A novel analytical procedure for assaying lysozymes - Develosil USA

Lysozymes (EC. are widely recognized for their contribution to the antibacterial defense in many animals and their use as a preservative in foods and pharmaceuticals. [1] The key characteristic for all lysozymes is their ability to hydrolyze the ß-(1-4)-glycosidic bond between the alternating residues of peptidoglycan, a unique bacterial cell wall polymer.[1] There are many approaches to study the lysozyme activity such as using chitooligosaccharides, but these approaches often require study for overall kinetic rates to later dissect into individual rates.[2] Ogata, et. al. were able to create a novel analytical procedure for studying lysozyme activity undergoing a single cleavage reaction, which simplifies the kinetic measurements.[3]

The research group was able to synthesize ß-D-galactosyl-chitotetraose derivative [Ga(GlCN)3D] from chitin tetrasaccharide [(GlcN)4] using chemical and enzymatic modifications. Subsequently, the hydrolytic action of lysozyme on Ga(GlCN)3D was then analyzed.[3] Overall, they were able to synthesize a novel compound that can serve as a basis for studying enzyme kinetics as well as develop a new assay method for quantifying lysozymes.[3]

Ogata, et. al. used quantitative HPLC-UV to aid this development of this novel analytical procedure. They used HPLC-UV to study the enzymatic-transglycosylation reaction for the synthesis of Gal(GlCN)3D, to measure the reaction products of chitin oligosaccharide derivatives, and to verify a portion of the lysozyme assay system using Gal(GlN3)D as the substrate.[3] The HPLC -UV system consisted of a Develosil ANIDIUS column and detection at 210 nm.[3]

Works cited:

  1. Callewaert, L. & Michiels, C.W. J Biosci (2010) 35: 127. https://doi.org/10.1007/s12038-010-0015-5
  2. Tamo Fukamizo, Takeya Minematsu, Yugi Yanase, Katsuya Hayashi, Sachio Goto, Substrate size dependence of lysozyme-catalyzed reaction, Archives of Biochemistry and Biophysics, Volume 250, Issue 2, 1986, Pages 312-321, ISSN 0003-9861, https://doi.org/10.1016/0003-9861(86)90732-0.
  3. Makoto Ogata, Megumi Matsui, Haruka Kono, Yuka Matsuzaki, Yuna Kato, Taichi Usui, A novel analytical procedure for assaying lysozyme activity using an end-blocked chitotetraose derivative as substrate, Analytical Biochemistry, Volume 538, 2017, Pages 64-70, ISSN 0003-2697, https://doi.org/10.1016/j.ab.2017.09.015