Dry heating of lysozyme: strong effects regarding antimicrobial activity

For food preservation as well as for medical applications, because of increasing bacterial resistance towards antibiotics, novel and natural antimicrobial molecules are request. The widely studied lysozyme is a promising candidate to develop such molecules. This protein is well known and already used for its enzymatic activity against Gram-positive bacteria, but it also presents some activity against Gram-negative bacteria, supposedly due to its capability to disturb bacteria membrane. Otherwise, molecules that provoke bacterial membrane disruption are generally positively charged, amphipathic, and hydrophobic, all characteristics which can be modified. Especially, we previously showed that dry heating is a safe and easy process resulting in slight chemical modifications of lysozyme, with strong consequences regarding the charge, the hydrophobicity, and finally the interfacial properties of the protein. The question then arose: could lysozyme dry heating be an opportunity to create a new efficient antimicrobial? We actually demonstrated that the activity of dry heated lysozyme (DH-L) against Escherichia coli was higher than that of native lysozyme (N-L). Using optical microscopy and atomic force microscopy (AFM), strong morphological modifications of the bacteria were observed, consistently with the higher membrane permeabilization when E. coli cells were treated with DH-L: either larger pores or more pores in the outer membrane, as well as more ion channels in the cytoplasmic membrane were obtained with DH-L as compared to N-L. Using a lipopolysaccharide (LPS) monolayer and a phospholipid (PL) mixture monolayer as models of the E. coli outer and cytoplasmic membranes, respectively, the interaction between lysozyme and these monolayer models has been investigated by biophysics techniques such as tensiometry, ellipsometry, Brewster angle microscopy and AFM. We could thus established that dry heating increases lysozyme affinity for the model monolayers and its insertion capacity; the resulting reorganization of the model monolayers was also more drastic. Finally, using similar investigations with each of the lysozyme isoforms produced by dry heating, we could show that the most positive, flexible and hydrophobic isoform shows the highest antimicrobial activity. However, it is noticeable that the lysozyme isoforms mixture, I;e; DH-L was the most efficient against E. coli, suggesting synergetic cooperation between lysozyme isoforms.