title: Purification and partial characterisation of a trypsin from the processing waste of the silver mojarra (Diapterus rhombeus).
abstract: An alkaline peptidase was purified from the viscera of the silver mojarra (Diapterus rhombeus) in a three-step process: heat treatment, ammonium sulphate fractionation and molecular exclusion chromatography (Sephadex® G-75), with final specific activity 86-fold higher than the enzyme extract and yield of 22.1%. The purified enzyme had an estimated molecular mass of 26.5kDa and NH2-terminal amino acid sequence IVGGYECTMHSEAHE. Higher enzyme activity was observed at pH 8.5 and between 50 and 55°C. The enzyme was completely inactivated after 30min at 55°C and it was significantly more stable at alkaline pH. Km, Kcat and Kcat·Km(-1) values, using BApNA as substrate, were 0.266mM, 0.93s(-1) and 3.48mM(-1)s(-1), respectively. Enzyme activity increased in the presence of the ions (1mM) K(+), Li(+) and Ca(2+), but was inhibited by Fe(2+), Cd(2+), Cu(2+), Al(3+), Hg(2+), Zn(2+) and Pb(2+) as well as by the trypsin inhibitors TLCK and benzamidine.
authors.affiliation: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife-PE, Brazil.
authors.forename: Janilson F
authors.initials: JF
authors.lastname: Silva
authors.affiliation: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife-PE, Brazil.
authors.forename: Talita S
authors.initials: TS
authors.lastname: Espósito
authors.affiliation: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife-PE, Brazil.
authors.forename: Marina
authors.initials: M
authors.lastname: Marcuschi
authors.affiliation: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife-PE, Brazil.
authors.forename: Karina
authors.initials: K
authors.lastname: Ribeiro
authors.affiliation: Laboratório de Piscicultura Marinha, Departamento de Pesca e Aquicultura, Universidade Federal Rural de Pernambuco, Recife-PE, Brazil.
authors.forename: Ronaldo O
authors.initials: RO
authors.lastname: Cavalli
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo 04044-020, Brazil.
authors.forename: Vitor
authors.initials: V
authors.lastname: Oliveira
authors.affiliation: Laboratório de Enzimologia (LABENZ), Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami (LIKA), Universidade Federal de Pernambuco, Recife-PE, Brazil. Electronic address: ransoube@uol.com.br.
authors.forename: Ranilson S
authors.initials: RS
authors.lastname: Bezerra
keywords: Alkaline peptidase; Diapterus rhombeus; Fish; Trypsin; Viscera