Abstract: A prolyl endopeptidase (PEP) was purified to homogeneity from the skeletal muscle of sea bass (Lateolabrax japonicus) by ammonium sulfate fractionation followed by column chromatography. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), 43 peptide fragments were obtained highly identical to the sequence of prolyl endopeptidase from Takifugu rubripes, confirming the purified protein to be a prolyl endopeptidase. Using Suc-Gly-Pro-MCA as a substrate, the optimal pH and temperature of the purified enzyme were pH 6.0 and 35 ℃, respectively. Circular dichroism (CD) spectroscopy showed that heating had a significant effect on the secondary structures of PEP, causing irreversible denaturation. The full-length cDNA sequence of PEP was cloned, which contained an open reading frame of 2 226 nucleotides, encoding a protein of 741 amino acid residues with a deduced molecular mass of 84.12 kDa. The molecular structure of PEP was obtained by homologous modeling. PEP exhibited a typical α/β hydrolase folding arrangement. Its catalytic triplets (His-711, Asp-672, and Ser-585) were covered with the central channel of the propeller region formed by β-folding. The stability of the catalytic domain of PEP could be essential for maintaining its activity and its unique structure could be the basis for its substrate specificity.

Key words: sea bass; prolyl endopeptidase; purification; characterization; circular dichroism; homology modeling