Journal
JOURNAL OF APPLIED GLYCOSCIENCE
Volume 65, Issue 3, Pages 31-36Publisher
JAPANESE SOC APPLIED GLYCOSCIENCE
DOI: 10.5458/jag.jag.JAG-2018_003
Keywords
lysozyme; enzyme assay; kinetics; chitin oligosaccharide derivatives; enzymatic synthesis
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Funding
- Ministry of Education, Culture, Sports, Science, and Technology of Japan [26450147]
- Grants-in-Aid for Scientific Research [26450147] Funding Source: KAKEN
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A novel substrate {Gal beta 1,4GlcNAc beta 1,4GlcNAc-beta-pNP [Gal(GlcNAc)2-beta-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via beta-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)2-beta-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)(2)-beta-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-beta-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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