The Study Of Purification, Gene Cloning And Expression Of Bacillus Subtilis BS-3 Fibrinolytic Enzyme

Thrombotic diseases have seriously endangered the health and the lives of mankind. In recent years which has been attracted a high degree of widespread concern and attention by medical profession. Microbial species are an important source of fibrinolytic enzyme. Not only has it can directly hydrolysis thrombus formatting from blood coagulation fibrin into small peptide and amino acids, and inhibit thrombosis, but also it has thrombolytic effective, not cause bleeding,non-toxic side effects, and advantages of long half-life. Therefore, the exploring of lobster sauce fibrinolytic enzyme has extremely attractive and broad prospects.This article is to screen a strain named BS-3 with higher fibrinolytic activity which was isolated from the soil. Physiological and biochemical responses showed that most characteristics of BS-3 strain was similar to Bacillus subtilis. Using NCBI Web site to compare The homology of the16S rDNA sequence, Drawing phylogenetic tree by the method of Neighbor-Joining,the results showed that the 16S rDNA sequence of BS-3 was 100% homology with Bacillus subtilis, so the physiological and biochemical reaction and 16S rDNA gene sequence analysis BS-3 as Bacillus subtilis.Through orthogonal experiments and single factor test, the optimal fermentation conditions of Bacillus subtilis BS-3 strain was determined, the results were as follow:the suitable main medUm was:corn flour 5 g/L, DouBing powder 10 g/L, CaCl2·2H2O 0.5 g/L, NaCl 0.5 g/L, NaH2PO4·2H2O 2 g/L, Na2HPO4·12H2O 4g/L, MgSO4·7H2O 0.2 g/L; In 250mL sharking flask the optimum fermentation conditions were:fermentation time is 60 hours, medUm initial pH 7.0, inoculation volume 4%, medUm volume 50mL, temperature 37℃, rotate speed 200r/min. The activity of fibrinolytic enzyme was 624.159U/mL, which increased 4.3 times than the initial fermentation conditions.The fibrinolytic enzyme from strain BS-3 was extracted by the ammonium sulfate sulfate fractional precipitation. Examined the isoelectric point by isoelectric focusing electrophoresis,Purified fibrinolytic enzyme by DEAE-Sepharose anion exchange and polyacrylamide gel electrophoresis. Obtained two single protein fraction with fibrinolytic activity (BsFE-1 and BsFE) from Bacillus subtilis BS-3. The BsFE-1 molecular mass was estimated to be about 48 kD by SDS-PAGE.The BsFE molecular mass was estimated to be about 43 kD by SDS-PAGE.The first 15 amino acids of the N-terminal sequence of the enzyme were determined to be AKLDETLTMLKDLTD. BsFE fibrinolytic activity was a serine protease including two sulphur keys and metal ions. Its optimum reaction temperature was 37℃and pH was 8.0. The activity was enhanced by Ca2+ and Na+. Pepsin,trypsin and bile salt were effect on BsFE, respectively, trypsin and bile salt had no effect on BsFE,but at pH 3.0 BsFE was hydrolyzed by pepsin and then its fibrinolytic activity was depressed a little.Using the kinetics of enzyme-catalyzed reaction measured the Km of BsFE was 2.6 mg/mL and Vmax was 1.0 mol/(L-min).Based on the homology of coding sequence to design degenerate primers. Also added EcoR I and Xho I restriction site on both ends. Using genomic DNA of BS-3 as template, PCR was proformed and obtained a complete fibrinolytic enzyme gene bsfe. The purified vector pET-30a and PCR product of gene bsfe were digested by EcoR I and Xho I. After purifying the digested products respectively, we ligated these two kinds of DNA by T4 DNA ligase and constructed the recombinant plasmid pET-30a-bsfe. Then bsfe gene was sequenced. The result indicated that this gene contained 1086 bp nucleotides and encoded 361 amino acid proteins.The recombinant plasmid pET-30a-bsfe were transformed into host bacterium of E.coli ER2566 respectively. SDS-PAGE analysis was performed after being induced by IPTG. The relative molecular weights of the expression products were 43 kD.The E.coli ER2566 was cultured with recombinant plasmid pET-30a-bsfe then the expression products were purified by Nickel-affinity chromatography column. SDS-PAGE showed that there was a single band at 43 kD of expression product.Express product conform with theory and had fibrinolytic enzyme activity.