Purification and kinetic characterization of trypsin from the intestine and pyloric caeca of the white grunt, Haemulon plumierii, (Lacepède, 1801)

Abstract

Trypsin is one of several animal digestive proteases whose activities collectively breakdown ingested proteins to facilitate gut amino acid absorption. The goals of this research were to purify and characterize trypsin from intestinal and pyloric caecal tissues of white grunt, Haemulon plumierii (Lacepède, 1801). Trypsin was purified 11-fold from intestinal and pyloric caecal tissues of the white grunt, through the sequential application of ammoniun sulfate fractionation, size exclusion chromatography, and affinity chromatography. The enzyme displayed optimal activity at pH 8 and 40 oC. It was completely inhibited by the presence of soybean trypsin inhibitor at a concentration of 100 %. SDS-PAGE analysis of the purified enzyme revealed a single band with an estimated molecular weight of 24 kDa. This white grunt trypsin was similar to those from other fishes in terms of its molecular weight, kinetic properties, and response to the presence of soybean Trypsin is one of several animal digestive proteases whose activities collectively breakdown ingested proteins to facilitate gut amino acid absorption. The goals of this research were to purify and characterize trypsin from intestinal and pyloric caecal tissues of white grunt, Haemulon plumierii (Lacepède, 1801). Trypsin was purified 11-fold from intestinal and pyloric caecal tissues of the white grunt, through the sequential application of ammoniun sulfate fractionation, size exclusion chromatography, and affinity chromatography. The enzyme displayed optimal activity at pH 8 and 40 oC. It was completely inhibited by the presence of soybean trypsin inhibitor at a concentration of 100 %. SDS-PAGE analysis of the purified enzyme revealed a single band with an estimated molecular weight of 24 kDa. This white grunt trypsin was similar to those from other fishes in terms of its molecular weight, kinetic properties, and response to the presence of soybean trypsin inhibitor.

Tripsina es una de las varias proteasas animales digestivas que rompen colectivamente las proteínas ingeridas, para facilitar la absorción de aminoácidos en el intestino. Las metas de esta investigación fueron purificar y caracterizar tripsina del tejido intestinal y ciego pilórico del pez cachicata, Haemulon plumierii (Lacepède, 1801) mediante la aplicación secuencial de fraccionamiento con sulfato de amonio, cromatografía de exclusión por tamaño y cromatografía de afinidad. La enzima desplegó actividad óptima a pH 8 y a 40 oC. Tripsina fue completamente inhibida por la presencia de inhibidor de tripsina de soya a concentración de 100 %. El análisis de SDS-PAGE de la enzima purificada reveló una banda con un peso molecular estimado de 24 kDa. Esta tripsina de cachicata fue similar a aquellas obtenidas de otros peces en términos de su peso molecular, propiedades cinéticas y respuesta a la presencia de inhibidor de tripsina de soya.