title: Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).
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, NH(2)-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 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees 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.
authors.affiliation: Laboratório de Enzimologia, Departamento de Bioquímica CCB and Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitária, 50670-910 Recife, Pernambuco, Brazil.
authors.forename: Marina
authors.initials: M
authors.lastname: Marcuschi
authors.forename: Talita S
authors.initials: TS
authors.lastname: Espósito
authors.forename: Maurício F M
authors.initials: MF
authors.lastname: Machado
authors.forename: Izaura Y
authors.initials: IY
authors.lastname: Hirata
authors.forename: Marcelo F M
authors.initials: MF
authors.lastname: Machado
authors.forename: Márcia V
authors.initials: MV
authors.lastname: Silva
authors.forename: Luiz B
authors.initials: LB
authors.lastname: Carvalho
authors.forename: Vitor
authors.initials: V
authors.lastname: Oliveira
authors.forename: Ranilson S
authors.initials: RS
authors.lastname: Bezerra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005399:Fishes / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014105:Tosyl Compounds / Q000494:pharmacology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014357:Trypsin / Q000737:chemistry* / Q000302:isolation & purification; D014361:Trypsin Inhibitors / Q000494:pharmacology
chemical_list: D011480:Protease Inhibitors; D014105:Tosyl Compounds; D014361:Trypsin Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; C003614:4-toluenesulfonyl fluoride; D014357:Trypsin