Purification and characterization of a novel leucine aminopeptidase from the earthworm Eisenia foetida

Aminopeptidases play important physiological roles and are widely distributed throughout organisms. In the present study, an aminopeptidase was identified and purified to homogeneity from the earthworm (Eisenia foetida) by ammonium sulfate fractionation and column chromatography, including Phenyl Sepharose, Sephacryl S-200, HITrap-DEAE, hydroxyapatite, and HITrap-Heparin resins. The enzyme is a 98-kDa single chain protein. It displayed optimal activity at a temperature of 45 °C and a pH of 7.0 and preferentially hydrolyzed the fluorogenic substrate Leu-MCA with a kinetic coefficient (kcat/Km) of 2.3 × 106 M−1 s−1 and an activation energy (Ea) of 48.7 kJ M−1. The aminopeptidase was greatly inhibited by metal-chelating agents, and Zn2+ appeared to be its metal cofactor. In addition, the specific aminopeptidase inhibitor bestatin strongly inhibited its activity. A polyclonal antibody was raised in rabbit, and Western blotting suggested that the antigenic determinants of the enzyme are unique. According to MALDI-TOF MS analysis, no known protein matches this enzyme in the NCBI Non-redundant Protein Sequence Database. This is the first report of the purification and characterization of an aminopeptidase, which was quite possibly a novel leucine aminopeptidase, from the earthworm.