Journal Article

Expression of BSN314 lysozyme genes in Escherichia coli BL21: a study to demonstrate microbicidal and disintegarting potential of the cloned lysozyme

Aslam, Sadar, Chan, Malik Wajid Hussain, Yin, Xian, Xu, Baocai, Naveed, Muhammad, Wen, Sai, Wang, Fenghuan, Ullah, Asad

2024

Overview

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. Highlights To achieve the highest lysozyme production, the genes were expressed in E. coli BL21. Genes, Lyz-1 (N-acetylmuramoyl-L-alanine amidase) and Lyz-2 (D-alanyl-D-alanine carboxypeptidase), were introduced. Lyz1 showed the highest antimicrobial activity, whereas Lyz2 had the lowest. The disintegrating effect of bacteria and yeast has been indicated by AFM analysis. Lyz1 can cleave amide bond between glycan and peptide components of the peptidoglycan. Graphical abstract

• Publisher: Springer International Publishing,Springer Nature B.V
• Subject: Acids / Alanine / Amidase / Ammonium / Ammonium sulfate / Anti-Infective Agents - pharmacology / Antiinfectives and antibacterials / Antimicrobial activity / Antimicrobial agents / Atomic force microscopy / Bacillus subtilis - genetics / Bacteria / Biomedical and Life Sciences / Biotechnology / Biotechnology and Industrial Microbiology - Research Paper / Carboxypeptidase D / Cell culture / Cloning / Culture media / D-Alanine / D-Alanine carboxypeptidase / D-Alanyl-D-alanine / Dialysis / Disintegration / E coli / Enzymes / Escherichia coli / Fermentation / Food Microbiology / Genes / Genetic engineering / Glycan / Gram-negative bacteria / Gram-positive bacteria / L-Alanine / Life Sciences / Lysozyme / Medical Microbiology / Microbial Ecology / Microbial Genetics and Genomics / Microbicides / Microbiology / Microorganisms / Microscopy / Minimum inhibitory concentration / Muramidase - genetics / Muramidase - metabolism / Muramidase - pharmacology / Mycology / N-Acetylmuramoyl-L-alanine amidase / Pathogens / Peptidoglycans / Pharmaceutical industry / Proteins / Ultrafiltration / Yeast