A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as substrate

Ogata, Makoto; Matsui, Masayuki; Kono, Haruka; Matsuzaki, Y; Kato, Y.; Usui, Taichi

doi:10.1016/j.ab.2017.09.015

Cited by 6 publications

(3 citation statements)

References 23 publications

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“… 7) In addition, we have recently reported that an end-modified galactosyl chitotetraose derivative [Gal(GlcNAc) 3 DGN] binds to the –3 to +2 subsites of lysozyme in a structurally selective manner. 10) In the interaction of HEWL and chitin oligosaccharide, the C1,3-diaxial C-H hydrogens of the ‒3 GlcNAc residue have a face-to-face stacking interaction with the indole side chain of Trp-62, but the 2- N -acetyl group of the ‒3 GlcNAc residue has little involvement in such stacking interactions. Therefore, the Gal residues of Gal(GlcNAc) 3 DGN with the C1,3-diaxial C-H hydrogens and without the 2- N -acetyl group preferentially binds to the ‒3 subsites.…”

Section: Resultsmentioning

confidence: 99%

“… 16) Furthermore, we compared the interactions in the Gal(GlcNAc) 2 -β- p NP-HEWL complex with those in the Gal(GlcNAc) 3 DGN-HEWL complex. 10) Figure 1A shows the docking of Gal(GlcNAc) 2 -β- p NP to lysozyme, and Fig. 1B shows a superimposition of the Gal(GlcNAc) 2 -β- p NP and Gal(GlcNAc) 3 DGN substrates bound to lysozyme.…”

Section: Resultsmentioning

confidence: 99%

“…We have recently reported the synthesis of the substrate {4 4 - O -β-D-galactosyl-β-tri- N -acetylchitotriosyl 2-acetamide-2,3-dideoxy-gluc-2-enopyranose [Gal(GlcNAc) 3 DGN]} for lysozyme, which binds to the –3 to +2 subsites of HEWL and is hydrolyzed between Gal(GlcNAc) 2 and GlcNAc-DGN. 10) As a result, an assay method for quantifying lysozyme was established which utilizes the Morgan-Elson reaction based on the generation of product DGN (2-acetamide-2,3-dideoxy-gluc-2-enopyranose), formed from Gal(GlcNAc) 3 DGN by lysozyme through a conjugated reaction involving β- N -acetylhexosaminidase. Our objective in this study was to design a novel substrate for assaying lysozyme activity that liberates only a chromophore, such as p NP, thereby simplifying the assay system.…”

Section: Introductionmentioning

confidence: 99%

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Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

2018

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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

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

2018

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2017–2018

Harvey

2021

Mass Spectrometry Reviews

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This review is the tenth update of the original article published in 1999 on the application of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2018. Also included are papers that describe methods appropriate to glycan and glycoprotein analysis by MALDI, such as sample preparation techniques, even though the ionization method is not MALDI. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, new methods, matrices, derivatization, MALDI imaging, fragmentation and the use of arrays. The second part of the review is devoted to applications to various structural types such as oligo‐ and poly‐saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Most of the applications are presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. The reported work shows increasing use of combined new techniques such as ion mobility and highlights the impact that MALDI imaging is having across a range of diciplines. MALDI is still an ideal technique for carbohydrate analysis and advancements in the technique and the range of applications continue steady progress.

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Study of stability, kinetic parameters and release of lysozyme immobilized on chitosan microspheres by crosslinking and covalent attachment for cotton fabric functionalization

Cerón

Costa

Imbernon

et al. 2023

Process Biochemistry

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A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as substrate

Cited by 6 publications

References 23 publications

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2017–2018

Study of stability, kinetic parameters and release of lysozyme immobilized on chitosan microspheres by crosslinking and covalent attachment for cotton fabric functionalization

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