4.3. Kinetic Characterization of Aminopeptidases

The activity of pAPN (K_M = 0.52 mM) was determined as described in the literature [31]. The activity of aminopeptidase from barley seeds was determined at 37 °C in 50 mM Tris-HCl, pH 8.0, containing 50 mM NaCl and 10 mM β-mercaptoethanol using substrate L-leucine-p-nitroanilidine (L-Leu-pNa) dissolved in DMSO. The kinetics parameters of the purified enzyme were measured for L-Leu-pNa at ten final concentrations (ranging from 0.1 to 1.0 mM) and being repeated two times. The K_M value of barley seeds aminopeptidase was determined by using the Lineweaver–Burk weighted regression method (see Table S7 and Plot 1 in the Supplementary Information).