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.

Covalently immobilized chemically modified lysozyme as a sorbent for bacterial endotoxins (lipopolysaccharides).

Chemical modification of lysozyme was carried out using benzaldehyde and anisaldehyde. It was shown that chemical modification affects only 1-2 amino groups of the protein molecule which does not prevent further covalent immobilization of lysozyme using the remaining free amino groups. The bacteriolytic activity of lysozyme is preserved after chemical modification and after subsequent covalent immobilization. As a result of chemical modification immobilized lysozyme more effectively adsorbs bacterial lipopolysaccharides (endotoxins). Adsorption of immunoglobulin G does not increase after modification. The sorbents obtained in this work can be used for the future development of new medical material for the extracorporeal treatment of sepsis. The proposed scheme for the modification and immobilization of lysozyme can be used with various aldehydes for the preparation of sorbents with different properties.

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.

Quantitative turbidimetric assay of enzymatic gram-negative bacteria lysis.

In this Technical Note, the quantitative turbidimetric assay for determination of the bacteriolytic activity of enzymes with gram-negative bacteria is proposed. The reactivity of hen white-egg lysozyme toward gram-negative E. coli intact cells was studied. It was found that the highest lysis rate occurred at pH 8.9 in the system containing 0.03 M NaCl. The mechanism of the reaction is discussed and applied for the quantitative evaluation of the reaction rate. The proposed method enables fast, reliable, and reproducible analysis of bacteriolytic activity of lysozyme with gram-negative bacteria.

A robust method of determination of high concentrations of peptides and proteins.

In this paper, we pioneer application of a unique method of protein determination by coloring peptide bonds for analysis of a variety of biomolecules with different grades of purity (e.g., oligopeptides, membrane, and glycol proteins). We demonstrated that the calibration curve for all studied molecules is universal and linear within 0.1 to 1.2mg protein content range. The assay thus can be used to analyze peptides without preliminary dilutions and calibration in up to 1g/ml solutions of peptides, which is crucial for many biotechnological processes, such as development of coatings, scaffolds, and biocompatible materials.