A SEPERATION METHOD OF PROTEASE INHIBITOR FROM GLASSFISH(LIPARIS TANAKAI) EGGS

摘要

A separation method of protease inhibitor derived from Liparis tanakai eggs is provided to reduce the purification time and improve purification yield by simplifying the purification process into one step by using CNBr(cyanogens bromide)-activating sepharose 4B-papain affinity column, and inhibit heat-resistant protease causing quality lowering of fish meat. A separation method of protease inhibitor derived from Liparis tanakai eggs comprises the steps of: homogenizing Liparis tanakai eggs in sodium phosphate buffer A(pH 7.0) containing NaCl, EDTA(ethylenediamine tetraacetic acid) and 2-mercaptoethanol; centrifuging the solution to remove homogenized suspension; culturing the supernatant at 80 deg.C for 10 minutes, cooling to room temperature and centrifuging it at 10,000g for 25 minutes; fractionating the extract with ammonium sulfite; dissolving precipitated fraction in buffer A and dialyzing the solution with sodium acetate buffer B(pH 5.5) containing NaCl, NaCl, EDTA and 2-mercaptoethanol for overnight; subjecting the dialyzed fraction to the CM sepharose column using NaCl gradient; dialyzing big fractions with sodium phosphate buffer C(pH 7.5) containing NaCl, NaCl, EDTA and 2-mercaptoethanol; ultracentrifuging the fractions with a 10-kDa membrane; subjecting the concentration to the sephacryl column to obtain the crude extract; washing aliquot of the CNBr-activating sepharose 4B column and swelling it on a glass filter with HCl to prepare gel; mixing the gel with solution containing papain in coupling buffer(pH 8.3) containing NaHCO3 and NaCl and stirring it at 4 deg.C for overnight; adding the gel into a blocking agent(pH 8.0) containing glycine and stirring at 4 deg.C for overnight; washing the gel with acetate buffer(pH 4.0) containing NaCl and coupling buffer, sequentially; pouring the gel into the column having equilibrium with buffer A; passing aliquot of Liparis tanakai egg crude extract through the affinity chromatography and washing it with buffer A; and eluting trisodium phosphate buffer(pH 10) containing NaCl to the affinity chromatography in a flow rate of 0.3 mL/min.