Studies On Nattokinase Fermentation By Bacillus Subtilis

Nattokinase(NK) is a fibrinolytic serine enzyme that is produced by Bacillus subtilis natto during the production of a traditional fermented food natto from Japan. It is reported that NK not only have a strong thrombustic function, but also is safer and easier to be absorbed by human body and has more direct and persistent effect on thrombosis. It is most important that nattokinase can be fermented by bacteria, so it can lower the production cost. So nattokinase can be developed as a new fibrinolytic medicine.Directional isolation of Bacillus natto strains from different natto products was conducted according to the aimed bacterium's properties such as heat-resisting ability and notable thrombustic function. In this way, a strain named NT-6 that can produce nattokinase was isolated. After NT-6 was mutagenized by the mutiple treat with HCl·NH4OH and UV, the mutant strain NU9012 was obtained by the screening of anti-rifampin plate and then screening of shaking flask fermentation. Its ability producing NK was more remarkable than NT-6 by 68%. The activity of NK produced by NU9012 in the liquid fermentation medium that hasn't be optimized can reach to 648IU/mL.Based on the results of single-factored and orthogonal experiments, a Plackett–Burman design was used to evaluate the influence of factors relating to NK liquid fermentation by NU9012 in the first optimization step. It can be deduced that xylose concentration influenced NK production positively while the pitching rate and K2HPO4 concentration negatively. And then, Response surface methodology was used to optimize the parameters of the 3 factors above. In conclusion, the liquid fermentation optimization demonstrated that the best composition of liquid medium is xylose 1.88%, pancreas protein peptone 2%, CaCl2 0.03%, MgSO4 0.05%, K2HPO4 0.21% and KH2PO4 0.1%. The best pH of the liquid medium is 7.0. The other parameters for the production of NK are as follows: the best temperature is 30℃, the best age of inoculum is 18h while the pitching rate 3.51%. The best volume of the liquid fermentation media for NK's synthesis is 100mL/250mL and the propriate period of fermentation is 3 days.