Partially Unfolded Lysozyme at Neutral pH Agglutinates and Kills Gram-Negative and Gram-Positive Bacteria through Membrane Damage Mechanism
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- 作者：Hisham R. Ibrahim ; Shinji Higashiguchi ; Mamoru Koketsu ; Lekh R. Juneja ; Mujo Kim ; Takehiko Yamamoto ; Yasushi Sugimoto ; and Takayoshi Aoki
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：1996
- 出版时间：December 1996
- 年：1996
- 卷：44
- 期：12
- 页码：3799 - 3806
- 全文大小：232K
- 年卷期：v.44,no.12(December 1996)
- ISSN：1520-5118
文摘
The antimicrobial mechanism and structural changes of hen egg whitelysozyme irreversiblyinactivated at 80 
C and at different pHs were investigated. Wefound that heat denaturation oflysozyme at increasing temperatures for 20 min at pH 6.0 results inprogressive loss of enzymeactivity while greatly promotes its antimicrobial action toGram-negative bacteria. Interestingly,lysozyme devoid of enzyme activity (heated at 80 C and pH 7.0 or atpH 6.0 over 90 C) exhibitedstrong bactericidal activity against Gram-negative and -positivebacteria, suggesting actionindependent of catalytic function. The most potent antimicrobiallysozyme to either Gram-negativeor -positive bacteria was that heated at 80 C and pH 6.0 (HLz80/6),retaining 50% of the nativeenzymatic activity, which exhibited a 14-fold increase in surfacehydrophobicity, with two exposedthiol groups. HLz80/6-induced agglutination coincided with severereduction in colony-forming abilityof the susceptible bacteria in a dose-dependent manner. Denaturedlysozyme HLz80/6 showedpromoted binding capacity to peptidoglycan of Staphylococcusaureus and lipopolysaccharide ofEscherichia coli as assessed by ELISA. Addition ofHLz80/6 to E. coli phospholipid vesiclesresultedin a blue shift in the intrinsic tryptophan fluorescence accompanied byan increase in the size ofthe vesicles, indicating enhanced protein-membrane binding andsubsequent fusion of liposomes.Direct membrane damage of E. coli membrane by HLz80/6was revealed by electron microscopyobservation. Thus, the results introduce an interesting findingthat partial unfolding of lysozymewith the proper acquisition of the hydrophobic pocket to the surfacecan switch its antimicrobialactivity to include Gram-negative bacteria without a detrimental effecton the inherent bactericidaleffect against Gram-positive ones. The data suggest that theunique antimicrobial action of unfoldedlysozyme attributes to membrane binding and subsequent perturbation ofits functions.Keywords: Lysozyme; conformational changes; antimicrobialaction; agglutination; membraneinteraction and fusion
C and at different pHs were investigated. Wefound that heat denaturation oflysozyme at increasing temperatures for 20 min at pH 6.0 results inprogressive loss of enzymeactivity while greatly promotes its antimicrobial action toGram-negative bacteria. Interestingly,lysozyme devoid of enzyme activity (heated at 80 C and pH 7.0 or atpH 6.0 over 90 C) exhibitedstrong bactericidal activity against Gram-negative and -positivebacteria, suggesting actionindependent of catalytic function. The most potent antimicrobiallysozyme to either Gram-negativeor -positive bacteria was that heated at 80 C and pH 6.0 (HLz80/6),retaining 50% of the nativeenzymatic activity, which exhibited a 14-fold increase in surfacehydrophobicity, with two exposedthiol groups. HLz80/6-induced agglutination coincided with severereduction in colony-forming abilityof the susceptible bacteria in a dose-dependent manner. Denaturedlysozyme HLz80/6 showedpromoted binding capacity to peptidoglycan of Staphylococcusaureus and lipopolysaccharide ofEscherichia coli as assessed by ELISA. Addition ofHLz80/6 to E. coli phospholipid vesiclesresultedin a blue shift in the intrinsic tryptophan fluorescence accompanied byan increase in the size ofthe vesicles, indicating enhanced protein-membrane binding andsubsequent fusion of liposomes.Direct membrane damage of E. coli membrane by HLz80/6was revealed by electron microscopyobservation. Thus, the results introduce an interesting findingthat partial unfolding of lysozymewith the proper acquisition of the hydrophobic pocket to the surfacecan switch its antimicrobialactivity to include Gram-negative bacteria without a detrimental effecton the inherent bactericidaleffect against Gram-positive ones. The data suggest that theunique antimicrobial action of unfoldedlysozyme attributes to membrane binding and subsequent perturbation ofits functions.Keywords: Lysozyme; conformational changes; antimicrobialaction; agglutination; membraneinteraction and fusion