Other lytic enzymes
Apart from lysozyme, two other types of hydrolytic enzymes may work bacterio-lytically. Firstly, as already covered above, the N-acetylmuramoyl-L-alanine amidases catalyse cleavage of the junction between the peptidoglycan polysaccharide backbone and the peptide crosslinks; additionally certain endopep-tidases are known to catalyse the hydrolysis of the peptide sidechains. Knowledge is lacking on enzymatic cleavage of the interpeptide bridges in peptidoglycan, however. Streptomyces spp. express an array of cell wall degrading activities that appear to comprise N-acetylmuramoyl-L-alanine amidase activity and/or endopeptidases that presumably act in cooperation to lyse sensitive organisms (Hayashi et al., 1981; Bronneke and Fiedler, 1994). Notably, the so-called mutanolysin from Streptomyces globisporus, presumably comprised of L-alanine amidase and N-acetylmuramoyl-L-alanine amidase, exerts bacteriolytic activity on a number of bacteria in model systems and addition of bacterial cell walls to the Streptomyces globisporus growth medium has been shown to stimulate the production of the bacteriolytic activities (Bronneke and Fiedler, 1994). Recently, Streptococcus milleri was demonstrated to produce an endopeptidase, 'millericin B', which was shown to cleave the last residue in the interpeptide crosslink of peptidoglycan of susceptible strains, and that displayed bacteriolytic activity against several Gram-positive bacteria, except for Bacillus subtilis (Beukes et al., 2000). Clearly, these more recent data confirm that hydrolytic enzyme activities other than lysozyme may show promise as preservative agents in foods. However, much more research remains to be done before any firm conclusions can be drawn regarding safety and efficacy of these enzyme systems in genuine food products.
4.3 Lactoperoxidase
As the name 'lacto' implies, the enzyme lactoperoxidase (EC 1.11.1.7) occurs in milk, where it contributes to milk's natural, antibacterial defence system by catalysing the net formation of hypothiocyanite (OSCN~) via oxidation of thiocyanate (SCN~) by hydrogen peroxide (H2O2) - see reaction, below. Lactoperoxidase activity has also been detected in other animal secreta, e.g., in saliva, tears, and nasal fluid (Ekstrand, 1994). Since the presence of lactoperoxidase in milk can be exploited for milk preservation, the milk lactoperoxidase system has been intensively studied and several reviews exist (see e.g., Daeschel and Penner, 1992; Ekstrand, 1994). The antibacterial reaction occurs by direct lactoperoxidase catalysed oxidation of SCN~ to thiocyanogen
(SCN)2 that in turn hydrolyses spontaneously to hypothiocyanite (OSCN~) as schematised below. At low pH the hypothiocyanous acid, HOSCN, is produced. Lactoperoxidase may also catalyse the direct oxidation of SCN~ to OSCN~ or HOSCN (Daeschel and Penner, 1992):
Lactoperoxidase: 2 SCN~ + H2O2 + 2 H+ —> (SCN)2 + 2 H2O (SCN)2 + H2O —> OSCN~ + SCN~ + 2 H+
Although the thiocyanate concentration in milk varies depending on the feed, SCN~ is ususally naturally present in cow's milk in sufficient concentrations to enter as the principal electron donor in the enzymatic reaction (Bjorck et al., 1979). H2O2 is generated by catalase negative lactic acid bacteria, and may therefore also be naturally present in milk (Ekstrand, 1994), but, as discussed below, the antibacterial effect of the lactoperoxidase system in milk can be enhanced by co-addition or individual addition of one of the substrates SCN~ and H2O2. The antibacterial effect is presumably caused by OSCN~, which oxidises protein sulfhydryl groups to disulphides, e.g., of accessible cysteine groups in bacterial proteins. This oxidising effect is assumed to result in inactivation of vital bacterial enzyme systems, notably of enzymes having cysteine residues in their active sites, leading to inhibition of bacterial metabolic functions and consequently cell death (Daeschel and Penner, 1992). The antimicrobial potency of OSCN~ is higher than that of H2O2. However, proteinaceous systems rich in oxidisable protein groups will scavenge the activity of the lactoperoxidase system (Fuglsang et al., 1995).
At pH values below 5 the HOSCN may exert an inhibitory effect against microorganisms by entering cells as the undissociated acid. In the cytoplasm of the microbial cell, the equilibrium will favour the undissociated acid, and - in analogy to the mechanism behind the the ability of organic acids to inhibit microbial growth - this generation of protons inside the cells is then assumed to be responsible for the antibacterial activity of lactoperoxidase at low pH (Ekstrand, 1994). Furthermore, the bovine milk lactoperoxidase has an apparent activity maximum at pH 5, and this combined with the relatively elevated concentrations of HOSCN at this pH value are in accordance with practical experience that the lactoperoxidase system works optimally at ~ pH5 (Ekstrand, 1994; Fuglsang et al., 1995).
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