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Objective To construct a recombinant Escherichia coli strain for the expression of the bacteriophage-derived lytic enzyme Lys162, an efficient and broad-spectrum recombinant enzyme, thus providing a technological foundation for developing novel antimicrobial agents. Methods On the basis of the whole-genome sequencing data of bacteriophage pEC.M2929.1AR.1, the protein structure was predicted via bioinformatics tools, and molecular docking analysis was performed to evaluate the substrate-binding affinity. The expression vector pET28a(+)-Lys162 and the engineered E. coli BL21(DE3) expression system were constructed. Lys162 was further assessed for its environmental stability, in vitro antibacterial activity, and lytic spectrum. Results Structural analysis predicted that Lys162 was an N-acetylmuramidase-type lytic enzyme containing a conserved catalytic domain. Molecular docking confirmed its high-affinity binding to peptidoglycan. The enzyme was expressed in a soluble form in E. coli BL21(DE3) and purified to reach a concentration of 1.89 mg/mL. In vitro assays demonstrated that Lys162 at 125 μg/mL exhibited significant lytic activity against E. coli M2929.1AR, along with potent lytic effects against multiple pathogenic bacteria including Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp. The enzyme retained stable activity within a pH range of 4.0-11.0 and at temperatures between 4 ℃ and 60 ℃. Conclusion Lys162 transcends the host specificity of its parental phage, demonstrating broad-spectrum antimicrobial activity and considerable environmental adaptability. Its synergistic effect with EDTA suggests a practical strategy for performance optimization. These results establish a foundation for developing novel enzymatic antimicrobials to address challenges associated with bacterial antibiotic resistance.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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