Pheasant Egg White Lysozyme vs. Hen Egg White Lysozyme: A Comparative Study of Enzymatic Activity

Hen egg white lysozyme (HEWL) is a well-characterized antibacterial enzyme widely used in research and industry. However, little is known about the functional properties of lysozyme from other bird species, such as pheasants. This study aimed to compare the muramidase and chitinase activities of HEWL with the lysozyme from pheasant egg white (PEWL). Egg white samples were obtained from ten hens and ten pheasants. Lysozyme activity was assessed by two methods: a turbidimetric assay using Micrococcus luteus cell suspension to measure bacteriolytic (muramidase) activity and a fluorescence polarization assay (FPA) using FITC-labeled tri- and chitopentaoside substrates to measure the chitinase activity. The observed dissociation rate constant (kobs) was derived from the time-dependent FP signal. Turbidimetric measurements showed that PEWL possessed bacteriolytic activity approximately 30% lower than that of HEWL under the same conditions. However, FPA revealed a striking functional difference: whereas HEWL only bound the chitotrioside substrate without hydrolyzing it, PEWL was able to hydrolyze chitotriose. Both enzymes readily hydrolyzed the chitopentaoside substrate. The hydrolysis rate of the chitopentaoside was 50–70 times higher than that of the chitotrioside for PEWL. Sequence comparison identified six amino acid substitutions in PEWL (Phe3 → Tyr, His15 → Leu, Gln41 → His, Asn77 → His, Gln121 → Asn, Ile124 → Thr) relative to HEWL, which likely alter the conformation of subsites A–C and enable productive binding of the shorter oligosaccharide. Thus, despite high sequence similarity, pheasant lysozyme exhibits a unique functional property—the ability to hydrolyze chitotriose into N-acetylglucosamine. These findings expand functions of avian lysozymes and highlight the potential of alternative bird species as sources of enzymes with novel properties.