Lysozyme

Abstract

Lysozyme was discovered in 1921 by Alexander Fleming (1881-1955), who described it as a “remarkable bacteriolytic principle” (Fleming, 1922). His subsequent discovery of penicillin occurred in the late 1920s. In recognition of his pioneering activities in antimicrobials and treatment of infectious diseases, Fleming was knighted in 1944 and shared the Nobel Prize in Physiology or Medicine in 1945 (MacFarlane, 1985). Fleming’s lysozyme research emerged from his demonstration that chemical antiseptics were ineffective in treating infections. He showed that his own nasal mucus inhibited the growth of a Micrococcus species, a fortuitous discovery because micrococci are among the most sensitive organisms to lysozyme. He first thought that the inhibitory factor was a bacteriophage, but later showed that it was an enzyme that lysed bacterial cells (Fleming, 1922). He established that lysozyme was an endogenous (innate) antimicrobial in the body, and his work supported the concept that an effective way to treat infections was to enhance the body’s own immune responses. Fleming found that lysozyme was present in nasal mucus and tears and that hen egg white had a particularly high level of the protein. It is noteworthy that Laschenko in 1909 and Rettger and Sperry in 1912 showed that egg white was capable of causing lysis of certain strains of bacteria and spores (Laschenko, 1909; Rettger and Sperry, 1912). It was Fleming, however, who actually isolated the lytic principle and showed that it occurred in human secretions. Lysozyme was the first enzyme whose primary amino acid sequence was determined and was also the first enzyme whose structure was determined by X-ray crystallography (Blake et al., 1965; Phillips, 1966). It has an ellipsoid structure with dimensions of approximately of 45 × 30 × 30 angstroms. Its catalytic activity was demonstrated in 1966 by John Rupley, who showed that lysozyme cleaved purified oligosaccharides of N-acetylglucosamine containing a β-(1-4) glycosidic bond (Rupley, 1967). Oligosaccharides with more than three sugar units were hydrolyzed, and the hexasaccharide was shown to be the optimum length substrate. Several families of lysozyme have been found, but they all share the characteristic property of cleaving a β-glycosidic bond between the C-1 of N-acetylmuramic acid and the C-4 on N-acetylglucosamine of bacterial peptidogylcan. Although traditionally associated with eggs of birds, especially those of domestic hens, lysozyme is widespread in nature and is found in many sources including certain vegetables, insects, plants, and fungi (Masschalck et al., 2001; Tranter, 1994; Jolles and Jolles, 1984). Lysozyme is present in human colostrum (Mathur et al., 1990) and mammalian tissues and fluids such as milk, saliva, mucus, blood, and tears. It is also present in high concentrations in macrophages, leukocytes, monocytes, and neutrophilic granulocytes. Lysozyme has important roles in the immune response of organisms in reaction to infections and inflammation (Osserman et al., 1974; Sava, 1996). The major families of naturally occurring lysozyme that differ markedly in amino acid primary structure and structure are as follows: C for chicken or classical, G for goose, bacterial lysozyme (autolysins), phage lysozyme, and plant lysozyme (Jolles, 1996), but only the C enzyme from the hen egg whites is currently used in food preservation. Approximately 3.5% of the total protein content in hen egg white is lysozyme (Alderton et al., 1945; Sofos et al., 1998; Yamamoto et al., 1997). Lysozyme is present at concentrations of about 0.1, 0.13, and 0.25 μg/ml in milk from sheep, cows, and goats, respectively (Chandan et al., 1968), while human milk contains 0.4 μg/ml lysozyme (Reiter, 1978). Lysozyme is considered to be one of the most important factors of nonspecific immunity in human breast milk (Hennart et al., 1991). It also occurs at much higher concentrations in colostrum than in milk and may have positive attributes on the intestinal flora of nursing infants (Barbara and Pellegrini, 1976). Lysozyme has been proposed for use in various clinical applications, including antibacterial, antiviral, and antiinflammatory treatments in humans and animal species (Sava, 1996; Proctor and Cunningham, 1988). As described in detail in the following sections, lysozyme is also used to control microbial growth in foods such as cheese and wine and has potential uses as a preservative in other food systems. Lysozyme serves as a good model of an ideal food preservative in many respects because it is an innate component of the human immune system and thus would be expected to have low toxicity; it is an enzyme that acts catalytically and can be used at low concentrations in foods; it is specific for bacterial peptidoglycan and does not react with human tissue; and it has certain desirable resistance properties to heat, pH, and other intrinsic and extrinsic factors of foods.