soy serum 1000mL (protein content 40 g/L), fibrin 5,corn serum 3, KH₂PO₄1, K₂HPO₄ ·3H₂O 2.5, MgSO₄·7H₂O 5, CaCO₃ 4, natural pH7.0, It gave 3,300 IU/mL DCFE activity when it was fermented in 25L cultivator for 72h using the above-proposed medium at 37℃ with inoculum size 2%, air flow 14L/min.The process for purification of the DCFE was carried out, in which DCFE was separated from supernatant of UγD8 culture broth using ammonium sulfate fraction precipitation and ion-exchange chromatography on CM-sepharose FF and Sephacryl S-200 gel filtration.The specific activity of purified DCFE is 10,898IU/mg, 35.5 times as high as the crude one with a yield of 3.7%. Single band from SDS-PAGE showed the subunit Mw is 31.0kD. Similar molecular weight (30.2 kD) was obtained by gel chromatography suggesting DCFE from UγD8 had no subunits.The properties of the enzyme were investigated and the optimal growth conditions were found (temperature: 50°C, pH value: 7.5-8.0). The enzyme, a serine alkaline proteinase, was quite stable at pH 8.0;Crude enzyme was stable: after incubation at 37°C for lh the remnant activity was 93% of its original activity while that for purified enzyme was 50%. Crude enzyme retains 80% of its original activity compared to the purified enzyme only 40% during incubation at 50°C for lh. While after incubation at 60"C for lh, it retains 5.8% with the comparison to the purified enzyme almost lost all its avtivity. The crude enzyme is stable with the remnant activity at 100% of its original activity after incubation at -20 °C for 6d compared to the purified enzyme which retain 94% of its original activity. Crude enzyme retains 10% of its original activity after incubation at 4°C for 6d while the purified enzyme lost its activity completely. Crude enzyme lost its activity completely after incubation 6d at 25 °C while purified enzyme lost their activity completely after incubation 4d at 25 °C. Crude enzyme lost its activity completely after incubation 3d at 37°C while purified enzyme retains activity 7% after incubation 2d at 37°C, and lost activity completely after incubation 3d at 37°C.Lyophilized DCFE lost the activity less than 3.7% after lOd at 40"C, but no activity lost afterl0dat25°C.The enzyme should be kept in a dry form in a desiicator.Metal ions, such as K+(5mmol/L) and Na+(5mmol/L) have little influence on DCFE activity. Ions like Al3+(5mmol/L)> Zn2+(5mmol/L)x Cd2+(lmmol/L) and Cr6+(lmmol/L) inhibited DCFE activity almost completely. Whereas Mg2+(5mmol/L) and Mn2+(5mmol/L) activated DCFE activity at 15.3% and 18.5%, respectively.Bovine serum albumin and glutin can improve the stability of DCFE obviously. DFP and trysinase had the inhibition rate of 95% and 88%, respectively. DCFE is identified as a serine protease. TPCK, EDTA, PCMB and p-mercaptoethanol inhibited DCFE activity a little, meaning that DCFE is not a metalloproteinases and disulfide bridge were not in the active region of DCFE. DCFE was also stable at the imitating gastroenteric environments.Amidolytic activity was measured spectrophotometrically by using chromogenic substrates. The results showed that DCFE has more highly speciality on the synthetic substrate for plasmin than any other synthetic substrate such as that for trypsin and thrombin. It can not decompose the synthetic substrate for urokinase. DCFE has higher fibrinolytic activity than any other protease, which is 1.7 n 3.8 and 13.5 times as high as that of CK11^ Subtilisin BPN' > Subtilisin Carlsberg, respectively.The apparent Km and Vmax of DCFE obtained from Lineweaver-Burk plot was 2.3><103ug /mLand 5.12ug/(minmL), respectively.Modification and substrate protection experiments showed that the enzyme was inhibited by EDC, PMSF and DEPC, IAA has little influence on DCFE. SH" may not be needed forDCFE activity, while COO" and Ser were needed. Furthermore, It was important in maintaining enzyme activity but not in the activity region.The antithrombotic effect of DCFE: DCFE showed strong fibrinolytic activity which did not rely on stimulating plasminogen. DCFE promoted the fibrinolytic activity and inhibited blood coagulation. Compared with the urokinase standard, platelet aggregation induced by ADP was inhibited by DCFE group. DCFE possessed anti-thrombosis and thrombolysis activity. The TT> P^ APTT and the content of fibrinogen were assayed. The TT\ PT and APTT were lagged and the content of fibrinogen decreases. The level of t-PA was enhanced when rats were injected with DCFE and PAI was not changed. It shows effective antithrombosis function in rabbit at orally administered dosage of 4,000IU/kg.wt.