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.

Characterization and Purification of Alkaline Proteases From Viscera of Silver Carp (Hypophthalmichthys molitrix) Fish

“…Similar results were obtained from trypsin activity extracted from the viscera of Monterey sardine Kishimura et al (2006) or true sardine (Yanez et al, 2005) and alkaline proteases extracted from digestive system of carp (Catla catla) (Khangembam et al, 2012). In this reported, (Marcuschi et al, 2010) pointed out that trypsin extracted from the digestive system of fish is normally active in the alkaline region (from pH 7-12) using BAPNA as a model of substrate.…”

Metal-sensitive and thermostable trypsin from the crevalle jack (Caranx hippos) pyloric caeca: purification and characterization

“…Fish trypsins have molecular masses between 23 and 28 kDa, e.g., Oreochromis niloticus (23.5 kDa) [1], Colossoma macropomum (23.9 kDa) [5], Gadus macrocephalus (24 kDa) [19], Pseudupeneus maculatus (24.5 kDa) [6], Sardina pilchardus (25 kDa) [10], Diapterus rhombeus (26.5 kDa) [3], Salaria basilisca (27 kDa) [20], Pterygoplichthys disjunctivus (27.5 kDa) [21], Arapaima gigas (28 kDa) [2], Pomatomus saltatrix (28 kDa) [22], and Lutjanus synagris (28.4 kDa) [4]. …”

Trypsins from fish processing waste: characteristics and biotechnological applications – comprehensive review

“…Marcuschi et al (2010) reported the purification of trypsin from the pyloric caecum of tambaqui (C. macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex-G-75 and p-aminobenzamidineeagarose affinity chromatography with a purification yield of 30.0%. In the same context, Silva et al (2011) purified a trypsin 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 that of the enzyme extract and yield of 22.1%.…”