biostudies-literatureUnknown14(1)Miri M<h4>Background</h4>To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a <i>Bacillus subtilis</i>-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in <i>Escherichia coli (E. coli)</i>.<h4>Methods</h4>The sequences of <i>MurNAc-LAA</i> genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in <i>E. coli</i>. Furthermore, the structure and function of LysM2 was evaluated <i>in silico</i>. The optimized gene was synthesized, subcloned in the pET28a, and expressed in <i>E. coli</i> BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied.<h4>Results</h4><i>LysM2</i>, on 816 <i>bp</i> gene encoding a 33 <i>kDa</i> protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 <i>hr</i> in <i>E. coli</i>. <i>In silico</i> analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against <i>Proteus</i> (sp) by turbidity, and an antibacterial activity against <i>Klebsiella pneumoniae, Salmonella typhimurium</i>, and <i>Proteus vulgaris</i> in agar diffusion assays.<h4>Conclusion</h4>Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria.Avicenna journal of medical biotechnology46-53https://www.ebi.ac.uk/biostudies/studies/S-EPMC9017466biostudies-literatureExogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function.PMC9017466Miri MDarvish Alipour Astaneh SYazdianpour SAbolmaali SfalseExogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function.<h4>Background</h4>To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a <i>Bacillus subtilis</i>-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in <i>Escherichia coli (E. coli)</i>.<h4>Methods</h4>The sequences of <i>MurNAc-LAA</i> genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in <i>E. coli</i>. Furthermore, the structure and function of LysM2 was evaluated <i>in silico</i>. The optimized gene was synthesized, subcloned in the pET28a, and expressed in <i>E. coli</i> BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied.<h4>Results</h4><i>LysM2</i>, on 816 <i>bp</i> gene encoding a 33 <i>kDa</i> protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 <i>hr</i> in <i>E. coli</i>. <i>In silico</i> analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against <i>Proteus</i> (sp) by turbidity, and an antibacterial activity against <i>Klebsiella pneumoniae, Salmonella typhimurium</i>, and <i>Proteus vulgaris</i> in agar diffusion assays.<h4>Conclusion</h4>Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria.2022-01-01T00:00:00Z2022 Jan-Mar2025-04-18T12:11:18.232Z2025-04-06T21:49:23.936ZS-EPMC90174663550936410.18502/ajmb.v14i1.8169