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A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc) 2 -β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc) 2 -β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc) 2 -β-pNP and related compounds using hen eggwhite lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc) 2 and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc) 2 -β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc) 2 and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate. ). ＊ Megumi Matsui and Haruka Kono, contributed equally to this work. Abbreviations: Gal(GlcNAc) 2 -β-pNP, Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP; HEWL, hen egg-white lysozyme; pNP, p-nitrophenol; DGN, 2acetamide-2,3-dideoxy-gluc-2-enopyranose; β4GalTI, β-1,4-galactosyltransferase I; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight. RESULTS AND DISCUSSION Molecular design and enzymatic synthesis of Gal(GlcNAc) 2 -β-pNP.HEWL is known to have six subsites for sugar residue binding which correspond to hexa-N-acetyl-chitohexaose and these are termed -4 to +2. 11)12)13) The enzyme cleaves the glycosidic linkage between the sugar residues at subsites -1 and +1 with the aid of Glu-35 and Asp-52, which are acidic amino acid residues. 14)15) Nanjo et al. have reported that when lysozyme acts on (GlcNAc) 3 -β-pNP, multiple