General characteristics of the influence of surfactants on the bacteriolytic activity of lysozyme based on the example of enzymatic lysis of Lactobacillus plantarum cells in the presence of Tween 21 and SDS.

The general characteristics of the effect of surfactants on the activity of lysozyme were demonstrated. The kinetics of bacterial cell lysis is consistent with the Michaelis-Menten equation and the presence of surfactants does not shift the pH-optimum of activity. Surfactants do not change the Km value but instead, affect the Vmax value. The experimental dependencies are well described by theoretical equations, which assume three surfactant binding sites on the lysozyme molecule. The dependencies of the activity of lysozyme on the surfactant concentration are either a step type (i.e., a higher plateau becomes a lower plateau), or a dependency with a maximum and continuation of the curve in the form of a plateau but with an increase in the surfactant concentration. It can be assumed that there is a mechanism for the regulation of lysozyme activity by an unknown natural factor that has a suitable hydrophobic radical capable of binding to the surface of lysozyme.

The bacteriolytic activity of native and covalently immobilized lysozyme against Gram-positive and Gram-negative bacteria is differentially affected by charged amino acids and glycine.

The emergence of new antibiotic-resistant bacterial strains means it is increasingly important to find alternatives to traditional antibiotics, such as bacteriolytic enzymes. The bacteriolytic enzyme lysozyme is widely used in medicine as an antimicrobial agent, and covalent immobilization of lysozyme can expand its range of possible applications. However, information on the effect of such immobilized preparations on whole bacterial cells is quite limited. Here, we demonstrate the differential effects of glycine and charged (basic and acidic) amino acids on the enzymatic lysis of Gram-positive and Gram-negative bacteria by soluble and immobilized lysozyme. Glycine and basic amino acids (histidine, lysine, and arginine) significantly increase the rate of lysis of Gram-negative Escherichia coli cells in the presence of soluble lysozyme, but they do not substantially affect the rate of enzymatic lysis of Gram-positive Micrococcus luteus. Glutamate and aspartate significantly enhance enzymatic lysis of both E. coli and M. luteus. When using immobilized lysozyme, the effects of amino acids on the rate of cell lysis are significantly reduced. For immobilized lysozyme, the presence of an external diffusion mode on cell lysis kinetics at bacterial concentrations below 4 × 108 colony-forming units·mL-1 was shown. The broadening of the pH optimum of lysozyme activity after immobilization has been demonstrated for both Gram-positive and Gram-negative bacteria. The Michaelis constant (Km) values of immobilized lysozyme were increased by 1.5-fold for E. coli cell lysis and 4.6-fold for M. luteus cell lysis compared to soluble enzyme. A greater understanding of the effect of amino acids on the activity of native and immobilized lysozyme is important for both the development of new materials for medical purposes and elucidating the interaction of lysozyme with bacterial cells. Of particular interest is our finding that lysozyme activity against Gram-negative bacteria is enhanced in the presence of glycine and charged amino acids over a wide range of concentrations.

Effect of high pressure and reversed micelles on the fluorescent proteins.

Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent chromophore protonation changes resulting in a fluorescence decrease of ECFP and EYFP. In contrast to the remarkable stability of DsRed, the highest sensitivity of EYFP fluorescence under pressure and in micelles is attributed to its chromophore structure.