Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function.
| Autor: | Miri M; Department of Biotechnology, Faculty of Biotechnology, Semnan University, Semnan, Iran., Yazdianpour S; Department of Biotechnology, Faculty of Biotechnology, Semnan University, Semnan, Iran., Abolmaali S; Department of Biology, Faculty of Basic Sciences, Semnan University, Semnan, Iran., Darvish Alipour Astaneh S; Department of Biotechnology, Faculty of Biotechnology, Semnan University, Semnan, Iran. |
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| Jazyk: | angličtina |
| Zdroj: | Avicenna journal of medical biotechnology [Avicenna J Med Biotechnol] 2022 Jan-Mar; Vol. 14 (1), pp. 46-53. |
| DOI: | 10.18502/ajmb.v14i1.8169 |
| Abstrakt: | Background: 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 Bacillus subtilis -hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in Escherichia coli (E. coli) . Methods: The sequences of MurNAc-LAA 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 E. coli . Furthermore, the structure and function of LysM2 was evaluated in silico . The optimized gene was synthesized, subcloned in the pET28a, and expressed in E. coli BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied. Results: LysM2 , on 816 bp gene encoding a 33 kDa protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 hr in E. coli . In silico 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 Proteus (sp) by turbidity, and an antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium , and Proteus vulgaris in agar diffusion assays. Conclusion: Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria. Competing Interests: Conflict of Interest The authors declare that they have no competing interest. (Copyright© 2022 Avicenna Research Institute.) |
| Databáze: | MEDLINE |
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