Antibacterial and Antibiofilm Properties of Lysozyme Extracted from Catostylus perezi, an Invaluable Jellyfish Bloom

Abstract

To show the beneficial aspect of jellyfish bloom in Pakistan, this study was designed. Lysozyme was isolated from Catostylus perezi and then purified by multistep methodological procedures such as salt precipitation (ammonium sulfate), dialysis and ultrafiltration. The lysozyme obtained at a 70 % concentration of (NH₄)₂SO₄ had the highest yield of 14.58 mg/mL, while the lowest yield of 1.06 mg/mL was obtained at a 20 % concentration of (NH₄)₂SO₄ solution. Using the ultrafiltration method, a yield of 68 % with a specific enzyme activity of 1952 U/mg was obtained. The lysozyme obtained by ultrafiltration was used for the evaluation of its bactericidal potential against various Gram-positive and Gram-negative species. The results of the antibacterial activity, e.g. the zones of inhibition (ZOI) and the minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC), showed that lysozyme has a higher bactericidal potential against Gram-positive than against Gram-negative bacterial strains. The results of antimicrobial index (AMI) and percent activity index (PAI) showed that lysozyme had the highest bactericidal potential against S. aureus ATCC 25923. The AMI value of Lysozyme against S. aureus ATCC 25923 was 1.30 and the PAI value was 130. Lysozyme had higher anti-biofilm activity against Gram-positive than Gram-negative species. The lysozyme was purified by gel column chromatography, resulting in a yield of 15.07 % with a specific activity of 1606 U/mg. The resulting lysozyme was further processed by native-PAGE (Polyacrylamide gel electrophoresis) technique and the protein/lysozyme obtained had an enzymatic activity of 1732 U/mL. This indicated that the lysozyme still retained its enzymatic potential at this stage. The purified lysozyme was also further processed by SDS-PAGE (Sodium-dodecyl sulfate polyacrylamide gel electrophoresis) technique. The results indicated, the lysozyme sample obtained by 70 % (NH₄)₂SO₄ concentration had a prominent protein band at 20 kDa. The scanning electron microscopic (SEM) technique was used to confirm antibacterial activity for two Gram-positive (M. luteus and S. aureus) and two Gram-negative species (E. coli and K. pneumoniae). The bactericidal potential of lysozyme was analyzed by scanning electron microscopy (SEM). The results showed that the bacterial cultures treated with lysozyme exhibited a significant inhibition against biofilm producing bacteria. The antibacterial activity of lysozyme against these bacterial species was: 31 mm ZOI (zone of inhibition) for M. luteus ATCC 4698 and 26 mm ZOI for S. aureus ATCC 25923, while 18.5 mm ZOI for E. coli ATCC 25922 and 16 mm ZOI for K. pneumoniae ATCC 13883. It was concluded from this study that the isolation and purification of the potential lysozyme from the jellyfish provided fundamental information for the future use of jellyfish blooms as food and in the pharmaceutical and food industries.