Although pirarucu farming is still incipient, the species has shown great potential because of its peculiar characteristics, such as: excellent quality of meat, free of thorns, large consumer acceptance,

rusticity, air-breathing capacity and high growth rates, which can range from 7–10 kg in the first year of farming establishment ( Imbiriba, 2001 and Pereira-Filho et al., 2003). Since pirarucu is a large fish, its processing generates a considerable amount of waste, including viscera. In the light of this fact, the objective of this study was to establish a purification protocol, and to characterise and evaluate the possibility of using the digestive tract (pyloric caeca) of A. gigas as a potential source of trypsin. Specific substrate, inhibitors, check details Sephadex® G75 and DMSO were purchased from Sigma (St. Louis, MO, USA). Benzamidine–Sepharose was purchased from GE Healthcare (Buckinghamshire, UK). All salts and acid solutions were purchased from Merck (Darmstadt, Germany) and all SDS–PAGE reagents and molecular mass marker were from Bio-Rad Laboratories (Ontario, Canada). The Universidade Federal Rural de Pernambuco (Recife-PE, Brazil) kindly donated cultivated juvenile specimens Small molecule library of A. gigas for this study. The experimental cultivation of A. gigas was conducted in excavated tanks (250 m2)

located in the Estação de Aquicultura Continental Johei Koike – Universidade Federal Rural de Pernambuco, Recife-PE, Brazil. The animals were fed a commercial diet provided by Purina S/A, Brazil, containing 40% crude protein. Mean length of A. gigas specimens was 76.8 ± 12.2 cm

and mean weight was 4118 ± 1.8 g. After 40 days, three specimens were sacrificed in an ice bath Diflunisal for biometric measurements and tissue removal, according to standard methodology described by Bezerra et al. (2001). The pyloric caeca were dissected, carefully cleaned with deionized water, and kept at 4 °C during transportation to the laboratory (∼30 min). After this, the tissues (16 g) were homogenised in 0.1 M Tris–HCl pH 8.0 (200 mg of tissue/ml buffer), using a tissue homogenizer (4 °C) (IKA RW 20D S32, China). The homogenate was then centrifuged (Sorvall RC-6 Superspeed Centrifuge, North Carolina, USA) at 10,000g for 20 min at 4 °C. The supernatant (crude extract) was stored at −25 °C and used for further purification steps. Trypsin was purified, following a four-step procedure: heat treatment, ammonium sulphate precipitation, molecular size exclusion chromatography (Sephadex® G-75) and affinity chromatography (benzamidine-agarose). Crude extract (60 ml) was incubated at 45 °C for 30 min and centrifuged at 10,000g for 10 min at 4 °C. The supernatant was collected and fractionated into three fractions with ammonium sulphate (F1, 0–30%, F2, 30–90% of saturation and SF, final supernatant) for 2 h at 4 °C. Afterwards, the precipitate containing trypsin activity was collected by centrifugation and dialysed against 0.1 M Tris–HCl, pH 8.0.