噬菌体裂解酶Lys162基因重组菌株的构建及重组酶抗菌活性评价

Development of a recombinant bacterial strain for the expression of bacteriophage lytic enzyme Lys162 and assessment of the antibacterial activity of the recombinant enzyme

摘要:

目的 构建一株能够表达噬菌体裂解酶Lys162的重组大肠杆菌工程菌，以获得一种高效、广谱的重组裂解酶，为开发新型抗菌制剂提供技术支撑。方法 基于噬菌体pEC.M2929.1AR.1的全基因组测序数据，利用生物信息学工具预测其蛋白结构，并通过分子对接分析评估其与底物的结合能力。构建pET28a(+)-Lys162表达载体及大肠杆菌BL21(DE3)工程菌表达体系。进一步对噬菌体来源的重组裂解酶Lys162进行环境稳定性、体外抗菌活性及裂解谱评估。结果 预测结果显示，Lys162为具有N-乙酰胞壁质酶活性的裂解酶，含有保守的裂解酶结构域；分子对接证实其与肽聚糖结合紧密。该酶在BL21(DE3)中以可溶形式表达，纯化后浓度达1.89 mg/mL。体外实验表明，125 μg/mL的Lys162对大肠杆菌M2929.1AR具有显著裂解活性，对肺炎克雷伯菌、铜绿假单胞菌、不动杆菌属等多种病原菌也表现出良好的裂解能力。该酶在pH 4.0-11.0及4-60 ℃范围内均能保持稳定的活性。结论 Lys162突破了原噬菌体的宿主范围限制，表现出广谱的抗菌活性与环境适应性。其与EDTA的协同作用为实际应用提供了策略优化方向。本研究结果为开发应对细菌耐药挑战的新型抗菌制剂奠定了基础。

Abstract:

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.