Purification and characterization of lysozyme from Chinese Lueyang black-bone Silky fowl egg white

Abstract

Lysozyme, an important antibacterial protein, is an enzyme that cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan in cell walls. The novel lysozyme was purified and characterized from Chinese Lueyang black-bone silky fowl (CBSF) egg white, and its N-terminal amino acid sequence, enzymatic properties, and antibacterial activity were investigated. The CBSF lysozyme was purified using adsorption chromatography, ammonium sulfate precipitation, ion exchange chromatography, and size-exclusion chromatography. The purification fold and yield were 3.28 and 14.69%, respectively. The purified lysozyme was revealed as a single protein band with SDS–PAGE and had a MALDI-TOF/TOF molecular weight of 14305.57 Da and a final specific activity of 3.49 × 10⁵ U/mg protein using Micrococcus lysodeikticus as a substrate. The optimum temperature and pH of the lysozyme were 50 °C and 6.0, respectively. The 20 N-terminal amino acid residues of the purified lysozyme were determined to be KVFGRCELAAAMKRHGLDNY, showing some homology to the N-terminus of the odontophoridae egg white lysozyme. The purified lysozyme exerted a potent antimicrobial activity toward indicator microorganisms, including Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. However, its inhibition of gram-negative activity was weaker than that of the Gram-positive bacteria.

Keywords:

• Activity
• characterization
• Chinese Lueyang black-bone silky fowl
• egg white
• lysozyme
• purification

Acknowledgments

The authors thank Dr. S. Ganesh, Post-Doctoral Researcher, College of Veterinary Medicine, Northwest A&F University, for the assistance of revising this article.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [grant number 31402071]. Shaanxi Provincial Key Research and Development Program [grant number 2015 NY-070 and 2017 NY-180]; the scientific research program of the Shaanxi Provincial Education Department [grant number 17JK0137] and the Construction Project of Shaanxi Collaborative Innovation Center [grant number BXT-Z(Z)-15-21].