Partial purification and characterization of Lys-N from Grifola frondosa using a novel, specific assay

Abstract In the present study, a novel and specific HPLC-based assay was established for the enzyme activity determination of the peptidyl-lysyl metalloendopeptidase (Lys-N, EC 3.4.24.20). The substrate hippuryl-Lys-Val-OH (hip-KV) was cleaved at the amino side of lysine due to the hydrolysis action of Lys-N and the hippuric acid (HA) released could be determined via RP-C18 HPLC at 228 nm. The biochemical parameters of side-activity free Lys-N from Grifola frondosa were characterized using this assay after partial purification. The pH optimum determined was 9.5 using CHES/NaOH buffer. The temperature optimum was 55 °C. Almost no inactivation of Lys-N was observed even after 4 d of incubation at 55 °C. The kinetic parameters for Lys-N were determined with 189.5 nkat/mg for V max and 2.04 mM for K m using hip-KV as the substrate. The enzyme was fully inhibited using the metal chelators 1,10-phenanthroline (1 mM) or EDTA (10 µM).