Studies On The Submerged Fermentation Process And Purification Of Nattokinase

The dissertation focuses on isolation of strains, optimization of liquid fermentation condition producing nattokinase by Bacillus natto, purification of nattokinase from liquid fermentation and enzyme characteristics.Directional isolation on Bacillus natto strains from Japanese natto were conducted according to the Bacterium s properties such as heatproof ability, salt resistance, notable proteinase activities and (V_H)^-. Eight fibrinolytic enzyme producing strains were screened from Japanese natto, which were named NK-1～NK-8. With nattokinase activity as target, the high-procing NK-4 with highly genetic stability was selected by shaking flask liquid cultivation.The seed medium composition, shaking flask batch fermentation medium compositions and culture conditions were determined through single factor experiment and orthogonal experiment. The maximum nattokinase yield was 1865.59U/mL, which was improved from 467.18U/mL in original fermentation conditions.The batch fermentation kinetics of nattokinase produced by Bacillus Natto were studied based on the experimental data from fermentation in shaking flask liquid cultivation. The kinetic models of the fermentation were established by MATLAB software.Purification process of nattokinase was explored and established. The nattokinase was purified using salting out, ultrafiltration, CM Sepharose Fast Flow ion exchange and Sephadex G-75.Based on the protocol 35.8-fold purification of the enzyme was obtained, with a final yield of 59.8 percents.The molecular weight of the enzyme was 28000Da, determined by SDS-PAGE, on which the isolated enzyme showed a single sharp band. The enzyme hydrolyzed fibrin, and it also activated plasminogen to plasmin. The enzyme had an optimal temperature of 45℃, it is stability in the temperature range from 25 ℃ to 50℃. and it rapidly declined by treating the enzyme at 55℃. The enzyme had an optimal pH of 7～8, kept 80% of the initial activity during pH6～8, out of which fibrinolytic activity reduced quickly. Hg²⁺, Zn²⁺, Al³⁺could inhibit the activity, Ca²⁺ and Mg²⁺ could...