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. 2023 Jan 20;28(3):1058.
doi: 10.3390/molecules28031058.

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria

Muhammad Naveed  1   2 , Yadong Wang  1   2 , Xian Yin  1   2 , Malik Wajid Hussain Chan  3 , Sadar Aslam  4 , Fenghuan Wang  1   2 , Baocai Xu  1   2 , Asad Ullah  1   2   5
Affiliations

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria

Muhammad Naveed et al. Molecules. .

Erratum in

Abstract

In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive (Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative (Salmonella typhimurium and Pseudomonas aeruginosa) strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus (AMI/PAI; 1.01/101) and Bacillus subtilis 168 (AMI/PAI; 1.03/103), compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213 (N-acetylmuramoyl-L-alanine amidase) and A0A1Q9FRD3 (D-alanyl-D-alanine carboxypeptidase). The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database.

Keywords: Bacillus subtilis BSN314; LC-MS; SDS-PAGE; antibacterial activity; atomic force microscopy; enzymatic activity; lysozyme purification.

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Conflict of interest statement

The authors indorse no conflicts of interest.

Figures

Figure 1
Figure 1
The standard curve for measuring the protein concentration (quantification) by the BCA (Bicinchoninic acid) method. X-axis; showing the concentration of protein mg/mL, Y-axis; showing the absorbance at 562 nm.
Figure 2
Figure 2
Antibacterial activity of lysozyme zone of inhibition (ZOI) (A,B): ZOI (A1,A2); of Bacillus subtilis 168 by lysozyme, (B); ZOI of antibiotic, and AFM images: (C,D): (C); the structure of Bacillus cereus, before the application of lysozyme, (D); after the application of lysozyme representing the disintegrating effect of lysozyme on the bacterial surface.
Figure 3
Figure 3
Antibacterial activity of lysozyme zone of inhibition (ZOI) (A,B): ZOI (A1,A2); of Bacillus cereus by lysozyme (B); ZOI of antibiotic, and AFM images: (C,D): (C); the structure of Bacillus cereus, before the application of lysozyme, (D); after the application of lysozyme representing the disintegrating effect of lysozyme on the bacterial surface.
Figure 4
Figure 4
Antibacterial activity of lysozyme zone of inhibition (ZOI) (A,B): ZOI (A1,A2); of Micrococcus luteus by lysozyme (B); ZOI of antibiotic, and AFM images: (C,D): (C); the structure of Micrococcus luteus, before the application of lysozyme, (D); after the application of lysozyme representing the disintegrating effect of lysozyme on the bacterial surface.
Figure 5
Figure 5
Antibacterial activity of lysozyme zone of inhibition (ZOI) (A,B): ZOI (A1,A2); of Pseudomonas aeruginosa by lysozyme (B); ZOI of antibiotic, and AFM images: (C,D): (C); the structure of Pseudomonas aeruginosa, before the application of lysozyme, (D); after the application of lysozyme representing the disintegrating effect of lysozyme on the bacterial surface.
Figure 6
Figure 6
Antibacterial activity of lysozyme zone of inhibition (ZOI) (A,B): ZOI (A1,A2); of Salmonella typhimurium by lysozyme (B); ZOI of antibiotic, and AFM images: (C,D): (C); the structure of Salmonella typhimurium, before the application of lysozyme, (D); after the application of lysozyme representing the disintegrating effect of lysozyme on the bacterial surface.
Figure 7
Figure 7
SDS-PAGE (sodium-dodecyl sulfate polyacrylamide gel electrophoresis), three protein bands, indicating that BSN314 lysozyme was purified to a monomeric protein. Molecular weight of the extracted Bacillus subtilis BSN314, where M is the molecular marker, and 1 indicates the protein bands.
Figure 8
Figure 8
LC–MS (liquid chromatography–mass spectrometry) chromatogram, showing the detected protein components.
See this image and copyright information in PMC

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