Study On Solid Fermentation Of Natto And The Catalytic Characteristics Of Nattokinse

Natto is a kind of Asia Oriental traditional fermentation product, and nattokinase (NK) can dissolve blood clots. Being a kind of protein kinase of Bacillus subtilis, NK can be taken by oral and obviously reduce the time needed to dissolve globulin; Compared with the traditional medicines such as the urokinase and streptokinase, NK has the advantage of fast dissolving clots, long half-life, low side effect, etc. The enzyme strongly catalyzes fibrinolysis, suggesting that it is possible to treat patients of embolisms or prevent the disease as a new drug.In this paper, the BN-3(Bacillus natto) strain was used to study the effect of liquid fermentation and solid fermentation on the production of NK. The optimum conditions for solid state fermentation of natto were as following:inoculum volume of8%, ferment time24h and with the soybean being cut in half, thus the highest activity of NK was arrived to976IU/g. The optimal technical condition for liquid fermentation was48h, thus the protein concentration of NK was178.7mg/L and the enzyme activity of NK was174.7IU/mL. The experimental data show that there were good correlations among the concentration of living Bacillus natto cells, NK activity and the protein concentration of NK in the liquid fermentation. The fermentation process can be monitored by measuring the concentration of living cells.The purification of NK was modified according to the traditional method. The NK was purified by ammonium sulfate fractionation, DEAE-Sepharose Fast Flow and Sephadex G-100, respectively. The purified enzyme showed two protein bands in SDS-PAGE. The specific Activity of NK was increased to7.5times of that of original sample with47%activity recovery.This experiment using skim milk as raw materials to analyze the protein hydrolysis mechanism of NK on solid plate and in liquid system, and to study the influence of conditions such as substrate concentration, ratio of enzyme to substrate concentration (E/S) and hydrolysis time that affect the enzyme hydrolysis characteristics. We have studied the enzymatic hydrolysis conditions of NK and the method to determine the activity of NK.The results show that on the solid plate, a greater degree of hydrolysis was achieved under the following conditions:hydrolysis temperature37℃, initial substrate concentration2%, ratio of enzyme to substrate concentration (E/S) being4～14and hydrolysis time16h. In liquid system optimism conditions for the enzymic hydrolysis of milk proteins were as following:hydrolysis temperature37℃, initial substrate concentration2%, ratio of E/S being6-10, and hydrolysis time2.5h, and the hydrolysis degree were from40%to70%.