Purification And Characterization Of Nattokinase Produced By Bacillus Subtilis

The separation and purification techniques of nattokinase from the fermenation broth of Bacillus subtilis were explored in the present work to obtain high pure nattokinase. The optimized purification process includes the following steps: removing cells by the centrifugation, 20%~60% saturation ammonium sulfate precipitation, gel filtration chromatography with Superdex 75and ion exchange chromatography with SP Sepharose Fast Flow. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the purification effect, and the results indicated that the final product obtained was homogeneous and had a molecular weight around 27.7kD. The fibrinolytic activity of nattokinase was measured by means of fibrin plate method. The purification factor and activity recovery of nattokinase was 8.4 and 49%, respectively. We used dialytic bag of 6,000MW to purify nattokinase under room temperature. After changing the water several times, the recovery of activity was 83.8% and the purification factor was 1.05. Then we use ion exchange chromatography for further purification with SP Sepharose Fast Flow. The flow rate was 5mL/min and the injection volumn was 15mL, the final purification factor was 2.4 and the recovery of Nattokinase was 71%. There were some impurities in the final product.The optimal reaction temperature and pH of nattokinase was 55℃ and 9.4 respectively. From pH6 to pH12, nattokinase exhibits high stability. Under 37℃, after 5h the losing of activity was not visible. There was a little deactivate under 45 ℃. When temperature increased to 50℃, Nattokinase lost its activity in few minutes. Nattokinase lost 37% activity in a week under 4℃. Nattokinase contained 85% activity under 37℃ after18h and 50% after 2d. The 0.1mM PMSF can totally deactivate Nattokinase. DTT and EDTA cannot influence the activity. lmM SDS cannot restrain the activity of Nattokinase. However the l0mM SDS can decrease the activity by 32.5%. Mg2+ can activate Nattokinase. Ca2+, Ba2+s' influence were not apparent. Cu2+ can deactivate Nattokinase greatly. Other ions, such as Al3+, K+, Fe2+, Fe3+, Zn2+, Mn2+, Co2+, Ni2+, Cr3+, Pb2+, Ag+ also have some deactivation effect. Skimmed milk powder, glutin, sodium alginate, chitosan, BSA, starch, glycerin, dextrine, mannitolum can protect Nattokinase under high temperature. Nattokinase can directly decompose the fibrin and it account for 78.2% of total decomposition effect. Nattokinase decomposed the a chain first then β chain and y chain. The decomposition rate of γ-γ chain is the highest.We used calcium alginate and CAP to produce the enteric coated capsules. For calcium alginate system, under simulated gastric juice, the release rate of Nattokinase was 8.1% in 2h. Under simulated enteric juice, 82.6% of Nattokinase released within 2h. The recovery of Nattokinase was 75.8%. Using CAP to produce the enteric coated capsules was apractical way. Under simulated gastric juice, the release rate of Nattokinase was less than 10% in 2h. However under simulated enteric juice, Nattokinase can totally release within 60min. However, the final recovery of Nattokinase was only 26.3%. Except starch, sodium alginate, chitosan, BSA, glycerin, dextrine, mannitolum and glutin have some protection effect The skimmed milk powder can increase the final recovery of Nattokinase to 63.4%.