{"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":1,"journal":{"name":"Accounts of Chemical Research","volume":null,"pages":null},"PeriodicalIF":16.4000,"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\":1,\"journal\":{\"name\":\"Accounts of Chemical Research\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":16.4000,\"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\":\"Accounts of Chemical Research\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.5458/jag.jag.JAG-2018_003\",\"RegionNum\":1,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2018/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q1\",\"JCRName\":\"CHEMISTRY, MULTIDISCIPLINARY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Accounts of Chemical Research","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.5458/jag.jag.JAG-2018_003","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2018/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","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.
期刊介绍：
Accounts of Chemical Research presents short, concise and critical articles offering easy-to-read overviews of basic research and applications in all areas of chemistry and biochemistry. These short reviews focus on research from the author’s own laboratory and are designed to teach the reader about a research project. In addition, Accounts of Chemical Research publishes commentaries that give an informed opinion on a current research problem. Special Issues online are devoted to a single topic of unusual activity and significance.
Accounts of Chemical Research replaces the traditional article abstract with an article "Conspectus." These entries synopsize the research affording the reader a closer look at the content and significance of an article. Through this provision of a more detailed description of the article contents, the Conspectus enhances the article's discoverability by search engines and the exposure for the research.
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