Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity.

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-NP [Gal(GlcNAc)-β-NP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)-β-NP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)-β-NP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc) and -nitrophenol (NP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)-β-NP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc) and NP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of NP liberated from the substrate.