柱色谱法纯化藻蓝蛋白的优化研究与机理分析  

Optimization and Mechanism Analysis of Purification of Phycocyanin by Column Chromatography

作　　者：武康 张发宇[1] 赵冰冰[1] 方艳 汪家权[1] 

机构地区：[1]合肥工业大学资源与环境工程学院,安徽合肥

出　　处：《生物医学》2019年第2期81-88,共8页Hans Journal of Biomedicine

基　　金：感谢国家“十二五”科技重大专项(2012ZX07103-004);博士专项科研基金(JZ2018HGBZ0145)资助。

摘　　要：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5mL/min、进样浓度1mg/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 ofphycocyanin is beneficial to its research and popularization as a drug andfluorescent reagent.In this paper,the phycocyanin was purified by columnchromatography using cyanobacteria from Chaohu Lake.The single-factorexperiment was carried out to optimize the operating conditions of columnchromatography.The mechanism of the whole purification process was analyzed byUV-Vis absorption spectroscopy and infrared absorption spectroscopy.Studieshave shown that the optimum single factor conditions for Cellufine A-500purified phycocyanin experiments are pH 7.0,ionic strength 0.25 mol/L,elutionrate 5 mL/min,and injection concentration 1 mg/mL.Ultraviolet-visibleabsorption spectroscopy showed that a small amount of heteroprotein was removedby one-step salting-out process,a large amount of nucleic acid andheteroprotein were removed by two-step salting-out,and the heterologousprotein was further removed by column chromatography to obtain reagent-gradephycocyanin.The infrared absorption spectrum indicated that the secondarystructure of the hetero protein removed by the whole process was mainlyβ-sheet,and the additives such as ammoniumsulfate were effectively removed.

关 键 词：藻蓝蛋白 柱色谱 机理分析 

分 类 号：R28[医药卫生—中药学]