A catalytically inactive mutant of hen egg white lysozyme was constructed by site-directed mutagenesis to elucidate the role of enzymatic activity on its antimicrobial activity against Gram-positive bacteria. The catalytic residue aspartic acid at position 52 of lysozyme was substituted with serine (D52S-Lz) and the mutant cDNA was inserted into a yeast expression vector, pYES-2. Western blot analysis indicated that the mutation did not affect secretion of the D52S-Lz lysozyme into the medium of the expressing Saccharomyces cerevisiae, INVSC1. In addition, circular dichroism and fluorescence spectral analysis revealed no change in the structure of D52S-Lz compared to that of wild-type (Wt-Lz) lysozyme. The mutation (D52S) abolished the catalytic activity of lysozyme. Antimicrobial tests against Staphylococcus aureus and Bacillus subtilis revealed that the catalytically inactive D52S-Lz was as bactericidal as the Wt-Lz lysozyme. Heat treatment leading to enzyme inactivation had no effect on the bactericidal activity of either wild-type or the mutant D52S-Lz lysozyme. The binding affinity of D52S-Lz to the isolated peptidoglycan of S. aureus was unaffected. Our results provide the first demonstration of direct genetic evidence that the antimicrobial activity of lysozyme is operationally independent of its muramidase activity, and strongly suggest the antimicrobial action of lysozyme is due to structural factors. ß

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The antimicrobial mechanism and structural changes of hen egg white lysozyme irreversibly inactivated at 80 °C and at different pHs were investigated. We found that heat denaturation of lysozyme at increasing temperatures for 20 min at pH 6.0 results in progressive loss of enzyme activity while greatly promotes its antimicrobial action to Gram-negative bacteria. Interestingly, lysozyme devoid of enzyme activity (heated at 80 °C and pH 7.0 or at pH 6.0 over 90 °C) exhibited strong bactericidal activity against Gram-negative and -positive bacteria, suggesting action independent of catalytic function. The most potent antimicrobial lysozyme to either Gram-negative or -positive bacteria was that heated at 80 °C and pH 6.0 (HLz80/6), retaining 50% of the native enzymatic activity, which exhibited a 14-fold increase in surface hydrophobicity, with two exposed thiol groups. HLz80/6-induced agglutination coincided with severe reduction in colony-forming ability of the susceptible bacteria in a dose-dependent manner. Denatured lysozyme HLz80/6 showed promoted binding capacity to peptidoglycan of Staphylococcus aureus and lipopolysaccharide of Escherichia coli as assessed by ELISA. Addition of HLz80/6 to E. coli phospholipid vesicles resulted in a blue shift in the intrinsic tryptophan fluorescence accompanied by an increase in the size of the vesicles, indicating enhanced protein−membrane binding and subsequent fusion of liposomes. Direct membrane damage of E. coli membrane by HLz80/6 was revealed by electron microscopy observation. Thus, the results introduce an interesting finding that partial unfolding of lysozyme with the proper acquisition of the hydrophobic pocket to the surface can switch its antimicrobial activity to include Gram-negative bacteria without a detrimental effect on the inherent bactericidal effect against Gram-positive ones. The data suggest that the unique antimicrobial action of unfolded lysozyme attributes to membrane binding and subsequent perturbation of its functions. Keywords: Lysozyme; conformational changes; antimicrobial action; agglutination; membrane interaction and fusion

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The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.

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Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.

Food proteins were phosphorylated by heating in a dry state in the presence of phosphate. When casein, whey protein isolate (WPI), and egg white proteins (EWP), which were lyophilized from their solutions in a phosphate buffer, were dry-heated at various temperatures and pH levels for 1-5 days, EWP was more highly phosphorylated than casein and WPI. Phosphorylation of EWP was promoted with a decrease of pH from 7.0 to 3.0 when the incubation temperature was raised from 55 to 100 degrees C. The phosphorus content of EWP increased from 0.08 to 0.64% by dry-heating at pH 3.0 and 85 degrees C for 5 days in the presence of phosphate. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The heat-induced polymerization of EWP by dry-heating was not affected by the presence of phosphate. Although the solubility of EWP decreased by dry-heating at pH 3.0-5.5, the phosphorylation depressed the insolubilization at low pH. The phosphate bonds in phosphorylated EWP (P-EWP) were stable at pH 2.0-10.0 and were more acid-labile and base-stable than phosphoesters of egg riboflavin-binding protein (RfBP). (31)P NMR spectral data suggested that besides phosphoesters, phosphodiester and polyphosphate bonds were introduced in P-EWP. Heat stability of EWP was improved, and calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation.