, 2009), food processing ( Shahidi & Kamil, 2001) and pharmacology ( Jónsdóttir, Bjarnason, & Gudmundsdóttir, 2004). The silver mojarra (Diapterus rhombeus) is a marine finfish from the northeastern

Brazilian coast, of economic and ecological importance that can be used to extract proteases for biotechnological applications. This fish PCI-32765 mw belongs to the family Gerreidae and is found in coastal estuaries throughout the tropical waters of the Atlantic Ocean ( Austin, 1973). According to the Brazilian Environmental Agency IBAMA (2008), 2080 tons of mojarras were captured in northeastern Brazil in 2006, which generated an estimated annual discharge of about 100 tons of viscera. Therefore, the investigation into enzymes present in this type of byproduct may help optimise the use of these resources, by adding value to these industrial

segments. The aim of the present study was to purify a trypsin from the digestive tract of the silver mojarra and characterise its physical and biochemical properties, such as the effect of temperature, pH, ions, inhibitors, substrate concentration and NH2-terminal amino acid sequence. Specimens of D. rhombeus were obtained from a fishing community in Itapissuma, Pernambuco, HTS assay Brazil. Fish were packed in ice and transported to the laboratory. Average weight and length was 350 ± 20 g and 28 ± 2 cm, respectively. The intestine and pyloric caeca of ten fish (about 30 g) were removed and stored in a freezer at −25 °C until analysis. Fish intestines and pyloric caeca were mixed together and homogenised at a concentration of 40 mg ml−1 (w.v−1) of tissue in a solution of 0.01 M Tris–HCl, pH 8.0, with 0.9% NaCl, using a tissue homogeniser (Bondine Electric Company, Chicago, IL) at 300 rpm for 60 s. The homogenate was then centrifuged (Herolab Unicen MR Centrifuge, Germany) at 10,000g for 25 min at 4 °C for the removal of insoluble

particles. The supernatant (enzyme extract) was collected and stored in a freezer at −25 °C for subsequent use in the purification Oxymatrine steps. Enzyme activity was measured using BApNA (Nα-benzoyl-l-arginine-p-nitroanilide) prepared with dimethylsulphoxide (DMSO), as substrate specific for trypsin. The assay was carried out by mixing 30 μl of sample with 140 μl of 0.1 M Tris–HCl, pH 8.0 and 30 μl of 8 mM BApNA (final concentration of 1.2 mM) for 10 min at 25 °C. The formation of p-nitroaniline (product) was measured at 405 nm with a microplate reader (Bio-Rad X-Mark spectrophotometer, California, USA). A blank control was prepared by replacing sample with 0.1 M Tris–HCl, pH 8.0 (Souza, Amaral, Santo, Carvalho, & Bezerra, 2007). One unit (U) of enzyme activity was defined as the amount of enzyme capable of hydrolysing 1 μmol of BApNA per min under the established conditions, using a molar coefficient of 9100 mM−1 cm−1.