Optimization and Mechanism Analysis of Purification of Phycocyanin by Column Chromatography

Abstract

藻蓝蛋白的提取纯化有利于其作为药品和荧光试剂的研究和推广应用。本文以巢湖蓝藻为原料，利用柱色谱法纯化藻蓝蛋白，开展单因素实验优化柱色谱的运行条件，并使用紫外–可见吸收光谱与红外吸收光谱分析整个纯化工艺的机理。研究表明，CellufineA-500纯化藻蓝蛋白实验的最适单因素条件分别为pH 7.0、离子强度0.25 mol/L、洗脱速度5 mL/min、进样浓度1 mg/mL。紫外–可见吸收光谱表明一步盐析工艺去除了少量杂蛋白，二步盐析去除了大量核酸和杂蛋白，柱色谱工艺进一步去除杂蛋白，得到试剂级藻蓝蛋白。红外吸收光谱表明整体工艺去除的杂蛋白二级结构以β-折叠为主，硫酸铵等添加物会被有效去除。 The extraction and purification of phycocyanin is beneficial to its research and popularization as a drug and fluorescent reagent. In this paper, the phycocyanin was purified by column chromatography using cyanobacteria from Chaohu Lake. The single-factor experiment was carried out to optimize the operating conditions of column chromatography. The mechanism of the whole purification process was analyzed by UV-Vis absorption spectroscopy and infrared absorption spectroscopy. Studies have shown that the optimum single factor conditions for Cellufine A-500 purified phycocyanin experiments are pH 7.0, ionic strength 0.25 mol/L, elution rate 5 mL/min, and injection concentration 1 mg/mL. Ultraviolet-visible absorption spectroscopy showed that a small amount of heteroprotein was removed by one-step salting-out process, a large amount of nucleic acid and heteroprotein were removed by two-step salting-out, and the heterologous protein was further removed by column chromatography to obtain reagent-grade phycocyanin. The infrared absorption spectrum indicated that the secondary structure of the hetero protein removed by the whole process was mainly β-sheet, and the additives such as ammonium sulfate were effectively removed.