Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).
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.
Marcuschi
Marina
M
Laboratório de Enzimologia, Departamento de Bioquímica CCB and Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitária, 50670-910 Recife, Pernambuco, Brazil.
Espósito
Talita S
TS
Machado
Maurício F M
MF
Hirata
Izaura Y
IY
Machado
Marcelo F M
MF
Silva
Márcia V
MV
Carvalho
Luiz B
LB
Oliveira
Vitor
V
Bezerra
Ranilson S
RS
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
D011480:Protease Inhibitors; D014105:Tosyl Compounds; D014361:Trypsin Inhibitors; D014107:Tosyllysine Chloromethyl Ketone; C003614:4-toluenesulfonyl fluoride; D014357:Trypsin