Study On The Optimization Of Submerged Fermentation And Enzyme Stability Of Nattokinase

Nattokinase (NK) is a fibrinolytic enzyme that is extracted from natto(a traditional fermented food in Japan). in fact it has enzymatic property of protease. Compared with the thrombolytic agents such as urokinase and streptokinase, Nattokinase is safer, easier to be absorbed by body because of its low molecular weight, and has more direct and more persistent effect on thrombosis, and It is more important that Nattokinase can be fermented by bacteria, which will lower the production cost. So nattokinase can be developed as a new fibrinolytic medicine. This paper investigated the liquid fermentation of NK. The main results were as follows. 1 .Using Bacillus sp.stored in lab, which can produce NK, the liquid fermentation condition was optimized: The best nitrogen source is the defatted soybean, the best carbon source is maize powder, C N==l 1 was the best choice; Ca2~ was the positive ion which had the greatest effect on NK抯 synthesis, while whether Mg2~ was added or not was not very important, 0.02% of Ca2~ and Na2HPO4 0.2% NaH2PO4 0.1% were the optimal conditions for NK~s yield; Mn2~ inhibit NK抯 biosynthesis; Surface-active agents could not increase the secretion of NK: The bacteria grew the fast in 37(2, but 300(2 was the most suitable temperature for the production of NK; The strain had the biggest speed of growth and the biggest yield of NK under PH7; This strain was oxybiotic, So less volume of fermentation medium fit its growth and sythesis of NK better; But because of the volatilization of water was more when volume of media is too little, lOOml/500m1 was choosen for the biggest gross production of NK; 2% of 24h-old inoculum was the best condition for NK抯 synthesis. 2. The optimum reaction temperature of NK was 45 ~?50~C and the stability of NK was very good at 250(2 and 3TC. The relative activity of NK is V Abstract 95% after 24h at 25C and is 78% at 370C; NK inactivated quickly above 55C. The PH7.O~?.5 was the optimal PH and PHTh-8 is the best condition for NK抯 stability; Hg2~ could inhibit NK抯 activity and Mg2~ promoted it , Serum albumin, glycerol, propylene glycol, sodium alginate, peptone, gelatin could improve the stability of NK. When used casein as substrate, Vm=7.76 u gIml mm, Km10.87X iO%t g/ml.