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

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.

Diversity of bacterial communities in the Sundarbans mangrove, Bangladesh, with special focus on pathogens affecting aquatic organisms

“…The previous research showed the high abundance of Bacilli in some stations of mangroves could be signatures of human interferences often cause infections in humans [73] . Virulence genes identified from the Sundarbans resistance to antibiotics confirmed by high abundance of ARGs.…”

“…DNA extraction in conventional way and kit based techniques [ 39 , 73 ] was used to obtain the DNA from the isolated 11 specimens (FS2, FS3, FS4, SS1, SS3, SS4, SS5, WS1, WS3, WS4 and WS5) from the different samples of the Sundarban following the manufacturer's instruction. The PureLink Microbiome DNA Purification Kit minimized the need to order individual kits for specific sample types because it has been optimized for use with a wide range of biological samples.…”

Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology

“…Bacillus subtilis exhibits enormous potential as a suitable host for biochemical production, primarily due to its excellent growth characteristics and adept bioresource utilization 16 . It is often employed as a host carrier for the production of industrially important enzymes such as lysozyme, cellulase, and amylase as well as for the synthesis of specific substances, including selenium nanoparticles, and other essential compounds 17 , 18 . Accordingly, this study shows a keen interest in cellulase-producing Bacillus subtilis strains and further aims to obtain the target strain through systematic screening experiments and optimization of the cultivation conditions.…”

Evaluation of cell disruption methods for protein and coenzyme Q10 quantification in purple non-sulfur bacteria

“…Their cell envelope consists of the cytoplasmic membrane holding the cell contents; the peptidoglycan layer, which consists of lipoproteins, phospholipids, porin proteins, lipopolysaccharide, and other proteins; and an external outer membrane that provides mechanical strength ( Weckesser et al, 1995 ; Rojas et al, 2018 ). This enables gram-negative bacteria to withstand lysozyme cell lysis better than gram-positive bacteria ( Naveed et al, 2023 ). Moreover, photosynthetic bacteria like PNSB have an additional intracytoplasmic membrane (ICM) adjoined to the inner membrane to facilitate energy harvest from light, potentially increasing the cells’ resistance to cellular disruption ( Oikonomou and Jensen, 2021 ).…”