Zaghloul Nourhan A, Gouda Mona K, Elbahloul Yasser, El Halfawy Nancy M
Department of Botany and Microbiology, Faculty of Science, Alexandria University, Alexandria, Egypt.
Sci Rep. 2025 Jan 30;15(1):3735. doi: 10.1038/s41598-025-86649-w.
Azurin, a bacterial blue-copper protein, has garnered significant attention as a potential anticancer drug in recent years. Among twenty Pseudomonas aeruginosa isolates, we identified one isolate that demonstrated potent and remarkable azurin synthesis using the VITEK 2 system and 16S rRNA sequencing. The presence of the azurin gene was confirmed in the genomic DNA using specific oligonucleotide primers, and azurin expression was also detected in the synthesized cDNA, which revealed that the azurin expression is active. Furthermore, crude azurin protein was extracted, precipitated using 70% ammonium sulfate, dialyzed, and subjected to purification using carboxymethyl-Sephadex in affinity chromatography as a cheap method for purification. The partially purified azurin protein was characterized using polyacrylamide gel electrophoresis, energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Notably, qualitative elemental analysis by EDX showed the presence of copper and sulfur, corresponding to the copper-core and disulfide-bridge, respectively, in the purified azurin fraction. Moreover, FTIR spectroscopy revealed characteristic amide I and II absorption peaks (1500-1700 cm), revealing the possible secondary structure of azurin. The results of NMR revealed the presence of characteristic amino acids such as methionine and cysteine, which confirmed the EDX results for sulfur-containing amino acids. Purified azurin exhibited antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Klebsiella pneumoniae. Additionally, its anticancer properties were determined using the MTT assay and cell cycle analysis, revealing a preference for inhibiting the MCF7 breast cancer cell line where breast cancer is most common in Egypt. Overall, the research findings suggest that the local isolate, P. aeruginosa strain 105, could be a potential source of azurin protein for incorporation into cancer treatment strategies.
天青蛋白是一种细菌蓝铜蛋白，近年来作为一种潜在的抗癌药物受到了广泛关注。在20株铜绿假单胞菌分离株中，我们通过VITEK 2系统和16S rRNA测序鉴定出一株能够高效合成天青蛋白的分离株。使用特异性寡核苷酸引物在基因组DNA中证实了天青蛋白基因的存在，并且在合成的cDNA中也检测到了天青蛋白的表达，这表明天青蛋白的表达是活跃的。此外，提取了粗制天青蛋白，用70%硫酸铵沉淀，透析，并采用羧甲基葡聚糖凝胶在亲和色谱中进行纯化，这是一种廉价的纯化方法。使用聚丙烯酰胺凝胶电泳、能量色散X射线光谱、傅里叶变换红外光谱和核磁共振光谱对部分纯化的天青蛋白进行了表征。值得注意的是，通过能量色散X射线光谱进行的定性元素分析表明，纯化的天青蛋白组分中存在铜和硫，分别对应于铜核心和二硫键桥。此外，傅里叶变换红外光谱显示了特征性的酰胺I和酰胺II吸收峰（1500 - 1700 cm），揭示了天青蛋白可能的二级结构。核磁共振结果显示存在蛋氨酸和半胱氨酸等特征性氨基酸，这证实了能量色散X射线光谱对含硫氨基酸的分析结果。纯化的天青蛋白对金黄色葡萄球菌、枯草芽孢杆菌、大肠杆菌和肺炎克雷伯菌具有抗菌活性。此外，使用MTT法和细胞周期分析测定了其抗癌特性，结果表明它更倾向于抑制埃及最常见乳腺癌的MCF7乳腺癌细胞系。总体而言，研究结果表明，本地分离株铜绿假单胞菌105菌株可能是天青蛋白的潜在来源，可用于癌症治疗策略。
