摘要
以巢湖新鲜蓝藻为处理对象,采用冻融破壁方法获得藻蓝蛋白粗提液,采用两步盐析结合两步柱层析的方法纯化藻蓝蛋白粗提液,以最终获得试剂级藻蓝蛋白。分别将纯化过程中各阶段得到的藻蓝蛋白溶液和杂质液进行紫外-可见光谱扫描。经光谱扫描发现:藻蓝蛋白溶液经一步盐析、二步盐析、一步柱层析和二步柱层析的四步纯化实验过程中,在250~300 nm吸收谱带中,吸收峰从260 nm红移至280 nm;在500~700 nm吸收谱带中,吸收峰从617 nm红移至620 nm,并表现为最大特征吸收峰。对四步纯化工艺过程中分别得到的不同杂质液同样进行250~700 nm全波段光谱扫描,分析判断得出:一步盐析杂质液成分主要含有杂蛋白和部分藻蓝蛋白;二步盐析的杂质液中主要成分为核酸和维生素类物质;一步柱层析分离的先出组分主要成分为藻红蛋白;二步柱层析分离的后出组分主要成分为别藻蓝蛋白。经四步纯化工艺后,最终得到了纯度大于4以上的试剂级藻蓝蛋白。由此可见,两步盐析的工艺主要作用在于去除杂蛋白、核酸和维生素类物质;两步柱层析工艺主要作用在于分离出和藻蓝蛋白性质接近的藻红蛋白和别藻蓝蛋白。
Fresh blue algal from Chao Lake was used in this study.The crude extracts of phycocyanin were obtained with freezethaw method.The purification of phycocyanin was performed by combining two-step salt precipitation and two-step column chromatography.The reagent grade phycocyanin was achieved.Phycocyanin and impurity solution were obtained respectively in various stages subjected to the UV-Vis absorption spectrum scanning.With the development of the four-step purification process,the absorption peak of phycocyanin solution was redshifted from 260 to 280 nm in the wavelength range from 250 to 300 nm,and the maximum absorption peak of phycocyanin was redshifted from 617 to 620 nm in the wavelength range from 500 nm to 700 run.In the wavelength range from 250 to 700 nm,it showed that the impurity solution mainly contained impurity proteins and part of the phycocyanin in the first salting out,and mainly contained nucleic acids and vitamins substance in the second salting out.The first outflow components mainly contained phycoerythrin separated by the first column chromatography.The last outflow components mainly contained allophycocyanin separated by the second column chromatography.After the four-step purification process,the final purity of phycocyanin（A_（620）/-A_（280）） is greater than 4,which met the standard of reagent grade.Thus it can be seen that two-step salt precipitation had a main function which was to remove impurity proteins,nucleic acids and vitamins substance,and two-step column chromatography main function was to remove phycoerythrin and allophycocyanin which were similar to phycocyanin.
出处
《光谱学与光谱分析》
SCIE
EI
CAS
CSCD
北大核心
2017年第3期806-810,共5页
Spectroscopy and Spectral Analysis
基金
国家"十二五"科技重大专项(2012ZX07103-004)资助
关键词
两步盐析
双水相萃取
吸收光谱
藻蓝蛋白
Two-step salt precipitation
Two-step column chromatography
Absorption spectrum
Phycocyanin
