Fermentation Of BN137 And Purification, Characterazation Of Nattokinase

The effect of liquid fermentation on enzyme activity of Nattokinase produced by Bacillus natto No. 137 strain was studied, and intensive studies on the purification and characterization of Nattokinase were conducted in this thesis.For the purpose of increasing the harvest of fermentation, cultivating condition and nutrient resources were considered. Initiative pH for about 8.0, incubating temperature at 35℃ and improved aeration were beneficial to enzyme produce . The correlation of enzyme-produce and pH was studied too, both varied simultaneously toward the same tendency during fermentation.Medium experiments were arranged under uniform design , and then an optimum medium was got accordingly .The culture liquid was centrifugalized at 3,500r/min for 30min,then ammonium sulfate was added into the supernatant to a final concentration of 30% to precipitate the others. After centrifugation, ammonium sulfate was increased to 70% to get the enzyme. The precipitated enzyme was redissolved with Tris-HCl buffer (pH7.8).The enzyme obtained in the above processing was applied to a column packed with DEAE-Sephadex A-50, then a column packed with Sephadex G-75. At last, we got the pure protein with the determination of electrophoresis.The molecular weight of the enzyme is estimated at 30903 through gel filtration and SDS-PAGE electrophoresis. The isoelectric point is 8.6?.3.The optimum pH for enzymatic reaction is 7-12.And the enzyme is sensitive to temperature.The fibrinolysis activity of Nattokinase was determined and analyzed in the thrombolysis experiment in vitro. Through some experiments, it is found that there are no metal ion having obvious activation, and the enzyme activity is not inhibited by Na₂S₂O₃.but by 0.1%SDS.