Purificação e caracterização de tripsina a partir do ceco-pilórico do Pacamã (Lophiosilurus alexandri)

Abstract

The Food and Agriculture Organization of the United Nations (FAO) indicate that until 2030, world population will rise from seven billion to 8.3 billion. Facing this reality, the commercialization of fish is an activity that has been increasing expressively in the production chain, especially those derived from aquiculture, represented by species grown in captivity. Brazil, for example, is a strong candidate to become the major producer and exporter in the category because of its wide diversity and proper geographic and climate conditions. One of the major obstacles faced by breeders is related to production cost, especially food costs that, depending on the species, can reach 70% of total expenses. In order to counterbalance the excessive costs of production, breeders should dispose all of the animals’ mass potential and not only the commercialization of muscle fiber since around 30% of the assessed weight corresponds to innards, majorly categorized as sub-products. These residues take on significant biotechnological potential providing, for example, a source of enzymes that can be applied in several industries. In the past few years, searching for proteases from different sources is an activity that has been increasing significantly; around 50% of total industrial sales of enzymes consist of proteases. We highlight the pioneer study of an isolated enzyme from Pacamã (Luphiosilurus alexandri), native fish from São Francisco River (Brazil). Trypsin of Luphiosilurus alexandri pyloric cecum was purified with only two purification processes: ammonium sulfate precipitation and anion exchange liquid chromatography in DEAE-Sepharose; with a recuperation of 86.97 and 44.91 %, respectively. Trypsin mass was estimated in 24 kDa through SDS-PAGE, presenting only one band when subject to silver coloration. The purified enzyme presented optimum temperature and pH at 50°C, and pH 9.0, respectively, using BApNA as substrate. Stability maintained high levels of activity up to pH 11 as well as high stability up to 50°C after a 60-minute incubation. The inhibition test verified strong inhibition by PMSF, Serine-proteases inhibitor, and absence of inhibition for EDTA and 2-Mercaptoethanol. Kinetic constants km and kcat for BApNA were 0.517 and 5.0, respectively. The purified enzyme also proved active in a zymography with casein. For these reasons, we consider it to be a promising enzyme for industrial processes for presenting a wide range of stability in pH and temperature as well as activity even for immobilization in solid matrices.