Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum) |
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Authors: | Marina Marcuschi Maurício FM Machado Marcelo FM Machado Luiz B Carvalho Jr Ranilson S Bezerra |
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Institution: | a Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica (CCB) and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670-910 Recife, Pernambuco, Brazil b Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo 04044-020, Brazil |
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Abstract: | An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH2-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 °C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 °C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 °C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry. |
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Keywords: | FRET peptide Processing waste Specific cleavage site Tropical fish Trypsin |
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