Identification And Optimization For The Production Of Fibrinolytic Enzyme By Strain Lw-72 And Purification And Characterization Of Fibrinolytic Enzyme

In recent years, thrombotic diseases seriously endanger hunman health, not only the high incidence but also high mortality and disability. According to the WHO, no less than 15 million people are suffering from thrombotic diseases all of the world, and the number is increasing. Developing high-specific thrombolytic drugs are the important means for the prevention and treatment of thrombotic diseases.At present, there are some disadvantages of the common thrombolytic agents: not specific, antigenicity, short half-life, large consumption, expensive and so on. It is necessary to find the thrombolytic agents which are high selectivity, little side effect and low price.Microorganism is an important source of thrombolytic drugs which grow fast ,control the conditions of growth easily. Therefore, it is more and more important to research the microbial metabolite as thrombolytic drugs (fibrinolytic active substances). Discovering the new thrombolytic drugs has become the research focus in medicine at home and abroad.In addition, ecological environment and species diversity of microorganism are the treasury of natural substances. The purpose of the research is to isolate strains which produce fibrinolytic enzyme and do research on them.A strain with higher fibrinolytic activity was isolated from the 60 strains which were isolated from the soil. In the paper, lw-72 strain was studied by strain identification, optimization of flask fermentation conditions, extraction of fibrinolytic enzyme, purification of fibrinolytic enzyme and characterization of fibrinolytic enzyme.Lw-72 was identified based on partial 16S rDNA gene sequence aligned with the GenBank database, morphological characteristics, physiological and biochemical characteristics.The results showed that the morphological characteristics, physiological and biochemical characteristics of lw-72 were similar to those of Bacillus licheniformis , nucleotide sequence was identity of 99.86% with Bacillus licheniformis. Therefore,strain lw-72 belongs to Bacillus licheniformis.The flask fermentation conditions of Bacillus licheniformis lw-72 was studied by single factor experiments and orthogonal experiments. The components of fermentation culture medium were as follows: corn flour 0.5%, soybean powder 2.0%, Na2HPO4 0.4%, NaH2PO4 0.2%, CaCl2·2H2O 0.01%, KCl 0.01%. Fermentation was performed for 72 h in a 250 mL shake flask containing 30 mL medium at inoculum volume and initial pH 7.0. The enzyme activity could reach 622 U/mL, a 2.4 fold higher than that under the original conditions.A fibrinolytic enzyme(BlFE) from Bacillus licheniformis lw-72 were purified by the ammonium sulfate fractionation and DEAE-Sepharose fast flow chromatography. It shows that a single protein band in the SDS-PAGE and relative molecular weight is 25 kD; pI is 8.3; fibrinolytic activity is stable between 4℃~50℃and pH6~10, decrease above 50℃and pH10.Its fibrinolytic activity is 0 when pH3. Metal cations Na+,K+,Ca2+,Mg2+,Zn2+,Mn2+ increase slightly and Fe2+,Cu2+ inhibit its activity. Urea is inhibit its activity stronger. SDS and Triton-X100 are in the role of weak,but they also inhibit the activity. Trypsin can increase the activity.Km is 2.237mg/mL.