Optimization Of Fermentation Conditions For Nattokinase Production By Bacillus Natto And Analysis Of Enzyme Quality

Nattokinase(NK)is a serine protease with thrombolytic function produced by Bacillus natto.It has the advantages of high activity,high safety,oral convenience and so on.It has great potential for application.However,at present,the fermentation unit of nattokinase is generally not high and the purity of the plasmin is not clear.In addition,vitamin K2(MK-7),the fermentation by-product of Bacillus natto,which has the blood coagulation function,has not been clearly analyzed for cell growth,which has become a key problem restricting the efficient preparation of high-quality nattokinase.In order to study and solve these key problems,a strain with plasmin activity was screened from fermented food and identified as Bacillus natto TN-02.By optimizing the fermentation process of the strain,the highest expression of nattokinase was 13978.3 U/mL in 10 L fermentor using a fed-batch process.The nattokinase gene aprN and men A gene in the vitamin K2(MK-7)synthesis pathway of Bacillus natto TN-O2 was inactivated by gene knockout technique.It was indicated that nattokinase is the only plasmin that produced by Bacillus natto TN-02,and MK-7 is an essential growth factor for growth and metabolism of Bacillus natto TN-02.The main results are as follows:(1)A strain of fibrinolytic enzyme was screened from the fermented food,and it was identified as Bacillus subtilis by its morphological observation,physiological and biochemical experiments and 16S rDNA identification.The strain was identified and named as Bacillus natto TN-02 by a nattokinase gene screening method.(2)The fermentation conditions of nattokinase produced by Bacillus natto TN-02 were optimized in shake flask fermentation.The optimal culture medium composition was glycerol 3%,soybean meal 1.25%,Na2HP04.12H20 0.4%,KH2P04 0.1%,MgSO4.7H20 0.06%,CaCl2 0.01%,methionine 0.02%,tryptophan 0.01%,phenylalanine 0.01%,tyrosine 0.01%.The expression level of nattokinase by this medium reached the highest at 48 h,which was 7748.1 U/mL.Based on the optimized conditions resulted from shake flask fermentation,scale-up fermentation was carried out and the highest expression level of nattokinase was 13978.3 U/mL,by using stage control of temperature and fed of main carbon and nitrogen sources,which was 1.8 times of that obtained from shake flask fermentation.(3)The nattokinase gene aprN of Bacillus natto TN-02 was partial replaced by kanamycin gene kan and inactivated through insertion events,which derived the recombinant strain TN-021 with mutated aprN.The recombinant strain TN-021 was cultured in shake flask and no fibrinolytic enzyme activity was detected in the product,which indicated that nattokinase is the only plasmin that produced by Bacillus natto TN-02.(4)The recombinant strain TN-022 was constructed by constructing the men AOneo fusion gene and inactiving the men A gene in Bacillus natto TN-02.The results showed that the recombinant strain TN-022 could grow normally only when MK-7 was added.It was indicated that MK-7 metabolized by Bacillus natto TN-02 is the key growth factor for its growth and reproduction.