Lysozyme assay using a rationally designed GN4G2 substrate with coupled β-glucosidase reaction

Lysozyme (EC 3.2.1.17) is a glycoside hydrolase that cleaves β-1,4-glycosidic linkages in peptidoglycan and chitin and plays a key role in host defense. Conventional lysozyme activity assays, such as turbidity-based methods using Micrococcus luteus (formerly M. lysodeikticus) cells, often suffer from poor reproducibility due to substrate heterogeneity and sensitivity to assay conditions. Consequently, there is a requirement to develop a simpler and more reliable assay for lysozyme with improved reproducibility. Here, we developed a novel lysozyme assay using a rationally designed substrate, N-acetylchitotetraosyl cellobiose (GN4G2), combined with a coupled β-glucosidase reaction. Molecular dynamics simulations were carried out, which suggested GN4G2 stably occupies the -4 to +2 subsites of hen egg-white lysozyme (HEWL). GN4G2 was enzymatically synthesized and analyzed. The rationally designed substrate exhibited approximately 250-fold higher water solubility than conventional chromogenic substrates. Moreover, kinetic analysis revealed that GN4G2 displayed approximately threefold higher catalytic efficiency (k_cat/K_m) than N-acetylchitopentaose (GN5). HEWL catalyzed hydrolysis of GN4G2 preferentially liberated cellobiose, which was subsequently converted by a coupled β-glucosidase into two molecules of glucose. Hence, this coupled β-glucosidase reaction amplified the signal output. The assay showed a linear increase in glucose concentration over time and a proportional increase with enzyme concentration, as well as good reproducibility (CV < 10%, n = 3). These results were consistent with high-performance liquid chromatography (HPLC)-based quantification. In conclusion, the methodology offers a robust, reproducible, high-fidelity assay for lysozyme activity, which should prove invaluable for biochemical characterization and enzyme analysis.