Studies On Liquid Fermenation, Purification And Characterization Of Nattokinase

Nattokinase(NK) is produced by Bacillus subtils natto in the process of the fermentation of Natto which is a fibrinolytic serine enzyme. It is extracted from a traditional fermented food of Japan and was firstly discovered by Sumi who is a Japanese scholar. With the growth in the living standard, the cardiovascular disease is threatening people's health and life more and more severely. So people have thought much of the developing of the medicament which can cure the cardiovascular disease. It is very necessary to research into a new-style oral medicament which has good security and low price because of the relative merits of the thrombolytic agents now used. Compared with the thrombolytic agents such as urokinase andtreptokinase, 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, health products and health foods.This paper study on the liquid fermentation of NK, purification, and character of enzyme. At last, the main results are as follows:1 From the 9 strains stored in the lab, screened out a strain of which the production of NK is highest.And detected the enzyme activity by fibrin plate method. Both the component of culture medium and the liquid fermentation condition were optimized. To fermentative medium, the best carbon source is xylose 1.5%,the best nitrogen source is soya septone 2.0%.The component of inorganic salts is that MgSO₄ 0.05%,CaCl₂ 0.02%,K₂HPO₄/KH₂PO₄ 0.1%/0.1%. 37℃ 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;The volume of media is 100ml/250ml which was choosen for the biggest gross productionof NK;2% of 18h-old inoculum was the best condition for NK's production.After the system optimization, in the fermenter enlarge test,the yield of NK of 1217IU/ml was obtained 60 hours later. Compared with shake-flask culture, the culture periods was shortened.2 Purification experiment result: Purifying the enzyme by the ammonium sulfate, SephadexG-100 Chromatography and so on, the purified enzyme showed a single protein band in the SDS-PAGE. Purified is 11.34,and the last yield is 37.10%,Specific activity is3543.41IU/mg.3 The primary study on the character of NK shows: The optimal pH of NK is pH7.0-8.0;the optimum reaction temperature of NK is 45-55°C.