Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity |
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| Authors: | Megumi Matsui Haruka Kono Makoto Ogata |
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| Affiliation: | 1. Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College |
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| Abstract: | 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-1,4-galactosyltransferase-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 egg-white 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. |
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| Keywords: | lysozyme enzyme assay kinetics chitin oligosaccharide derivatives enzymatic synthesis |
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