Screening Of Bacillus Coagulans Producing Fibrinolytic Enzyme And Study On The Characterizations Of Fibrinolytic Enzyme

Bacillus coagulans are widely used in various commercial food products as it exhibits characteristics of both the Bacillus and Lactobacillus genera.Thrombotic disease(TD)is one of the diseases that seriously affect mankind's health and even cause death.The incidence rate of TD has been increasing in recent years.Dissolving thrombin-forming fibrin with fibrinolytic enzymes is the main method for preventing and treating thrombotic diseases.Microorganisms is an important resource for the development of new fibrinolytic enzyme,because they are very various and easy to cultivation.Therefore,it is of great significance to screen out the Bacillus coagulans producing fibrinolytic enzyme form food.In this research,a strain which producing fibrinolytic enzyme was isolated from traditional fermented foods such as soybean meal and natto.The strain was studied by strain identification,biological characteristics.The fibrinolytic enzyme of the strain was concentrated and explored its properties.The amino acid sequence and gene sequence of the fibrinolytic enzyme were predicted at the same time.The main results are as follows:(1)A strain producing high fibrinolytic activity was screened by using skim milk plate and fibrin plate from traditional fermented foods,and named HQ-1.The enzyme activity of HQ-1 fermentation supernatan was 383.1U/mL.According to the morphological characteristics,physiological and biochemical characteristics and 16S rDNA sequence,the HQ-1 stain was identified as Bacillus coagulans,and the fibrinolytic enzyme of HQ-1 was named BCF.(2)Partial biological properties of HQ-1 was tested.The results showed that the strain HQ-1 was sensitive to 11 common antibiotics such as amoxicillin,and moderately sensitive to rifampicin and erythromycin,and it resistance to penicilin.The strain had certain antibacterial activities to Staphylococcus aureus,Escherichia coli and Salmonella,with the inhibition zone of 19.3�0.2mm,18.8�0.4mm and 16.6�0.1mm respectively.After processed with simulated gastric juice of pH=2,the survival rateis of HQ-1 was 64.3%and the number of viable cells was 5.1�10⁹ CFU/mL.After treated with 0.3%concentration of bile salt medium of pH=8,the survival rate of HQ-1was 85.7%and the viable count was4.0�10⁹ CFU/mL.(3)The crude enzyme solution was prepared by salting out,dialysis and concentrated.By comparing the results of active electrophoresis and inactive electrophoresis,it was shown that there was only one fibrinolytic enzyme active ingredient in the crude enzyme solution.The study of characterizations showed that BCF was a plasminogen activator which degraded fibrin by activating plasminogen and the optimum pH value is 6.0;the optimum temperature of BCF is 45?;Ca²⁺and Mg²⁺apparently promoted the enzyme activity,but Na⁺,K⁺,Fe²⁺,Cu²⁺and Ba²⁺not;Both inhibitors PMSF and EDTA showed significant inhibition on the activity and the center contains metals and serines.Thus,all results above indicated that the enzyme probably belongs to a metallo and serine proteases.(4)The activity electrophoresis band of BCF was recovered for the amino acid sequencing.The results showed that fibrinolytic enzyme BCF was consisted of 245 amino acid residues and it was low similarity to the other reported biologically derived fibrinolytic enzyme sequences.Designing degenerate primers based on the amino acid sequencing results and carry out PCR,the result of PCR sequeneing analysis shows that the bcf has 735bp by in length and it also has low similarity to the other gene sequence.In conclusion,a strain producing fibrinolytic enzyme was screened and named Bacillus coagulans HQ-1.The partial biological properties of HQ-1 were tested,and the crude fibrinolytic enzyme was concentrated.The characteristics of the enzyme was studied.The amino acid sequence of BCF and the sequence of the coding gene were predicted.This research provide scientific theoretical basis for the further development and utilization of the strain HQ-1and fibrinolytic enzyme BCF.