Lysozyme – bioactive component of the hyperimmune egg PC2: characterization, purification and antimicrobial activity

Abstract

Lysozyme obtained from PC2 hyperimmune egg whites, originating from hens immunized with a complex of bacterial and fungal antigens, was purified by ion exchange chromatography on Amberlite FPC 3500 resin. The purity of lysozyme was analysed by polyacrylamide gel electrophoresis in denaturing system (SDS-PAGE). Based on the migration pattern of the molecular marker, the presence of a single band with a molecular mass of 14.1 kDa was found. The agar gel immunodiffusion test (AGID) showed the presence of lysozyme which was tested in dilutions from 1/2 to 1/32 as compared to standard lysozyme (Sigma) and that obtained from eggs from chickens free of specific germs (SPF) and conventional (CV). The immunological identity between the standard lysozyme and the PC2 lysozyme was established by the AGID test. The PC2 lysozyme showed agglutination reactions with flaky clumps in the presence of Micrococcus lysodeikticus cultures and with granular clumps in the presence of Staphylococcus aureus cultures. The antimicrobial activity of lysozyme was intense against Gram-positive bacteria and less intense against Gram-negatives. The concentration of lysozyme was assessed by the lysoplate method against a Micrococcus lysodeikticus culture. Mean values (x̄ ± ds) of purified lysozyme were obtained, ranging between 12.5 mg/mL and 14,0 mg/mL. Lythic units (mg/mL) were determined in immunologically active products containing lysozyme (gels, emulsions, solutions, powders). The mean values (x̄ ± ds) were between 49.0±0.34 and 60.5±0.84. This study suggests that PC2 lysozyme exhibits an immunologic activity with an important role in the mechanisms of non-specific defense of organisms.

Keywords

• lysozyme
• purification
• ion exchange chromatography
• amberlite fpc 3500
• micrococcus lysodeikticus
• lysoplate method