Study On Purification, Characterization, In Vitro Digestion Of Lumbrokinas

Lumbrokinase is a serine protease kinase and has strong thrombolyticcharacteristics. It was used as novel thrombolytic drugs because it is non-toxic, sideeffects and anticoagulation. In this thesis, earthworm (Eisenia faetida) was used asraw materials to study its extraction and purification. The following methods wereapplied in order: ammonium sulfate, ultrafiltration, the gel filtrationchromatography and ion exchange chromatography, after acquiring a relatively highpurity of lumbrokinase, this thesis preliminary studied the properties of this enzym.The main results are ills follows:1) The extraction and purification of lumbrokinaseUsing earthworm (Eisenia faetida) as raw materials, The LK pure was obtainedby applying the advanced technology of tissue fragmentation, centrifugation,ammonium sulfate salting out, ultrafiltration, Sephadex G-75gel filtration,DEAE-Sepharose FF by ion exchange chromatography, Activity Was detected byfibrin plate method, After being treated with DEAE ion exchange chromatography,specific enzyme activity of LK Was up to4006.8IU/mg, purification factor reached10.1-fold. The molecular weight distribution of the product purified were between20KD~60KD，was learned through SDS-PAGE electrophoresis analysis.2) Study of enzymatic properties of LKStudy the effect of temperature, pH, and metal ions on the activity of thepurified lumbrokinase. The results showed that: LK is relatively stable between pH6to11; when the temperature is between20℃~60℃, The stability of LK is alsogood. The fibrinolytic eaperiment in vitro indicated that LK has a direct dissolutionof thrombosis and activation of plasminogen, indirect thrombolytic double effect.Hg2+could completely inhibit the LK activity, and Mg2+could activate the enzymeactivity significantly.3) Study of vitro digestion and the role of cell proliferation on LKThe two-step method in vitro digestion and Caco-2cell model simulating intestinal epithelial cells were studied that LK influence on the human digestivesystem and the rate of absorption and utilization. Through Pepsin-trypsin method,it was measured that LK of in vitro digestibility was about90%; Through Caco-2cells simulation method, it were leared that:1)concentration of0.1mg/ml and0.01mg/ml of enzyme solution promoted cell growth in12h and24h and inhibitedcell growth in48h before purifid;2)concentration of1mg/ml enzyme solutionalways inhibited cell growth in the three time points, and the trend has beenstronger;3)concentration of0.1mg/ml and0.01mg/ml of enzyme solution had beenpromoted cell growth after purifid;4) the effect of the enzyme solution of1mg/mlwas similar with pre-purified, but the trend of enhanced was bigger than beforepurification.