Moldes A B, Álvarez-Chaver P, Vecino X, Cruz J M
CINTECX (Research Center in Technologies, Energy and Industrial Processes), Chemical Engineering Department, University of Vigo, Vigo, Spain.
CACTI (Centro de Apoyo Científico y Tecnológico a la Investigación), Structural Determination and Proteomics Service, University of Vigo, Vigo, Spain.
Front Bioeng Biotechnol. 2023 Jun 6;11:1199103. doi: 10.3389/fbioe.2023.1199103. eCollection 2023.
Protocols to identify lipopeptide biosurfactant extracts contained in complex residual streams are very important, as fermented agri-food matrices are potential sources of these valuable compounds. For instance, corn steep liquor (CSL), a secondary stream of the corn wet-milling industry, is composed of a mixture of microbial metabolites, produced during the corn steeping process, and other natural metabolites released from corn, that can interfere with the purification and analysis of lipopeptides. Electrophoresis could be an interesting technique for the purification and further characterization of lipopeptide biosurfactant extracts contained in secondary residual streams like CSL, but there is little existing literature about it. It is necessary to consider that lipopeptide biosurfactants, like Surfactin, usually are substances that are poorly soluble in water at acidic or neutral pH, forming micelles what can inhibit their separation by electrophoresis. In this work, two lipopeptide biosurfactant extracts obtained directly from CSL, after liquid-liquid extraction with chloroform or ethyl acetate, were purified by applying a second liquid extraction with ethanol. Following that, ethanolic biosurfactant extracts were subjected to electrophoresis under different conditions. Lipopeptides on Tricine-SDS-PAGE (polyacrylamide gels) were better visualized and identified by fluorescence using SYPRO Ruby dye than using Coomassie blue dye. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of lipopeptide isoforms separated by electrophoresis revealed the presence of masses at 1,044, 1,058, and 1,074 m/z, concluding that Tricine-SDS-PAGE electrophoresis combined with MALDI-TOF-MS could be a useful tool for purifying and identifying lipopeptides in complex matrices.
鉴定复杂残留物流中所含脂肽生物表面活性剂提取物的方案非常重要，因为发酵的农业食品基质是这些有价值化合物的潜在来源。例如，玉米浆（CSL）是玉米湿法研磨工业的二次流，由玉米浸泡过程中产生的微生物代谢物混合物以及从玉米中释放的其他天然代谢物组成，这些物质会干扰脂肽的纯化和分析。电泳可能是一种用于纯化和进一步表征CSL等二次残留物流中所含脂肽生物表面活性剂提取物的有趣技术，但关于它的现有文献很少。有必要考虑到，像表面活性素这样的脂肽生物表面活性剂通常是在酸性或中性pH下难溶于水的物质，形成的胶束会抑制它们通过电泳分离。在这项工作中，通过用氯仿或乙酸乙酯进行液 - 液萃取后直接从CSL获得的两种脂肽生物表面活性剂提取物，通过用乙醇进行第二次液液萃取进行纯化。随后，将乙醇生物表面活性剂提取物在不同条件下进行电泳。与使用考马斯亮蓝染料相比，使用SYPRO Ruby染料通过荧光在Tricine - SDS - PAGE（聚丙烯酰胺凝胶）上能更好地可视化和鉴定脂肽。对通过电泳分离的脂肽异构体进行基质辅助激光解吸/电离飞行时间质谱（MALDI - TOF - MS）分析，结果显示在1044、1058和1074 m/z处有质量峰，得出结论：Tricine - SDS - PAGE电泳与MALDI - TOF - MS相结合可能是纯化和鉴定复杂基质中脂肽的有用工具。
