This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 μg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 μg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 μg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 μg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.
Keywords: Bacillus subtilis BSN314; Antimicrobial activity; D-Alanyl-D-alanine carboxypeptidase; Gene expression; Lysozyme production strategies; N-Acetylmuramoyl-L-alanine amidase.
© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.