Purification, Characterization, Gene Cloning And Expression Of Fibrinolytic Enzyme From Endophytic Paenibacillus Polymyxa

Thromboenbolism disease is a serious threat to our health and life. Thrombolytic therapy, which can significantly decrease the patients mortality and mutilation rate, has been extensively investigated as a means of medical treatment. However, it is difficult to widely used in clinical application for the reason of low specificity, short half-life, hemorrhagic risk, and higher rate of thrombus reforming. So, the research and developments of the novel thrombolytic drugs with high efficiency, high specificity, high safety, little side effect, low cost are needed urgently to solve problems described above. Many traditional Chinese medicines, especially those herbs with activiting blood circulation, can inhibit blood platelet aggregation and thrombosis, including some alkaloids such as alkaloids, flavonoids and saponin. Endophytes are widely existing inside the tissues of healthy plants. Co-evolving with medicinal plants, some endophytes have developed the abilities to produce same or similar bioactive substances as what their host plants do. Therefore, it is a huge potential to screen novel, highly active fibrinolytic enzyme from endophytes.In this study, strain EJS-3 with strongly fibrinolytic activity and thrombolytic effect test in vitro was screened from endophytes and later identified by morphological, physiological, biochemical tests and 16S rRNA gene sequence analysis. The fibrinolytic enzyme was purified and characterized. The gene encoding the fibrinolytic enzyme was cloned, sequenced, and overexpressed. Then, the recombinant enzyme was purified and characteried. All these results provided certain theoretical basis for developing and designing new thrombolytic drugs. Main study results as following:1. Using improved method,7 strains with fibrinolytic activity were screened from the root, stem and leave of Chinese medicinal plants such as Japanese Honeysuckle, Skullcap, Mongol dandelion, Coptis chinensis Franch, Weeping Forsythia, Japan creeper, Houttuynia, and Japan Stemona. The screening strategies included the first selection with the casein plate and the further with shaking culture. Among them, the strain EJS-3 from the root of Japan Stemona had the highest fibrinolytic activity and later was preliminary identified as Paenibacillus polymyxa by morphological, physiological, and biochemical tests. The 16S rDNA gene of EJS-3 was amplified by polymerase chain reaction (PCR) from the genomic DNA. The PCR product (1.5 kb) was purified and sequenced. A similarity search was performed using Genbank databases and the neighbor-joining phylogenetic tree was constructed. The results showed that a typical characteristion of the Paenibacillus polymyxa 16S rDNA sequences (GenBank accession number DQ120522) comparison study supported a strong relationship between strain EJS-3 and members of genus Paenibacillus and particularly revealed the highest homology (98%) with Paenibacillus polymyxa. The result further indicated that strain EJS-3 belonged to Paenibacillus polymyxa. No work on Paenibacillus polymyxa producing fibrinolytic enzyme has been reported so far.2. The purification of fibrinolytic enzyme from the endophytic strain EJS-3 was processed as the following procedure:centrifugal sterilization, fractional sedimentation with ammonium sulfate, Hiprep phenyl FF chromatography, RESOURCEM Q ion-exchange chromatography, Sephacryl S-300HR gel-filtration. The purified fibrinolytic enzyme PPFE-I was homogenous in SDS-PAGE electrophoresis, and a single peak in HPLC. Its purity was more than 94.1% estimated by HPLC. Compared with the crude enzyme extraction, the specific activity of the purified enzyme increased by 14.5 folds with a recovery of 3.3%, and 1.6mg of active proteins with an activity of 2096 IU·mg-1 could be obtained from 1L fermentation broth.3. Estimation of the molecular weight of PPFE-Ⅰby SDS-PAGE and MALDI-TOF gave the values of 63 kDa and 63.3KDa respectively. The optimum pH and temperature of PPFE-Ⅰwere pH7.5 and37℃, respectively. The PPFE-Ⅰexcreted by isolate EJS-3 showed a greater degree of pH and thermal stability. The fibrinolytic enzyme was inhibited by Cu2+ and Ca2+, while enhanced by Mg2+, Fe2+, and Zn2+. The PPFE-Ⅰwas inhibited completely by PMSF, partly inhibited by EDTA, indicating that the PPFE-Ⅰwas a serine metalloprotease.4. The proteolytic activity of PPFE-Ⅰwas identified and measured using synthetic substrates with arginyl bonds. The rusult showed that the optimum substrate of PPFE-Ⅰwas Suc-Ala-Ala-Pro-Phe-pNA, which was the same as that of subtilisin or chymotrypsin. The Michaelis constant Km value of PPFE-Ⅰusing N-Succinyl-Ala-Ala-Pro-Phe-pNA as substrate was 0.20mM, which showed that the enzyme had a great affinity with the substrate. Fibrin clots could be effectively degraded by PPFE-Ⅰvia direct fibrinolysis. The PPFE-Ⅰwas a kind of fibrinolytic enzyme but not plasminogen activator. Theα-subunits of fibrinogen were firstly cleaved, followed by theβ-chains, while theγ-chains were resistant to the enzyme digestion. Throniholysis was evaluated by constant temperature natural lysis in vitro. The dissolving rate of blood clots was 91.33% after incubation 24h showed that the PPFE-Ⅰhad significantly thrombolysis effect in vitro. The anticoagulant activity of the PPFE-Ⅰwas more significant than that of urokinase.5. The mature PPFE-Ⅰgene was amplified from purified strain EJ-3 genomic DNA by PCR. The PCR product was isolated and ligated into the pMD-19T vector and sequenced. The vector constructed above was pMD-PPFE-Ⅰ. The PPFE-Ⅰgene was cloned into pET-DsbA and integrated into E. coli BL21(DE3). Active recombinant fibrinolytic enzyme had been successfully expressed in Escherichia coli as protein fusions with DsbA by addition of IPTG. The fibrinolytic activity of recombinant enzyme was 228 IU·mL-1. The expression of the recombinant enzyme was cinfirmed by SDS-PAGE and western blotting. SDS-PAGE analysis showed the recombinant enzyme was soluble and was about 18.4% of total cell protein. The recombinant enzyme exhibited a special band on Western blotting, which showed that the recombinant protein was DsbA-PPFE-Ⅰ.6. The recombinant enzyme was purified from the supernatant by Ni affinity column, thrombin digestion, and sephadex G-100 gel-filtration. Its fibrinolysis activity was detected by fibrin plate method. The characterization of the purified recombinant enzyme such as optimal temperature, thermal stability, optimal pH, pH stability, substrate specificity, protease inhibitors, and metal ions were determined. There were no different characterization between the natural enzyme and recombinant enzyme.7. The PPFE-Ⅰgene was amplified by PCR and cloned to the Escherichia coli-Bacillus subtilis shuttle expression vector pHPQ constructed in our laboratory, and then integrated into Bacillus subtilis WB800. The fibrinolytic enzyme of recombinant enzyme whose activity in LB media containing 10μg·mL-1 chloramphenicol and 10μg·mL-1 erythromycin was 60 IU·mL-1 at 32h incubation time.