Screening Strains Producing High Nattokinase Activity And Characterazation Of Nattokinase

Nattokinase (NK) was a fibrinolytic enzyme that was extracted from a traditional fermented food of Japan. NK was a serine enzyme produced from Bacillus subtilis natto. It was reported that NK had strong thrombustic function. Compared with the thrombolytic medicines such as urokinase and streptokinase, NK was safer, easier to be absorbed by body because of its low molecular weight, and it had more direct and more persistent effect on thrombosis. And it was more important that NK could be obtained by fermentation with the effect of bacteria, which would lower the production cost. Consequently, many scientists believed that NK could be used as a new fibrinolytic medicine.BSN-3 strain belonging to Bacillus subtilis (natto) with high NK activity was screened through the UV-induced mutation, fibrinogen plate and shake-flask liquid fermentation in the paper. Intensive studies on the purification and characterization of NK were conducted in this thesis.It was 4 minites to induce spores on the base of spore fatality rate. On the base of comparision of results from fibrinogen plate and casin plate, it was indicated that results from casin plate was not credible.The optimization of NK liquid fermentation was investigated through single factor experiments and orthogonal tests in this paper. The results indicated that the optimal fermentation medium was tryptone 6g/L, xylose 30g/L, K₂HPO₄-3H₂O 1g/L, MgSO₄·7H₂O 0.2g/L, the optimal fermentation time was 72h, the optimal fermentation temperature was 30℃, the optimal pH was 8.0. Using this optimization, enzyme activity of strain BSN-3 could reach 1120IU/mL.NK production kinetics was studied through studying the relation between the growth curve of strain BSN-3 and the time of production NK. The result indicated that the type of NK synthesizing was lag synthesizing.The culture liquid was centrifugalized at 4800r/min for 10 min, then ammonium sulfate was added into the supernatant to a final concentration of 45% to precipitate the others.After centrifugation, ammonium sulfate was increased to 50% to get NK. The CM-52 was used to purify NK. Enzyme recovery was 39% during purification and enzyme fold was 10.3.The fibrinolysis activity of NK was determined and characterazation of NK was analyzed in the thrombolysis experiment in vitro. Results indicated:NK in liquid was not sensitive to temperature below 50℃and could be conserved at room tempreture.NK was stable when pH was between 3.0 and 11.0.Effect of different ions on NK stability was various. Zn²⁺ and Hg²⁺ could surpress NK activity. Cu²⁺ in low concentration could not surpress NK activity, but Cu²⁺ in high concentration could surpress NK activity.Effect of different materials on NK stability was various when NK was heated. It was found glutin in 3‰concentration protected NK best.The present study provided basic information for the production of NK. It also contributed to the preservation and transportation of NK. NK was proved to be a potential drug candidate for the treatment and prevention of thrombus.