Screening Of Efficient Thrombolytic Strains And Comparative Analysis Of Methods For Detecting Thrombolytic Activity

Natto, a kind of transitional fermented food that has been2000years of history injapan, is made by fermenting boiled soybean inoculated with bacillus subtilisorBacillus subtilis. For a long time, folk will also be used for preventing andtreatingcardiovascular drugs.In1987, the first Japanese researcher found that natto contains such a highfibrinolytic activity enzyme which can cure and prevent thrombotic diseases, andnamed nattokinase. Since then, researchers conducted series of researches onnattokinase, in particular a lot of researches on nattokinase activity assay. The majormethods of measuring the activity of nattokinase can be classified into two types:First, by biological methods, measured fibrinolytic activity through the fibrinolyticproperties of nattokinase, such as the fibrin plate method and the method of fibrindissolution block time. Second, measured fibrinolytic activity of nattokinase on thebasis of hydrolysis of NK basis to determine its activity, such as toluenesulfonyl.Arginine methyl ester (TAME) method, casein hydrolysis, tetra-peptide substratemethod. In this study, through a comparative study of fibrin plate method and thetetra-peptide substrate method for the determination of nattokinase activity of crudeenzyme solution, to find a sensitive and easy to operate, affordable, fast andaccurately nattokinase activity assay method, which can used for thrombolyticenzyme research.In this study, compared the linearity and precision of fibrin plate(FP)methodwith tetra-peptide (Suc-Ala—Ala—Pro—Phe—pNA) substrate (SS) method, andtheir results’ corespondency was also investigated．Results showed that: The activitylinear ranges were50IU/mL-800IU/mL for the fibrin plate method and0.03U/mL-2.60U/mL for the tetra-peptide substrate method respectively, while bothCVs were less than10％in within-day and day-to-day tests. The two results showed agood linear correlation and could be converted as: The fibrin plate method (IU)=420.22X the tetra-peptide substrate method(U)-368.51, R2=0.9920. therefore, fibrinplate method and tetra-peptide substrate method are both applicable for detecting Nattokinase activity．The result from FP method can directly reflect nattokinase’sthrombolytic activity and the tetra-peptide substrate method has the advantage forfast，sensitive and high-throughput screening.This study screened and solated the strains that produce fibrinolytic enzyme from10fermented foods.From the first step of casein flat screen,flat line drawings,we got113strainsthat. Aftert measured by tetra-peptide substrate methodhad, we got5strainsthat had enzyme activity. The enzyme activity produce from Hengtong nattocapsule,Luzhou liquor,Japan natto strans,Inner Mongolia Kumiss,Tsingtao Beercurd are590.6IU/mgprotein,510.1IU/mgprotein,481.3IU/mgpro，461.7IU/mgprotein，338.3IU/mgprotein respectively.Routine tests of H6including morphology, physiological and biochemicalcharacteristics, and comparison with Bacillus subtilis, preliminarily suggest that H6isBacillus subtilis.