Screening Of Fibrinolytic Enzyme Producing Lactic Acid Bacteria And Study On The Characterizations Of Fibrinolytic Enzyme

In recent years,the incidence of thromboembolic diseases has been increasing.Dissolving thrombus with fibrinolytic enzymes is one of the important methods for the treatment of thrombosis diseases.Microorganisms are important sources to produce fibrinolytic enzymes.There are many lactic acid bacteria in the fermented food,and most of the lactic acid bacteria are beneficial microbes in the intestines.Therefore,screening the lactic acid bacteria producing fibrinolytic enzyme from food and identifying the characteristics of fibrinolytic enzyme are of great significance for developing health food to prevent thrombotic diseases.In this research,the strains with fibrinolytic activity were screened by using fibrin-plate from the traditional fermented food,and identified by detecting partial 16S rDNA gene sequence,morphological characteristics,physiological and biochemical characteristics;the safety assessment and biological characteristics of the strains were also studied;the fibrinolytic enzyme was purified and explored its properties.The main results are as follows:?1?Two fibrinolytic strains were screened by using fibrin-plate from the traditional fermented food,and named HYD09 and HYN06.Enzyme activity of HYD09 and HYN06fermentation supernatant were 115.3 U/mL and 107.2 U/mL,respectively.According to the cell morphology,physiological,biochemical characteristics and 16S rDNA sequence analysis,the two strains were identified as Enterococcus faecalis.?2?HYD09 and HYN06 were tested for safety,and the results of indole test,nitrate reductase assay,amino acid decarboxylase test and hemolysis test of HYD09 and HYN06were negative.There was no multiple antibiotic resistance of HYD09 and HYN06 to 12 kinds of antibiotics.Virulence genes were not detected in strain HYD09,while virulence gene ESP was detected in strain HYN06.All of the results showed that HYD09 was a safe strain.Further studies showed that the HYD09 had the ability of acid resistance and bile salt tolerance.?3?The fermentation supernatant of strain HYD09 was subjected to fractionated precipitation experiments with different ammonium sulfate saturations,and it was determined that the optimum ammonium sulfate saturation for protein extraction of the strain HYD09fermentation broth was 60%.Two proteins were obtained after 60%ammonium sulfate saturation,dialysis desalting,DEAE-Sephadex A-25 exchange chromatography.The detection results showed that,only one protein was determined to have fibrinolytic activity and its molecular weight was 31.018kDa.?4?The characteristics of fibrinolytic enzyme HYD09 showed that itsoptimum reaction temperature was 35?,and optimal pH value was 7.5;PMSF,IAM and?-mercaptoethanol on inhibition of the enzyme were relatively weak,the inhibition rates were2.3%?3.7%and 5.8%,respectively;EDTA inhibited the enzyme activity completely.The results suggested that the fibrinolytic enzyme HYD09 might be a metalloproteinase.In addition,Ca²⁺promoted the enzyme activity significantly,and the enzyme activity increased by 12.2%.Fe²⁺and Ba²⁺promoted the enzyme activity slightly,and the enzyme activity increased by 3%and 1.8%.Zn²⁺inhibited the enzyme activity significantly,and the enzyme activity decreased by 86.8%.?5?The single factor experiment method was used to determine the factors which had a significant influence on the enzyme production activity of strain HYD09.Then the fermentation conditions were optimized by response surface method to finally determine the best conditions for enzyme production:lactose 9 g/L,yeast extract 5 g/L,inoculation quantity2.96%,pH value is 7.The enzyme activity was 394.74 U/mL in this condition,the enzyme activity increased by 3.4 times.A strain producing fibrinolytic enzyme was screened and named Enterococcus faecalis HYD09.The safety of the strain HYD09 was evaluated,and it was identified as a safe strain.The enzyme was isolated and purified,and the characteristics of the enzyme were identified preliminarily.It provides a scientific basis for the further development and utilization of the enzyme.