A STRUCTURAL PHASE OF HEAT-DENATURED LYSOZYME WITH NOVEL ANTIMICROBIAL ACTION

The structure and antimicrobial function of hen egg white lysozyme was investigated by means of thermal denaturation at 80 degrees C (pH 7.2 ), which leads to irreversible denaturation. With an increase in the h eating time (up to 30 min) of lysozyme, the soluble fraction showed pr ogressive decrease in its enzyme activity that coincided with the form ation of a slower migrating band on the acid PAGE. Fluorescence spectr a revealed that, as the extent of denaturation increases, the surface hydrophobicity and the exposure of tryptophan residues were greatly pr omoted. In parallel to these conformational changes of lysozyme there has been consistent increase in its antimicrobial activities against G ram-negative bacteria, with no detrimental effect on its inherent acti on to Gram-positive bacteria. Interestingly, lysozyme heated for 20 mi n, devoid of enzyme activity (HDLz), killed Escherichia coli K12 in a dose-dependent manner, while its bactericidal activity to Staphylococc us aureus was almost similar to that of the native lysozyme. The bindi ng capacity of HDLz to membrane fractions off. coli K12 was greatly pr omoted, particularly to the inner membrane, as determined by ELISA. Th e HDLz permeabilized liposomal membranes made from E. coli phospholipi ds, as demonstrated by calcein efflux, in a protein concentration-depe ndent manner. Good correlations between the degree of heat inactivatio n of lysozyme (or dimerization), increased hydrophobicity, and enhance d bactericidal activity against Gram-negative E. coli K12 were observe d. The results of this study, first of all, suggest that susceptibilit y of Gram-negative or even Gram-positive bacteria to lysozyme is indep endent of enzymatic activity. It is likely that denatured lysozyme, e. g., the dimeric form, has an intrinsic structural motif which is gener ally lethal to the bacteria through membrane perturbation.