A Structural Phase of Heat-Denatured Lysozyme with Novel Antimicrobial Action

Abstract

The structure and antimicrobial function of hen egg white lysozyme was investigated by means of thermal denaturation at 80 °C (pH 7.2), which leads to irreversible denaturation. With an increase in the heating time (up to 30 min) of lysozyme, the soluble fraction showed progressive decrease in its enzyme activity that coincided with the formation of a slower migrating band on the acid PAGE. Fluorescence spectra revealed that, as the extent of denaturation increases, the surface hydrophobicity and the exposure of tryptophan residues were greatly promoted. In parallel to these conformational changes of lysozyme there has been consistent increase in its antimicrobial activities against Gram-negative bacteria, with no detrimental effect on its inherent action to Gram-positive bacteria. Interestingly, lysozyme heated for 20 min, devoid of enzyme activity (HDLz), killed Escherichia coli K12 in a dose-dependent manner, while its bactericidal activity to Staphylococcus aureus was almost similar to that of the native lysozyme. The binding capacity of HDLz to membrane fractions of E. coli K12 was greatly promoted, particularly to the inner membrane, as determined by ELISA. The HDLz permeabilized liposomal membranes made from E. coli phospholipids, as demonstrated by calcein efflux, in a protein concentration-dependent manner. Good correlations between the degree of heat inactivation of lysozyme (or dimerization), increased hydrophobicity, and enhanced bactericidal activity against Gram-negative E. coli K12 were observed. The results of this study, first of all, suggest that susceptibility of Gram-negative or even Gram-positive bacteria to lysozyme is independent of enzymatic activity. It is likely that denatured lysozyme, e.g., the dimeric form, has an intrinsic structural motif which is generally lethal to the bacteria through membrane perturbation. Keywords: Lysozyme; denaturation; conformational changes; antimicrobial action; membrane interaction; liposome