Isolation, Identification, Fermentation Optimization Of Strain 6-1 And Purification Characterization Of Fibrinolytic Enzyme From Stain 6-1

With the modern diet structure change and life rhythm speeding up, the morbidity and the mortality of thrombotic disease is rising year by year, meanwhile onset age has been reduced.Thrombotic diseases cause death and invalidism became one of main reasons. Surgery, anticoagulation and thrombolytic drug treatment were the main method of thrombosis. But now, the most widely used and clinically effective treatment methods are still thrombolytic drug. Therefore, the focus of the research is still the research of new drugs.Microbial variety breeding fast speed and easily teaim, so it is an important source of fibrinolytic enzyme.We used the method of fibrin flat screen separation to 41 strains produced fibrinolytic activity material strains from the soil.After many times, finally got the strain 6-1 which is the highest yield fibrinolytic enzyme.We used the strain 6-1 as material the study contained identify the species , the optimization of fermentation conditions , the extraction the separation and the purification of fibrinolytic enzyme , the study of the enzymology character . The main results are as follow: the strain 6-1 was similar to the Bacillus subtilis strains on the form and physiological-biochemical characteristic .The result of 16S rDNA phylogenetic tree of homology analysis showed that the homology of the two strains is 99.76%, which suggested the strain 6-1 was belong to Bacillus subtilis. By the single factor and orthogonal experimental analysis, the optimum medium composition was : maltose 10g/L, peptone 10 g/L, Na2HPO4 2.0 g/L, NaH2PO4 1.0 g/L, FeSO4·7H2O 0.5 g/L, CaCl2·2H2O 0.5 g/L. The optimum process conditions of fermentation are: PH 7.0 and 50mL fluid triangle flask 250mL each, fermentation temperature is 37℃, the bed speed is l80r/min, fermentation period is 48h. Under this condition the maximum quantity of enzyme production is 647.11±11.19 U/mL, which is much higher than before.Ammonium salt precipitation to the fermented filtrates of 6-1 strain, and DEAE-Sepharose Fast Flow anion exchange chromatography by extraction of protein of 6-1 strain, we get 6-1-1 fibrinolytic enzyme components, whose molecular weight is 19.0 KD.The enzyme properties of 6-1-1 fibrinolytic enzyme research shows that: the optimal temperature is 37℃, the optimal pH is 8.0, it have extensive adaptability and acid-base temperature adaptability. The enzyme can be advanced by Ca2+,Mg2+,Na+,K+, but can be inhibited by Cu2+,Fe3+ completely. EDTA, PMSF and DTT can inhibit the activity of the enzyme which suggested that the activity centre of the enzyme had the structure of hydrosulfuryl,metal and serine. It meybe belong to serine protease.