Enhancing the Thermo-Stability and Anti-Bacterium Activity of Lysozyme by Immobilization on Chitosan Nanoparticles

Abstract

The recent emergence of antibiotic-resistant bacteria requires the development of new antibiotics or new agents capable of enhancing antibiotic activity. Lysozyme degrades bacterial cell wall without involving antibiotic resistance and has become a new antibacterial strategy. However, direct use of native, active proteins in clinical settings is not practical as it is fragile under various conditions. In this study, lysozyme was integrated into chitosan nanoparticles (CS-NPs) by the ionic gelation technique to obtain lysozyme immobilized chitosan nanoparticles (Lys-CS-NPs) and then characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), which showed a small particle size (243.1 ± 2.1 nm) and positive zeta potential (22.8 ± 0.2 mV). The immobilization significantly enhanced the thermal stability and reusability of lysozyme. In addition, compared with free lysozyme, Lys-CS-NPs exhibited superb antibacterial properties according to the results of killing kinetics in vitro and measurement of the minimum inhibitory concentration (MIC) of CS-NPs and Lys-CS-NPs against Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Escherichia coli (E. coli), and Staphylococcus aureus (S. aureus). These results suggest that the integration of lysozyme into CS-NPs will create opportunities for the further potential applications of lysozyme as an anti-bacterium agent.

Keywords: anti-bacterium activity; chitosan nanoparticles; immobilization; lysozyme.

Figure 1

Characterization of nanoparticles: (A) schematic illustrations of the preparation process of Lys-CS-NPs; (B) particle size distribution of Lys-CS-NPs; and (C) zeta potential distribution of Lys-CS-NPs.

Figure 2

Analysis of lysozyme immobilized chitosan nanoparticles (Lys-CS-NPs): (A) lysozyme loading capacity of Lys-CS-NPs; and (B) lysozyme loading efficiency of Lys-CS-NPs.

Figure 3

Biochemical characterization of free (blue circles) and immobilized (red squares) lysozyme. (A) Optimal temperature of the free lysozyme and immobilized lysozyme. The activities were determined in 20 mM sodium phosphate buffer (pH 6.5) for different temperatures from 10 to 80 °C. (B) The effect of pH on the activities of free lysozyme and immobilized lysozyme. Enzymatic activity assay was processed at 50 °C in various buffers as follows: 20 mM sodium acetate buffer (pH 3.7–5.0), 20 mM phosphate buffer (pH 6.0–7.0), and 20 mM glycine-NaOH buffer (pH 7.6–8.9). (C) Thermal stability of free lysozyme and immobilized lysozyme. Enzyme was incubated at various temperatures without substrate for 30 min and then its residual activities assayed at 50 °C and pH 6.5. (D) Thermo-stability of free and immobilized lysozyme at 37 °C for incubation at different times. The enzymatic activity of a fresh sample of free or immobilized lysozyme measured in 20 mM sodium phosphate buffer and at 50 °C was defined as 100%.

Figure 4

Reusability of Lys-CS-NPs. Activity for each cycle was compared with the initial activity.

Figure 5

Antimicrobial activity kinetics of CS-NPs (red filled circles), free lysozyme (blue filled square), free lysozyme + CS-NPs complex (green filled triangle), and Lys-CS-NPs (black hollow triangle) for: P. aeruginosa (A); K. pneumoniae (B); E. coli (C); and S. aureus (D). lg, log10.