Study On Purification And The Enzymatic Characterization Of Spirulinakinase

OBJECTIVE:To research the purification of Spirulinakinase method and provide some experimental evidence for the industrial production of Spirulina kinase drugs and lay the foundation for the enzymatic characterization. To research the the enzymatic characterization and provide some reference for the preservation, transportation and other conditions and new Thrombolytic drugs.METHODS:1. Purification of Spirulinakinase:(1) Determine the fibrin plate method as a method of detecting the activity of Spirulinakinase, draw the standard curve. (2) Determine the Coomassie brilliant blue method of protein content and respectively do sensitivity 100μg/ml and 10μg/ml standard curve. (3) Preparation of crude extract of Spirulinakinase. The Spirulinakinase powder was soaked in distilled water and mixed, placed at 4℃overnight. Discard precipitate, the supernatant was the crude extract of Spirulinakinase. (4) Ammonium sulfate salting-out. Set 6 fractions, the precipitate and supernatant were collected to detect activity after salting-out. Drawn curve according to the relative activity and ammonium sulfate saturation. (5) Dialysis. Treated dialysis bags, placed the product in bags and soaked in distilled water, changed the water every other 1,2,4 h, the final dialyzed overnight. (6) Gel Permeation Chromatography. Loaded Sephadex G-75 gel chromatography and placed the Spirulinakinase on the column. Eluted with PBS buffer to UV 280nm detection of synchronous detection eluent and collected the penetration peaks to detect activity by the fibrin plate method. Collected the active penetration peak. (7) SDS-PAGE electrophoresis. Installed vertical electrophoresis tank and gel preparation, Verified the purity of Spirulinakinase and determined molecular weight by SDS-PAGE electrophoresis.2. The enzymatic characterization of Spirulinakinase. (1) The optimum temperature for Spirulinakinase. Put Spirulinakinase into the hole in the fibrin plate, then placed the plate in different temperatures and measured dissolution ring size. Calculated the relative activity of Spirulinakinase on every temperature point by the highest activity of 100%. Set temperature as horizontal ordinate and relative activity as vertical ordinate and calculated optimum temperature for Spirulinakinase. (2) Temperature stability for Spirulinakinase.6 tubes of Spirulinakinase were placed in different temperatures. Sampled every 30min and removed the enzyme solution immediately then placed under-20℃cryopreservation. Determined all samples simultaneously in a fibrin plate. Calculated the relative activity of Spirulinakinase on every temperature point by the highest activity of 100%. Set temperature as horizontal ordinate and relative activity as vertical ordinate and calculated temperature stability for Spirulinakinase. (3) The optimum pH Spirulinakinase. Fibrin plate was cut into small pieces and soaked in a series of pH concentration range of pH4～pH12 buffer solution overnight so that the liquid inside the fibrin pieces to exchange with the buffer. Measured the maximum activity of Spirulinakinase as 100% at 37℃and calculated the relative activity. Set pH as horizontal ordinate and relative activity as vertical ordinate and calculated the optimum pH Spirulinakinase. (4) PH stability of Spirulinakinase. Spirulinakinase would be dissolved in a series of pH concentration range of pH4～pH12 buffer solution. Measured the maximum activity of Spirulinakinase as 100% at 37℃and calculated the relative activity. Set pH as horizontal ordinate and relative activity as vertical ordinate and calculated PH stability of Spirulinakinase. (5) Mode of action of Spirulinakinase. Half of a fibrin plate was cut out at 85℃for 30min, the other half was not treated. Put Spirulinakinase and urokinase into the hole in the two fibrin plates and observed effect at 37℃. (6) Inhibitors and denaturant on the activity of Spirulinakinase. To prepare solution by a certain percentage of enzymes and inhibitors and the mixture of denaturants with the mixture of distilled water and Spirulinakinase as control, measured activity at 37℃for 1h.RESULTS:1. Purification of Spirulinakinase:(1)Spirulinakinase crude separation. Spirulinakinase was synthesized crude enzyme solution, and then by ammonium sulfate salting-out. The method was determined which used of 45% saturation of ammonium sulfate to remove hybrid protein and 75% saturation of ammonium sulfate to collect the enzyme by salting-out curves. Then the enzyme was saved by freeze-drying after dialysis. (2) Gel Permeation Chromatography. The penetration peaks were detected activity by the fibrin plate method. Only the second penetration peak wash activity, the others not. So collected the peak. (3) The result of SDS-PAGE. Protein impurities were almost divisible and obtained pure protein. Molecular weight of Spirulinakinase was 40KD.2. The enzymatic characterization of Spirulinakinase. (1) The optimum temperature for Spirulinakinase. The activity of Spirulinakinase will be highest when 45℃, it's optimum temperature.45℃-55℃is the optimal temperature range. (2) Temperature stability for Spirulinakinase. Below 50℃, the thermal stability of Spirulinakinase is strong and half-life are more than 1h. (3) The optimum pH Spirulinakinase. Spirulinakinase's optimum pH is 8, the optimum pH value range of pH8-pH9. (4) PH stability of Spirulinakinase. The stability of Spirulinakinase is strong in the range of pH6-pH10 and less stable below pH6 or above pH10. The activity will be highest at pH9. So the spirulinakinase should be kept under slightly alkaline. (5) Mode of action of Spirulinakinase. The role of spirulinakinase which dissolve fibrin is different from urokinase. Spirulinakinase has a direct role of fibrin degradation. (6) Inhibitors and denaturant on the activity of Spirulinakinase. Spirulinakinase is a serine protease and the structure does not contain disulfide bonds.CONCLUSION:Enzyme recovery reached 42.3% and purification factor 6.1 after Spirulinakinase sample through two ammonium sulfate salting-out and Sephadex G-75 gel chromatography. The optimum temperature is 45℃, strong thermal stability below 50℃. The optimum pH is 8 and the enzyme should be kept under slightly alkaline. Spirulinakinase has a direct role of fibrin degradation. Spirulinakinase is a serine protease and the structure does not contain disulfide bonds.