Purificação e caracterização da tripsina isolada do Coryphaena hippurus (dourado do mar)

Abstract

The use of enzymes as industrial catalysts has been growing in recent years and proteases are the class of enzymes with the highest employment in these processes, accounting for about 60% of the enzyme market. Among the proteases the trypsin that are important digestive enzymes in animals are highlighted, they act by hydrolyzing the C-terminal peptide bonds of lysine and arginine residues. The sources of enzymes are diverse: animals, vegetables and microorganisms, but with the advancement of sustainable development, the use of organic wastes from fishing have become important sources of enzymes. Due to the characteristics that the digestive enzymes of marine animals present, such as high thermal stability and variations of pH, important characteristics for industrial processes, as additives in detergents, among others, these are increasingly studied. Our objective in this work was isolated and characterized the trypsin of the Coryphaena hippurus (common dolphinfish) medium-sized fish from Ponta Verde beach, Maceió-AL. Isolation was achieved with saline fractionation and S-100 HR gel-filtration chromatography. Purification can be confirmed with a 15% SDS-PAGE where only one band at 26 kDa, molecular mass compatible with other fish trypsins was visualized. The isolated trypsin had Km and Kcat of 0.035 mM and 241.037 S-1, respectively. With respect to temperature and pH, the enzyme had an optimum temperature of 40 ° to 50 ° C and stability to pH ranging from pH 5.0 to 10.0, a common feature among fish enzymes, mainly due to their adaptation to the marine environment and eating habits. Trypsin was totally inhibited by Benzamidine, SBTI and PMSF specific inhibitors for trypsin, the first two, and for the latter serine proteases. Some ions reduced the activity of the enzyme alone and Ca2+ did not reduce its active The purification and characterization of trypsin of Coryphaena hippurus (sea gold) showed important characteristics of the enzyme, considering it as promising in industrial processes, mainly due to high thermal stability and variations in pH, but further studies involving biotechnological applications should be developed.