Isolation And Identification Of Strains Producing Douchi Fibrinolytic Enzyme And Studies On Cloning And Expression Of Fibrinolytic Enzyme Gene In Prokaryote

Douchi Fibrinolytic Enzyme(DFE) is a newly discovered fibrinolytic enzyme which was contained in Douchi,a Chinese traditional fermented soybean.The DFE has the advantage of strong fibrinolytic activity, no toxicity, no endobleeding and long half-life, and so on. Therefore, DFE is worthy of developing as either thrombus medicine or health food.In this research,a bacterial strain producing fibrinolytic enzyme was isolated from Chinese traditional fermented soybeans, based on this ,its optimum fermentation conditions and the cloning and expressions of DFE gene was studied. The main results were as follows:1. Strains with higher fibrinolytic activity were isolated from the 42 strains which were preliminary isolated from Chinese traditional fermented soybeans using casein medium, and the one with the highest fibrinolytic activity of 613.8U/mL was screened using agarose-fibrin plate. HD4 was identified based on partial 16S rRNA 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 HD4 were similar to those of Bacillus subtilis, and its nucleotide sequence had similarity of 99% with Bacillus subtilis. Therefore, strain HD4 belongs to Bacillus subtilis .2. The flask fermentation conditions of Bacillus subtilis HD4 was studied by single factor experiments and orthogonal experiments. It was indicated that the optimal carbon source and nitrogen source were sucrose and yeast extract,respectively, the components of fermentation culture medium were as follows: sucrose 2%, yeast extract 2.5%, CaCl2 0.02%, K2HPO4 0.3%, KH2PO4 0.3%, MgSO4·7H2O 0.07%. Fermentation was performed for 48h in a 100mL shake flask containing 20 mL medium at inoculum volume and with the initial pH of 7.5. The fibrinolytic activity could reach 827.2U/mL, much higher than that under the original conditions.3. In this research,genome DNA of Bacillus subtilis HD4 was extracted,used as the PCR template,two DNA segments of 1059bP and 1491bP respectively were cloned using PCR, the larger segment was the complete DFE gene which encoding signal peptide, propeptide and mature peptide, the smaller segment was pro-DFE gene encoding propeptide and mature peptide. Then the complete DFE gene segments were cloned into the vector of pBluescript II KS, transferred into the host strain, multiplied, purified, and analyzed using electrophoresis and sequeneing, to confirm its accuracy. Alignment and homology analysis of the complete DFE gene had 99% homology with Nattokinase NAT.4. The complete DFE gene encoding pre-pro-fibrinolytic enzyme and pro-DFE gene encoding pro-fibrinolytic enzyme were cloned into pET-27b vector respectively and then transformed into Escherichia coli BL21(DE3). The recombinant of pro-DFE showed enzyme activity of 212.6U/mL in the fermentation supernant when induced with IPTG and glucose, and the protein expressed was about 28kDa. But no detectable enzyme activity could be found from the recombinant of complete DFE genes in the same condition, the protein expressed was 38kDa in the form of inclusion bodies.