Purification and characterization of trypsin and chymotrypsin from the di̇gesti̇ve trap of European seabass (Dicentrarchus labrax)

Fish digestive tract is very rich about hydrolytic enzymes. These enzymes are used as high value-added products in industries such as food, cosmetics, leather and textile in developed countries. Fort his reason, it was aimed to determine the resistance of trypsin and chymotrypsin enzymes to different environmental conditions by purifying the pyloric ceaca of cultured sea bass (Dicentrarchus labrax), which is the most produced fish species in our country. Trpsin enzyme was purified 52.71 fold with a yield of %8.60. The molecular weight of the purified trypsin was estimated to be 24 kDa by Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page) which showed single band in bromphenol blue stanning. The optimum temperature and pH for the trypsin activity were 55 ºC and pH 8.0, respectively. The enzyme was extremely stable in the pH range of 7.0-10.0 and highly (%70) stable up to 50ºC and non stable at 70ºC after 30 minutes incubation. Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) was used as a substrate for all activity and stability analyzes. The chymotripsin enzyme was purified 67.05 fold with a yield of %8.46 from same fish. The molecular weight of the purified chymotrypsin was estimated to be 29 kDa by SDS-Page. Chymotrypsin enzyme showed optimal activity at pH 9.0 and 50ºC with used n-succinyl-ala-ala-pro-phe-p-nitroanilide (SAAPNA) as a substrate for all activity and stability. The enzyme was extremely stable in the range of pH 7.0-10.0 and highly stable (%60) up to 50ºC and non stable at 70ºC after 30 minutes incubation.