Arginine aminopeptidase from white shrimp (Litopenaeus vannamei) muscle: purification and characterization

Abstract

Aminopeptidases act on N-terminal of proteins and peptides produce free amino acids making an impact on the final flavor of foods. An arginine aminopeptidase (RAP) which preferred to hydrolyze basic amino acids from N-termini of peptides and proteins was purified to homogeneity from white shrimp (Litopenaeus vannamei) muscle. The molecular mass of RAP was estimated as 100 kDa on SDS-PAGE. Peptide mass fingerprinting analysis obtained 95 amino acid residues which was 100 and 77.9 % identical to puromycin-sensitive aminopeptidases from insect and zebrafish, respectively. Optimum pH and temperature of the RAP were 7.0 and 30 °C. RAP rapidly hydrolyzed fluorogenic substrates l-arginine 4-methylcoumaryl-7-amide (Arg-MCA) and Lys-MCA with Km values of 2.7 and 4.9 μM, respectively. The enzyme can be strongly inhibited by puromycin, bestatin, and 1,10-phenanthroline and partially inhibited by ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). Moreover, the competitive inhibition of puromycin for RAP was confirmed, and Ki value was calculated as 0.07 nM. Metal ions of Zn2+ and Mn2+ significantly reactivated the inactive apoenzyme activity dialyzed by EDTA. All these results indicated that the purified enzyme is a metalloaminopeptidase which would possibly contribute to flavor development in shrimp muscle.