Breeding And Fermentation Optimization Of Fibrinolytic Enzyme-producing Strains And Characterization Of Fibrinolytic Enzyme

Douchi Fibrinolytic Enzyme (DFE) is a new fibrinolytic enzyme which was discovered in Douchi, a traditional Chinese food of fermented-soybean. The DFE shows strong fibrinolytic activity, has no toxicity and side effect, never causes endobleeding, and has long half-life in the body. Therefore, DFE is worthy of developing as either medicine of cleavage thrombus or diet food to prevent thrombotic diseases. However, most of the present studies in this field were focused on screening of fibrinolytic enzyme-producing strains and optimizing fermentation conditions for producing DFE. There have been few reports about purification and biochemical characterization of DFE. The studies are even fewer on molecular cloning of DFE. To obtain a novel DFE, Several fibrinolytic enzyme-producing strains were isolated from Douchi; One of the strains with strong fibrinolytic activity was treated with ultraviolet rays and HNO2, respectively, and a mutant with high yield of fibrinolytic enzyme and good genetic stability was obtained; The optimal fermentation conditions for producing fibrinolytic enzyme were determined; The method of purification of fibrinolytic enzyme was well established; Addtionally, the fragment encoding fibrinloytic enzyme mature peptide was amplified from the total DNA of DC12-N8 by PCR; The sequence homologous analysis of DNA and the deduced amino acid was made; The biochemical characterization of the enzyme was examined systematically.Six fibrinolytic enzyme-producing strains with different colony morphology were isolated from Douchi, and one strain named DC-12 with the highest fibrinolytic activity was selected; The fibrinolytic activity of the fibrinolytic enzyme produced by the wild strain DC-12 was 200 IU/mL, and the specific activity was 101 IU/mL. DC-12 was identified as Bacillus subtilis by testing its morphology, physiology, and biochemistry, and the result was confirmed by Guangdong Detection Center of Microbiology.The strain DC-12 was treated with ultraviolet rays and HNO2, respectively; Three mutants DC-12N8, DC-12N10 and DC-12U8 with high yield of fibrinolytic enzyme were obtained, whose fibrinolytic activity is 720 IU/mL,740 IU/mL and 950 IU/mL, and their enzyme production increased by 3.6-fold, 3.7-fold and 4.75-fold as compared with those of the original strain, respectively; Their genetic stabilty was analysed and the result showed the fibrinolytic activity of DC-12N10 and DC-12U8 decreased after ten genetations, however, the production of fibrinolytic enzyme of DC-12N8 was very stable; Therefore, DC-12N8 was selected for further study.The optimal fermentation conditions for producing fibrinolytic enzyme were determined and the results are as follows. The compositions of fermentation medium are: 3% water-soluble starch, 3% peptone, 0.5% yeast extract, 0.6% K₂HPO₄, 0.1% KH₂PO₄, 0.075% MgSO₄, 0.02% CaCl₂, pH 7.0. The optimal cultivation temperature is 30 ℃, the optimal shaking speed is 150 r/min when 150 mL flask contains 30 mL medium, and the fermentation time is 96 h. Under the conditions the fibrinolytic...