Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin |
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Authors: | Augusto C.V. Freitas-Jú nior,Helane M.S. Costa,Marcelo Y. Icimoto,Izaura Y. Hirata,Marcelo Marcondes,Luiz B. Carvalho Jr.,Vitor Oliveira,Ranilson S. Bezerra |
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Affiliation: | 1. Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica, and Laboratório de Imunologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Cidade Universitária, 50670-420 Recife, PE, Brazil;2. Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, 04044-020 São Paulo, SP, Brazil |
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Abstract: | A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters Km, kcat and kcat/Km were 0.47 ± 0.042 mM, 1.33 s−1 and 2.82 s−1 mM−1, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe2+, Hg2+, Zn2+, Al3+, Pb2+, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries). |
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Keywords: | Arapaima gigas Air-breathing fish Fish processing waste Digestive enzymes Proteases Trypsin purification |
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