PARTIALLY UNFOLDED LYSOZYME AT NEUTRAL PH AGGLUTINATES AND KILLS GRAM-NEGATIVE AND GRAM-POSITIVE BACTERIA THROUGH MEMBRANE DAMAGE MECHANISM

The antimicrobial mechanism and structural changes of hen egg white ly sozyme irreversibly inactivated at 80 degrees C and at different pHs w ere investigated. We found that heat denaturation of lysozyme at incre asing temperatures for 20 min at pH 6.0 results in progressive loss of enzyme activity while greatly promotes its antimicrobial action to Gr am-negative bacteria. Interestingly, lysozyme devoid of enzyme activit y (heated at 80 degrees C and pH 7.0 or at pH 6.0 over 90 degrees C) e xhibited strong bactericidal activity against Gram-negative and -posit ive bacteria, suggesting action independent of catalytic function. The most potent antimicrobial lysozyme to either Gram-negative or -positi ve bacteria was that heated at 80 degrees C and pH 6.0 (HLz80/6), reta ining 50% of the native enzymatic activity, which exhibited a 14-fold increase in surface hydrophobicity, with two exposed thiol groups. HLz 80/6-induced agglutination coincided with severe reduction in colony-f orming ability of the susceptible bacteria in a dose-dependent manner. Denatured lysozyme HLz80/6 showed promoted binding capacity to peptid oglycan of Staphylococcus aureus and lipopolysaccharide of Escherichia coli as assessed by ELISA. Addition of HLz80/6 to E. coli phospholipi d vesicles resulted in a blue shift in the intrinsic tryptophan fluore scence accompanied by an increase in the size of the vesicles, indicat ing enhanced protein-membrane binding and subsequent fusion of liposom es. Direct membrane damage of E. coli membrane by HLz80/6 was revealed by electron microscopy observation. Thus, the results introduce an in teresting finding that partial unfolding of lysozyme with the proper a cquisition of the hydrophobic pocket to the surface can switch its, an timicrobial activity to include Gram-negative bacteria without a detri mental effect on the inherent bactericidal effect against Gram-positiv e ones. The data suggest that the unique antimicrobial action of unfol ded lysozyme attributes to membrane binding and subsequent perturbatio n of its functions.