Screening Strains Producing High Nattokinase Activity,Separation And Purification Of Nattokinase And Fibrinolytic Effect On Thrombotic Mice

Nattokinase (NK) from Bacillus subtilis natto is a protease with well fibrinolytic activity. Animal and human oral tests have proved NK has a strong dissolving thrombus function and good security, can be easily absorbed, works directly and immediately, and has a long duration of drug action. What’s more, NK can be produced in a large scale by fermenting, so the cost would be low. But NK healthy products in China always have low fibrinolytic activity, and some companies of producting natto often cut corners and do a shoddy job, then the NK products may have no pharmacological effects or even have side effects. Therefore, developing some safety NK products with well thrombolytic effect of industrialized production and low cost is of great significance.Bacillus subtilis natto A10-5 producing high fibrinolytic enzyme was screened from Bacillus subtilis natto BSN-3 by UV radiation. The original strain BSN-3 had been irradiated from mutagenesis of spores, so bacterium was elected as raw materials for UV mutagenesis here. The irradiation time was 19 s to induce mutagenesis on the base of death rate curve. After skimmed milk medium screening, fermentation broth screening and fibrinogen plate checking, A10-5 was isolated with the highest fibrinolytic activity. The fibrinolytic enzyme activity of fermentation broth was up to 1139 U/mL through fibrinogen plate, which was 88% higher than that of original strain BSN-3.A10-5 strain had stable capacity of producing NK with high enzyme activity. Using the optimal medium which carbon source was xylose and nitrogenous source was tryptone, enzyme activity of strain A10-5 could reach 1768 IU/mL.In this experiment, the extraction effects of several factors on NK were investigated. These factors included the type and density of inorganic salt, molecular weight and density of PEG, bacteria fluid volume, extracting pH and temperature. According to the results of single factor experiments, the aqueous two-phase system of PEG-phosphate was optimized as follows:PEG40012%(w/w), K2HPO415%(w/w), the broth loading was 20%. The room temperature was suitable for extraction and pH didn’t need to change. The fibrinolytic enzyme could be recovered with a yield 93.4% when the system was scale up to 20 fold 200 g. Up-phase solution was filtered using 10 kDa ultrafiltration centrifuge tube, pH 7.4 PBS was the mobile phase. SDS-PAGE was used to check the extracting enzyme, and it showed the enzyme had a high degree of purification.34 healthy KW mice, which were different in sex, were randomly divided into 4 groups by body weight to do pharmacodynamic study of NK:Normal control group (NC), model control group (MC), crude enzyme group, salting-out liquid group. NC was given distilled water, MC and the 3rd group were given crude enzyme liquid, and the 4th was given salting-out liquid. Intragastric 10 d in a row,1 times daily and 0.6 mL each time. Then all mice except MC were subcutaneously injected with 0.4% carrageenan on rump-back for thrombotic model, MC was injected with physiological saline.24 h later, observed thrombosis ratio, calculated the thrombosis relative length. In carrageenan induced tail-thrombosis model, tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group. This study showed the possibilities of NK to be used in thrombolytic therapy. NK could significantly prolonged the mice’s clotting time compared with NC group, but it would not prolong the bleeding time significantly and NK showed conspicuous clot lysis activity.