Purification and partial characterization of aminopeptidase from barley (Hordeum vulgare L.) seeds

Aminopeptidases (EC 3.4.11) are proteolytic enzymes, which hydrolyze one amino acid from N-terminus of peptidic substrates. Inhibitors of plant aminopeptidases can find an application in agriculture as herbicides. Isolation and partial characterization of aminopeptidase from barley (Hordeum vulgare L.) seeds has been described. The enzyme was purified to molecular homogeneity using a six-step purification procedure (precipitation with (NH4)2SO4, followed by chromatography on Sephadex G-25, DEAE-Sepharose, Sephacryl HR 300, Macro-Prep Q and Phenyl-Sepharose HP columns). The enzyme was purified 365-fold with recovery above 18%. The molecular weight of the purified enzyme was determined by SDS-PAGE and gel filtration as 58 kDa, and was found to be a monomer. Its pH and temperature optima were 7.5 and 52 °C, respectively. The enzyme behaves as standard leucine aminopeptidase by preferring bulky amino acids at the N-terminus, with phenylalanine being of choice.

► The enzyme was purified 365-fold with recovery above 18%.
► Optimal pH of enzyme action is 7.5.
► EDTA, 1,10-phenanthroline and cupferron inhibited enzyme activity.
► Co2+, Cu2+, Cd2+ and Zn2+ ions inhibited enzyme activity, while Ca2+, Mg2+ and Mn2+ slightly activated.
► The enzyme preferred substrates with Leu and Phe at the N-terminus.