Surprisingly, partially heat-denatured lysozyme exerts antibacterial activity on a number of microorganisms (Ibrahim et al., 1996a and b). The antimicrobial activity of such thermally inactivated lysozyme extends to Gram-negative organisms and appears independent of the lysozyme muramidase activity (Ibrahim et al., 1996a). Two mechanisms have been proposed for this non-enzymic action of lysozyme. The first is that lysozyme acts as a cationic protein that induces cell lysis via puncturing of the cell membrane through a protein-phospholipid interaction mechanism (Ibrahim et al., 1996a and b; Pellegrini et al., 1992). The second mechanism is that the lysozyme protein may activate so-called autolysin enzymes in the bacterial cell wall that in turn induce cell lysis (Ibrahim et al., 1996a). Autolysins include N-acetylmuramoyl-L-alanine amidase enzymes that catalyse the cleavage of the amide bonds between the N-acetylmuramic acid lactyl side chain and the amino acid residue at position 1 of the pentapeptide sidechain, i.e., the bond that links the short peptides to the NAG-NAM backbone in peptidoglycan (Bierbaum and Sahl, 1987; Harding et al., 2002). The peptidase induced hydrolysis of the peptidoglycan peptide sidechains have long been suspected to promote lysis of undesirable Grampositive organisms, and the influence of cationic peptides on the activity of N-acetylmuramoyl-L-alanine amidase was studied 15 years ago (Bierbaum and Sahl, 1987). Amidase enzyme activities are found among bacteriolytic enzymes in bacteriophages; the bacteriophage T7 lysozyme, for example, is actually a bifunctional protein incorporating such an amidase activity (Cheng et al., 1994). Furthermore, the ply genes encoding N-acetylmuramoyl-L-alanine amidase were recently identified in the Bacillus cereus bacteriophages 12826 and TP21 (Loessner et al., 1997). Despite this, detailed knowledge on the molecular enzymology and practical implications of the action of the peptide sidechain hydrolases in medicine and food science is still limited as substrate analogues for their assay have only recently been synthesised (Harding et al., 2002). Even though the antimicrobial actions of denatured lysozyme are well described (Ibrahim et al., 1996a and b; Pellegrini et al., 1992) and even though very recent data demonstrate that partial denaturation of lysozyme may enhance its activity against Listeria monocytogenes (Pszczola, 2002), the significance of these non-enzymic antibacterial effects of lysozyme in the applications where active lysozyme is added in food processes is unknown.
It has been speculated for some time, that chemical modification of lysozyme by covalent attachment of fatty acids might facilitate the penetration of lysozyme through the outer membrane of Gram-negative organisms. Recently, it was proven, that such lipophilisation of hen egg white lysozyme with caproic acid (C6:0), capric acid (C10:0), or myristic acid (C14:0) enhance the bacteriocidal activity of lysozyme against Escherichia coli K-12 in a phosphate buffered test medium (Liu et al., 2000). Furthermore, chemical reduction of disulphide bonds in hen egg white lysozyme by reaction with either cysteine or glutathione, has been demonstrated recently to increase the antimicrobial activity of lysozyme against Salmonella enteritidis in a phosphate buffer test system (pH 7.2, 30°C) (Touch et al., 2003). Evaluation of the leakage of liposomes obtained after contact with the chemically reduced lysozymes indicated that the bacteriocidal action was mainly attributable to the hydrophobic and cationic properties of the modified lysozymes rather than to the lysozymes' muramidase activity (Touch et al., 2003).
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