Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization

Background: Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.Results: A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-alpha-benzoyl-(DL)-arginine-p-nitroanilide (BApNA) as substrate. in addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50 degrees C, respectively. After incubation at 50 degrees C for 30 min the enzyme lost only 20% of its activity. K-m, k(cat), and k(cat)/K-m values using BApNA as substrate were 0.689 mM, 6.9 s(-1), and 10 s(-1) mM(-1), respectively. High inhibition of trypsin activity was observed after incubation with Cd2+, Al3+, Zn2+, Cu2+, Pb2+, and Hg2+ at 1 mM, revealing high sensitivity of the enzyme to metal ions.Conclusions: Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.Financiadora de Estudos e Projetos (FINEP/RECARCINE)Petroleo do Brasil S/A (PETROBRAS)Secretaria Especial de Aquicultura e Pesca (SEAP/PR)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao de Apoio a Ciencia e Tecnologia do Estado de Pernambuco (FACEPE)Univ Fed Pernambuco, Lab Enzimol LABENZ, Dept Bioquim CCB, BR-50670910 Recife, PE, BrazilUniv Fed Pernambuco, LIKA, BR-50670910 Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniv Fed Pernambuco, Lab Glicoprot, Dept Bioquim CCB, BR-50670910 Recife, PE, BrazilUniversidade Federal de São Paulo, Dept Biofis, Escola Paulista Med, BR-04044020 São Paulo, BrazilWeb of Scienc