Department of Dentistry, Zuoying Branch of Kaohsiung Armed Forces General Hospital, Kaohsiung 81357, Taiwan.
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan.
Molecules. 2019 Jul 9;24(13):2516. doi: 10.3390/molecules24132516.
The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in BL21(DE3). Peocin was identified as a DNA starvation/stationary phase protection protein, also called DNA-binding protein from starved cells (Dps), by gel overlay and LC-MS/MS analysis. For mass production of peocin, fed-batch cultivation of was performed using a pH-stat control system. Purification by simple nickel affinity chromatography and dialysis yielded 45.3 mg of purified peocin from a 20-mL fed-batch culture (49.3% recovery). The biological activity of the purified peocin was confirmed by determination of the MIC and MBC against diverse pathogens. Purified peocin exhibited antimicrobial activity against aquatic, food spoilage, clinical and antibiotic-resistant pathogens. In an in vivo challenge test, zebrafish treated with purified peocin exhibited significantly increased survival rates after challenge. The present study is the first to show the antimicrobial activity of Dps and provides an efficient strategy for production of bioactive peocin, which will aid the development of peocin as a novel antimicrobial agent with potential applications in diverse industries.
先前，本实验室报道了一株益生菌 NPUST1 能产生一种具有抗菌活性的细菌素样物质，命名为 peocin。本研究旨在通过在 BL21(DE3) 中的异源表达来鉴定 peocin 并提高其产量。通过凝胶覆盖和 LC-MS/MS 分析，将 peocin 鉴定为一种 DNA 饥饿/静止期保护蛋白，也称为饥饿细胞中的 DNA 结合蛋白（Dps）。为了大规模生产 peocin，使用 pH -stat 控制系统进行分批补料培养。通过简单的镍亲和层析和透析，从 20-mL 分批补料培养物中纯化出 45.3mg 的纯化 peocin（回收率为 49.3%）。通过测定对不同病原体的 MIC 和 MBC，确认了纯化的 peocin 的生物活性。纯化的 peocin 对水生、食品腐败、临床和抗生素耐药病原体均具有抗菌活性。在体内攻毒试验中，用纯化的 peocin 处理的斑马鱼在 攻毒后显示出显著提高的存活率。本研究首次表明了 Dps 的抗菌活性，并提供了一种生产具有生物活性的 peocin 的有效策略，这将有助于将 peocin 开发为一种具有潜在应用前景的新型抗菌剂，应用于多个领域。
