Purification And Characterization Research Of A Fibrinolytic Enzyme Produced By Bacteria Screened From Douchi

Objective: Douchi Fibrinolytic Enzyme (DFE) is an exoenzyme which is secreted by bacillus isolated from Douchi. It has similar characteristic with Japanese Nattokinase. Compared with these thrombolytic drugs usually used in clinical treatment, DFE has a lot of advantages, such as high potency, long half-life, none antigenicity, none toxicity and safe, low price, and may be developed into the next generation of thrombolytic drugs. We plan to isolate and screen bacterial strains with fibrinolytic activity from Douchi, collecting from different district in China. The study is focused on the DFE and the strains that produce DFE, including: strain identity, optimization of liquid fermention and purification of DFE.Method: 1.By casein plate prescreening and fibrin plate postscreening, to get bacterial strains with fibrinolytic activity. 2. To identify the bacteria by physiology, biochemistry and 16SrRNA test. 3. Adopt single factor method and Orthogonal experiment method to discuss the best culture medium and fermentation condition of liquid fermentation. 4. To get the purity powder by ammonium sulfate subsection precipitation, dialyzation to remove the salt, Sephadex G-50 chromatography and vacuum freezing desiccation. 5. The protein content and fibrinolytic activity was determined by coomassie brilliant blue G-250 dyeing method and fibrin plate method. Calculate the enzyme activity yield and specific activity. 6. SDS-PAGE electrophoresis, coomassie brilliant blue R-250 dyeing, confirm the molecular weight of the enzyme.Result: 1. Eight bacterial strains with fibrinolytic activity were isolated from Douchi. The strongest fibrinolytic activity strain naming XY-1 was selected as the research object. 2. XY-1 was identified as Bacillus amyloliquefaciens by testing its morphology, physiology, biochemistry and 16S rRNA. 3. The best composition of culture medium are: yeast extract 1%, lactose 3%, K2HPO4 0.1%, KH2PO4 0.6%, MgSO4 0.1%, NaCl 0.05%, pH 9.0. The best fermentation conditions are: temperature 35℃, 250ml flask contains 50ml medium, inoculation quantity 500μl/50ml, fermentation time 24h. 4. After all the purification steps, the DFE activity yield was 37%, protein yield was 19.8%, specific activity was 1388.27U/mg, and purification fold was 1.87. 5. We got purity DFE powder after vacuum freezing desiccation. The SDS-PAGE shows two electrophoretic bands, the molecular weight were about 12k and 20k.Conclusion: We could also isolate bacteria which can produce fibrinolytic enzyme from Chinese traditional food Douchi. After physiology and biochemistry test, we identified sp. XY-1 as Bacillus amyloliquefaciens. Associate application of single factor method and Orthogonal experiment method have remarkable effect to the optimization of liquid fermentation. The DFE production can reach 2089.3U/ml from 235.2U/ml. Although the enzyme yield was high, the purification fold was low, and two kinds of protein were still contained. The purification method needs to be investigated further.