Purification and properties of an aminopeptidase from the mid-gut gland of scallop ( Patinopecten yessoensis)

Abstract

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu- pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn 2+. Relative hydrolysis rates of amino acid- pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K m of 32.2 μM and k cat of 29.5 s −1 with Ala- pNA and a K m of 11.1 μM and k cat of 9.49 s −1 with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.