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Enzymes as Antimicrobials Application of Alternative Food-Preservation Technologies 59 Table 1: Different types of lysozyme. Lysozyme types Provenience References Conventional or chicken-type c-lysozyme -egg white of domestic chicken (Gallus gallus); -purified from various tissues and secretions of mammals including milk, saliva, tears, urine, respiratory and cervical secretions Other types of lysozyme: [3, 5, 9] g-type lysozyme egg white of domestic goose [4, 9-11] h-type lysozyme Plant i-type lysozyme Invertebrates b-type lysozyme Bacteria (Bacillus) v-type lysozyme Viruses Despite the variability in the amino acid composition and sequence of lysozyme molecules, amino acids of the catalytic centre of the active site are well conserved [9]. In 1965 the structure of lysozyme was solved by X-Ray analysis with 2 angstrom resolution by David Chilton Phillips. In particular, lysozyme structure is characterized by an α-helix which links two domains of the molecule; one is mainly β-sheet in structure, and the other primarly α-helical. The hydrophilic groups are mainly oriented inward, with most of the hydrophilic residues on the exterior of the molecule [12]. It has been proposed that the enzymatic action of the molecule is dependent on its ability to change the relative position of its two domains and cause large conformational changes in the molecule. In particular, glutamic acid and aspartic acid residues are directly involved in the breakdown of the glycosidic bond between N-acetylglucosamine and N-acetylmuramic and their presence in the catalytic centre is thus crucial for the hydrolytic activity of the enzyme. However, the amino acid sequence of known lysozymes reveals that aspartic acid is not consistently present in the active site of lysozyme molecules [8]. In contrast, the substitution of glutamic acid results in a complete inactivation of the enzyme [8], thus confirming the critical role of this amino acid in the enzymatic activity of lysozyme [9]. ANTIMICROBIAL EFFECT AND MODE OF ACTION OF LYSOZYME Lysozyme has been recognized to possess many physiological and functional properties, including important roles in surveillance of membranes of mammalian cells; it enhances phagocytic activity of polymorphonuclear leukocytes and macrophages and stimulates proliferation and antitumor functions of monocytes [7], but its high microbicidal activity remains, by far, the main virtue that explains the high attention of scientists and industrial stakeholders for its practical applications in medicine and food industry [9]. The antimicrobial activity of lysozyme has been extensively demonstrated in vitro or in physiological fluids and secretions including milk, blood serum, saliva, and urine [13]. Although lysozyme has been shown to have antimicrobial activities towards bacteria, fungi, protozoan and viruses [14-16], it is essentially known for its antibacterial activity and has been used, on this basis, in food preservation. Lysozyme has been demonstrated to be active throughout a wide pH range of 4–10; however, high ionic strength (>0.2 M salt) was shown to have an inhibitory effect on lysozyme activity [2]. Under physiological conditions, only a minority of Gram positive bacteria are susceptible to lysozyme, and it has been suggested that the main role of lysozyme is to participate in the removal of bacterial cell walls, after the bacteria have been killed by antimicrobial polypeptides present in egg albumin, insect hemolymph [17] or by complement in animal serum [18]. This is in line with the notion that the lytic action of lysozyme does not kill susceptible bacteria under physiological conditions, osmotically balanced [19]. The bacteriostatic and bactericidal properties of lysozyme have been the subject of many studies, and over the last 10 years, several authors have proposed a novel antibacterial mechanism of action of lysozyme that is independent of its muramidase activity [20, 21]. A non-lytic bactericidal mechanism involving membrane
60 Application of Alternative Food-Preservation Technologies D’Amato et al. damage without hydrolysis of peptidoglycan, has been reported for c-type lysozymes, including human lysozyme and hen egg white lysozyme (HEWL) [7, 22, 23]. Other studies observed that the denatured lysozyme deprived of muramidase activity has an unique and potent microbicidal property [7, 24]. Antimicrobial Action Towards Gram Positive Bacteria Lysozyme belongs to a class of enzymes that lyses the cell walls of certain Gram positive bacteria, as it specifically splits the bond between N-acetylglucosamine and N-acetylmuramic acid of the peptidoglycan in the bacterial cell walls. Extensive hydrolysis of the peptidoglycan by exogenous lysozymes results in cell lysis and death in a hypo-osmotic environment but some exogenous lysozymes can also cause lysis of bacteria by stimulating autolysin activity upon interaction with the cell surface [23]. According to this effect, lysozyme affects strongly Gram positive bacteria but not Gram negative ones, which are shielded by the lipopolysaccharide (LPS). Nevertheless, recent studies suggest that resistance of bacteria to lysozyme is not exclusively related to the presence of the lipopolysaccharidic layer. In fact, Gram positive bacteria are generally sensitive to lysozyme because their peptidoglycan is directly exposed, but some of them are intrinsically resistant due to a modified peptidoglycan structure [25]. The occurrence of resistant Gram positive bacteria indicates that the lack of the LPS does not expose de facto the bacterium to lysozyme hydrolysis [22, 26-28]. Various mechanisms of resistance in Gram positive bacteria have been suggested, as the exact mechanism of lysozyme resistance is not fully understood and may vary according to the bacterial strain or species. Fig. 1 reports some of the suggested mechanisms of resistance to lysozyme in Gram positive bacteria. Hindrance of lysozyme action by surface attachment polymers Deacetylation of the amino group of Nacetylglucosamine residues Modification of hexosamine residues of the glycan backbone, by O-acetylation or N-deacetylation High degree of peptide crosslinking Suggested mechanisms of resistance in Gram- positive bacteria Production of proteininhibitors specific to lysozyme Figure 1: Possible mechanisms of resistance to lysozyme in Gram positive bacteria. Teichoic acid content in the cell-wall N-deacetylation of the acetamido group of the hexosamine residues Incorporation of Daspartic acid in the bacterial peptidoglycan crossbridge Several studies provided evidence that the modification of hexosamine residues of the glycan backbone, by Oacetylation or N-deacetylation, is the primary mechanism of resistance to lysozyme in Gram positive bacteria. Clarke and Dupont [29] were the first to suspect a possible role of O-acetylation of the peptidoglycan muramic acid in lysozyme resistance, and this idea was confirmed by other studies [25, 27]. A different bacterial strategy to evade the bactericidal action of lysozyme is the production of inhibitors. In the streptococci belonging to group A, a protein, identified as an inhibitor of the complement system and designated as SIC (streptococcal inhibitor of complement), was also shown to inhibit lysozyme [30, 31]. Other protein inhibitors may be produced by Gram positive bacteria, but they remain to be identified and characterized. Therefore, it is clear that one or more mechanisms of resistance can be involved. For example, deacetylation of the amino group of NAG residues appears to be a common mechanism of resistance in Bacillus and streptococci [26], while other bacteria (e.g., Staphylococcus aureus and lactobacilli) would counteract lysozyme action essentially by means of O-acetylation [28].
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