| Nattokinase(NK) is a fibrinolytic enzyme that is extracted from a traditional fermented food of Japan. NK is serine enzyme produced from the Bacillus subtilis natto. It is reported that NK have a strong thrombustic function. Compared with the thrombolytic medicines such as Urokinase and Streptokinase, Nattokinase is safer, easier to be absorbed by body because of its low molecular weight, and it has more direct and more persistent effect on thrombosis. And it is more important that Nattokinase can be obtained by fermentation with the effect of bacteria, which will lower the production cost. Consequently, Nattokinase can be used as a new fibrinolytic medicine. The objective of this thesis is to study purification and characteristics of Nattokinase. The main results are as follows: 1. The fibrinolytic activity of NK, which was estimated by the fibrin plate method, was counted according to the UK. The relationship between the enzyme activity and the dissolved area of fibrin plate with activity of UK had a high regression coefficient when enzyme activity unit was below 200 IU/ml. 2. NK was purified from the supernatant of the broth by Bacillus subtilis natto. The purification steps included centrifugation, ammonium sulfate fractionation and Phenyl Sepharose. 3. The saturations of ammonium sulfate fractionation were 30% and 60%, respectively. It was shown that 63.17% of the existing protein impurity could be removed and the specific activity was 753.90 IU/mg. In step elution, the NK was eluted using Phenyl Sepharose with 0.9mol/L sodium chloride. The purified enzyme had a single protein band on the SDS-PAGE gel, and the molecular weight of the enzyme was 28KD. The isoelectric point was 8.6. Specific activity of the enzyme sample was 47,098.04 IU/mg, the purified factor 14.82, and the last yield 44.03%. 4. The chromogenix substrate method was improved for studying NK kinetics. It was found that a high relativity of two methods (chromogenix substrate method and fibrin plate method). When S-2251 was used as substance, at pH 7.4,37℃, the kinetic parameter Km was 0.3735 mmol/L and Vm was 0.0536 mmol/(min·L). 5. The optimum reaction temperature range of NK was in the range of 47-60℃. The optimum reaction temperature was 55℃. NK was stable below 37 ℃. At above 45 ℃, the activity of NK decreased with the temperature very fast. NK had 95% relative activity in the pH range of 7.0-9.0. The optimum reaction pH was 7.5. And pH 6.5-9.0, pH 8 especially, was the best condition for NK's stability. 6. PMSF inhibited completely the activities of Nattokinase. Aprotin, EGTA, EDTA inhibited partly the activity. Pepstatin, Leupeptin was ineffective. It was suggested that NK was a kind of serine proteinase. The present study provides basic information for the production and pharma-cological research of NK. It also contributes to the preservation and transportation of NK. NK is proved to be a potential drug candidate for the treatment and prevention of thrombus.
|
PDF Full Text Request