Purification And Some Properties Of Grass Carp (Ctenopharyngodon Idellus) Trypsin

Trypsin, chymotrypsin, and elastase occur in the alimentary canal of virtually all invertebrate and vertebrate organisms (Solomon et al., 1996) , they are all endopeptidases, which catalyze the hydrolysis of peptide bonds in the interior of a polypeptide chain or protein molecule .Trypsin is one of several animal digestive proteases whose activities collectively breakdown ingested proteins to facilitate gut ammo acid absorption. Trypsin is an enzyme specific for peptides and esters of the amino acids lysine and arginine. The goals of this research were to purify and characterize trypsin from hepatopancreas of grass carp (Ctenopharyngodon idellus).Purification of grass carp trypsin (GT)Enzyme was purified by using ammonium sulfate fractionation, CHOM sepharose 4B affinity column, two kinds of trypsin were isolated, called GTA and GTB .Trypsin was extracted from the hepatopancreas with 0.5 M NaCl-0.05 M CaC12-0.05 M, Tris-HCl extraction buffer (pH8.0), some guts were added in an effort to active trypsinogen. After actived, the crude enzyme extract was brought to 30% saturation with ammonium sulphate and subsequently centrifuged at 8, 000 rpm for 20 minutes at 0 ℃ , The 30% supernatant solution was brought to 70% saturation with ammonium sulphate, so that the 30% to 70% precipitate were collected. The precipitate was resolved with 0.5 MNaCl, 0.05 M CaCI2, 0.05 M, Tris-HCl buffer (pH7.0) . Afterwards, the crude enzyme solution was loaded on CHOM sepharose4B affinity column chromatograph, which was pre-equilibrated by 0.5 M NaCl-0.05 M CaC12-0.05 M, Tris-HCl buffer (pH7.0) fully. The column was washed with the same buffer to wash off all of the impurity proteins, then the column was eluted with 0.5 M glycine-HCl buffer (pH3.5) . The unique peak of trypsin activity was pooled , named GTA. Then the column was eluted with 0.5 M glycine-HCl buffer(pH3.0) and another enzyme with trypsin activity was obtained ,named GTB. As a result. GTA was 243-fold purified , with the specific activity of 61388 TAME units/mg protein and recovery of 30.3%, while GTB 5-fold purified, with the specific activity of 1447 TAME units/mg protein andrecovery of 0.2%. SDS-PAGE electrophoresis was performed as per the modified Laemmli method, using a discontinuous buffer system, 5 % stacking gel was made with a 1 MTris-HCI (pH6.8) buffer, while 15 % resolving ge11.5 M Tris-HCI (pH8.8) buffer. It is notable that lanes which containing proteins representing the highest level of trypsins-GTA and GTB purification obtained in this study, revealed a single band using silver stain. The purified trypsins themselves indicates an approximate molecular weight of 27 kDa.Some properties of two enzymes were studied.The pH optima for the grass carp trypsinsActivity of GT was measured in buffers of different pH value. It indicated that:The activities were influenced significantly by pH value. They were very low at pH below 5.5and increased rapidly in the range of pHS.S-8.0. But they also decreased rapidly when pH exceeded8.0. Their optimal activities were observed at approximately pH8.0.pH stability of GTStability of GT was measured in buffers of different pH value. It indicated that:Stability of GTA increased continuously in the range of pH5.5-9.0. It decreased gradually whenpH exceeded 9.0. GTA was most stable at pH9.0.Stability of GTB increased continuously in the range of pH5.5-9.5. It decreased gradually whenpH exceeded 9.0. GTA was most stable at pH9.5.Temperature optima for the grass carp trypsinsActivity of GT was measured at different temperature. It indicated that:The activity of GTA increased when temperature increased. The temperature optimum for GTAwas 60℃. But the activity decreased gradually when temperature exceeded 60℃. It decreasedrapidly when temperature exceed 70℃.The activity of GTB increased when temperature increased. The temperature optimum for GTAwas 60℃. But the activity decreased rapidly when temperature exceeded 60℃.Thermostabilities of GTGT was stable at temperature under 40℃. When maintained at 40℃ for 30 minutes, theremaining activity of GTA was 91.7 percent of the highest activity of the enzyme, while that of GTB80.6 percent. GT lost activity rapidly when temperature exceeded 40℃ .When it reached 60℃, theremaining activity of GTA was 2.6 percent of the highest activity of the enzyme, while that of GTB2.0percent. When it reached 80℃, the remaining activity of GTA was 1.0 percent of the highestactivity of the enzyme, while that of GTB 0.5 percent.Summary: The Michaelis constant of GTA is 0.37 mmolBAPA/ mL. Both GTA and GTBexhibited optimal activity at a pH of 8.0 and at a temperature of 60℃, using TAME as substrate.