{"title":"检测溶菌酶活性的新型底物的分子设计与合成","authors":"Megumi Matsui, Haruka Kono, Makoto Ogata","doi":"10.5458/jag.jag.JAG-2018_003","DOIUrl":null,"url":null,"abstract":"<p><p>A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-<i>p</i>NP [Gal(GlcNAc)<sub>2</sub>-β-<i>p</i>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)<sub>2</sub>-β-<i>p</i>NP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)<sub>2</sub>-β-<i>p</i>NP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)<sub>2</sub> and <i>p</i>-nitrophenol (<i>p</i>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)<sub>2</sub>-β-<i>p</i>NP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)<sub>2</sub> and <i>p</i>NP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of <i>p</i>NP liberated from the substrate.</p>","PeriodicalId":14999,"journal":{"name":"Journal of applied glycoscience","volume":"65 3","pages":"31-36"},"PeriodicalIF":1.2000,"publicationDate":"2018-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bf/a7/JAG-65-031.PMC8056892.pdf","citationCount":"0","resultStr":"{\"title\":\"Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity.\",\"authors\":\"Megumi Matsui, Haruka Kono, Makoto Ogata\",\"doi\":\"10.5458/jag.jag.JAG-2018_003\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-<i>p</i>NP [Gal(GlcNAc)<sub>2</sub>-β-<i>p</i>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)<sub>2</sub>-β-<i>p</i>NP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)<sub>2</sub>-β-<i>p</i>NP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)<sub>2</sub> and <i>p</i>-nitrophenol (<i>p</i>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)<sub>2</sub>-β-<i>p</i>NP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)<sub>2</sub> and <i>p</i>NP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of <i>p</i>NP liberated from the substrate.</p>\",\"PeriodicalId\":14999,\"journal\":{\"name\":\"Journal of applied glycoscience\",\"volume\":\"65 3\",\"pages\":\"31-36\"},\"PeriodicalIF\":1.2000,\"publicationDate\":\"2018-08-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bf/a7/JAG-65-031.PMC8056892.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Journal of applied glycoscience\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.5458/jag.jag.JAG-2018_003\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2018/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of applied glycoscience","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.5458/jag.jag.JAG-2018_003","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2018/1/1 0:00:00","PubModel":"eCollection","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity.
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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