Purificação e caraterização de uma protease alcalina do resíduo de processamento da Carapeba prateada (Diapterus rhombeus)

• Autor(a) principal: SILVA, Janilson Felix da
• Data de Publicação: 2009
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UFRPE
• Texto Completo: http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/6337
• topic: Carapeba prateada; Diapterus rhombeus; Protease; Tripsina; Protease; Trypsin; Slver mojarra; CIENCIAS AGRARIAS::RECURSOS PESQUEIROS E ENGENHARIA DE PESCA

The silver mojarra (Diapterus rhombeus) is specie of relevant representation among the fishing community in northeastern Brazil. Also in this region, the fishing is the most representative records despite growing carapeba in extensive systems. Among the parts of the fish are not consumed are the viscera which represent 5% of the total weight of the animals. Thus, to be discarded without treatment, these wastes pose serious environmental problem. However, because they are rich in digestive enzymes, viable for use in certain biotechnological processes these viscera represent an important alternative source of industrial enzymes. Based on this information, the purpose of this study was to purify and characterize an alkaline protease from the viscera of D. rhombeus. Therefore, viscera silver mojarra were used to obtain a crude extract and thereafter used for enzyme purification. The purification process was carried out in three steps: heat treatment (45°C for 30 minutes), precipitation with ammonium sulfate and molecular exclusion chromatography (Sephadex G-75). At the end of the purification process, achieved an increase of 86.80-fold in the specific activity and a yield of 22.34%. An aliquot of purified extract was applied to gel electrophoresis (SDS-PAGE) and its molecular weight was estimated at 24.5 kDa. The optimum pH and optimum temperature for enzyme activity were 8.5 and 55°C, respectively. The enzyme was shown to be sensitive to temperatures above 45°C, after incubation for 30 min, losing 100% of its activity. The values of Km and Kcat of the protease were 0.266 mM and 0.116 mM-1 s-1, respectively, using benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Its activity was increased in the presence of ions K+, Li+, Ca2+, Mn2+ and Ba2+ ions and inhibited by Fe2+, Cd2+, Cu2+, Al3+, Hg2+, Zn2+ and Pb2+. Tests with protease inhibitors showed that the enzyme was strongly inhibited by TLCK and benzamidine, classic inhibitors of trypsin. The sequence of the first 15 amino acid N-terminal protease was IVGGYECTMHSEAHE and showed high homology with trypsins of various species of fish. The data show that the purified enzyme is one trypsin-like with characteristics compatible to be used in the biotechnology industry.