Screening And Identification Of A New Strain—Enterococcus Faecalis EF608 And Purification And Characterization Of The Fibrinolytic Enzyme From The Strain EF608

In recent years, more and more people are sick of and died of thrombus. Using fibrinolytic enzyme clinically to dissolve blood clot is called thrombolytic therapy, and it is a safe and effective treatment and prevention for the thromboembolic diseases. There are many fibrinolytic enzyme donors occurred at nature, for example, in human, animals, plants and microbes, these natural enzymes have been found. Such as microbe, because they have various species, are easy to culture, and propagate quickly, we use them as sources to produce fibrinolytic enzyme, and such a method covers more characteristics: abundant source, low cost, short cycle, and without geographical restrictions.In this research, a strain with fibrinolytic activity was isolated and screened by using agarose-fibrinogen plate from the air. We use traditional methods of bacterial classification and identification (including morphological observation, and physiological and biochemical assay), and modern bioinformatics method (including 16S rDNA sequence analysis and phylogenetic tree construction), and identified and obtained a novel strain, which was named Ent.faecalis strain EF608. The results indicate that the strain was found for the first time. We optimized the fermentation conditions and established the fermentation medium under the experimental conditions of the strain EF608, by studying the strength of the vitality of production of fibrinolytic enzyme.65% was notarized the best ammonium sulfate saturation for crude extraction of proteins to fermented liquid of strain EF608 by experiment with ammonium sulfate precipitation. Therefore, we took 65% as ammonium sulfate saturation for crude extraction of proteins and obtained total crude proteins from the fermented liquid of strain EF608. A novel protein ( named as strain EF608 fibrinolytic enzyme) was separated and purificated from strain EF608 by three-step chromatographic methods━Phenyl Sepharose FF HIC, DEAE-Sepharose A-25 Exchange and Heparin-Sepharose affinity. The results of SDS-PAGE analysis under reduced and non-reduced conditions of the single component indicated that it may be a protein composed of a single peptide chain. HPLC analysis showed that there is only a single peak of protein.Molecular weight of strain EF608 fibrinolytic enzyme was assayed as 37.1KDa by SDS-PAGE method. The study of characterizations of strain EF608 fibrinolytic enzyme showed that the optimum pH value is 7.5; the optimum temperature is 35℃; Ba2+ apparently promoted the enzyme activity, but Na+ and Ca2+ not; and high concentration of Fe2+, Cu2+ and Zn2+ significantly inhibited the enzyme activity; PMSF, a specific inhibitor of serine proteases, showed relatively weak inhibition on the enzyme activity;β-mercaptoethanol showed less obvious inhibition on the enzyme activity; EDTA, a specific inhibitor of metalloproteinases, showed significant inhibition on the activity; Thus, all results above indicated that the enzyme probably belongs to a metalloprotease. The strain EF608 fibrinolytic enzyme degraded Aαchain and Bβchain of fibrinogen, did not itsγchain, and a positive correlation existed between the intensity of fibrinolytic activity and effect action time.In summary, we have isolated a new Enterococcus faecalis strain EF608, type with producing fibrinolytic activity, and screened the cultured conditions for producing strain EF608 fibrinolytic enzyme primordially, isolated and purified strain EF608 fibrinolytic enzyme, and conducted a preliminary study for characteristics of the enzyme. This research provide scientific theoretical basis for developing and using of the strain and the enzyme further.