ABSTRACT
Trypsins (E.C. 3.4.21.4) are digestive enzymes that catalyze the hydrolysis of peptide bonds containing arginine and lysine residues. Some trypsins from fish species are active at temperatures just above freezing, and for that are called cold-adapted enzymes, having many biotechnological applications. In this work, we characterized a recombinant trypsin-III from Monterey sardine (Sardinops caeruleus) and studied the role of a single residue on its cold-adapted features. The A236N mutant from sardine trypsin-III showed higher activation energy for the enzyme-catalyzed reaction, it was more active at higher temperatures, and exhibited a higher thermal stability than the wild-type enzyme, suggesting a key role of this residue. The thermodynamic activation parameters revealed an increase in the activation enthalpy for the A236N mutant, suggesting the existence of more intramolecular contacts during the activation step. Molecular models for both enzymes suggest that a hydrogen-bond involving N236 may contact the C-terminal α-helix to the vicinity of the active site, thus affecting the biochemical and thermodynamic properties of the enzyme.
Subject(s)
Fishes/metabolism , Mutation , Trypsin/chemistry , Trypsin/genetics , Animals , Cold Temperature , Enzyme Activation , Enzyme Stability , Fish Proteins/chemistry , Fish Proteins/genetics , Fishes/genetics , Hydrogen Bonding , Models, Molecular , Molecular Docking Simulation , Protein Structure, SecondaryABSTRACT
BACKGROUND: Trypsin from fish species is considered as a cold-adapted enzyme that may find potential biotechnological applications. In this work, the recombinant expression, refolding and activation of Trypsin I (TryI) from Monterey sardine (Sardinops sagax caerulea) are reported. METHODS: TryI was overexpressed in Escherichia coli BL21 as a fusion protein of trypsinogen with thioredoxin. Refolding of trypsinogen I was achieved by dialysis of bacterial inclusion bodies with a recovery of 16.32 mg per liter of Luria broth medium. RESULTS: Before activation, the trypsinogen fusion protein did not show trypsin activity. Trypsinogen I was activated by adding 0.002 U of native TryI purified from the sardine pyloric caeca (nonrecombinant). The activated recombinant trypsin showed three times more activity than the nonrecombinant trypsin alone. CONCLUSION: The described protocol allowed obtaining sufficient amounts of recombinant TryI from Monterey sardine fish for further biochemical and biophysical characterization of its coldadaptation parameters.
