条斑紫菜R-藻红蛋白提纯工艺研究

Extraction and Purification of R-Phycoerythrin in Porphyra yezoensis

对条斑紫菜叶状体R-藻红蛋白提纯方法进行了研究和分析。首先分别采用冻融法、化学试剂法、溶胀法等单一及组合细胞破碎方法对条斑紫菜细胞进行破碎,结果表明溶胀+组织捣碎法效果最佳。其次用25%～60%饱和度范围内的硫酸铵沉淀法粗提藻红蛋白,发现25%～45%硫酸铵分步沉淀法效果最好,再经结晶法盐析纯度(D565nm/D280nm)达到2.088。盐析液用SephadexG-25层析柱脱盐,再经羟基磷灰石(HA)柱层析,纯度可达到4.98。R-藻红蛋白的最大吸收峰在565nm,其室温荧光发射峰为578nm。SDS-PAGE结果显示,R-藻红蛋白可分为α、β两个亚基,Mr分别为17.0×103和19.0×103。

The extraction and purification of R-phycoerythrin（PE） in the thallus of Porphyra yezoensis was studied. The different cell fragmentation methods（freezing/unfreezing method, chemistry method, dissolving-bulging method, etc, and their admixture cell fragmentation methods） were applied and compared. The results showed that the combination of freezing/unfreezing adding tissue-trituration method was the optimal method for extracting phycoerythrin. The purification of crude extracts was performed by means of ammonium sulphate precipitation in different concentrations （25%-60%） and the phycoerythrin purification（D565nm/D280nm） was checked. The results showed that most phycoerythrin could precipitate between 25% and 45% concentration of （NH4）2SO4. So the 25% and 45% concentration of （NH4）2SO4 was applied to remove small protein and precipitated the phycoerythrin respectively. The purity of R-PE reached up to 1.09 and then increased to 2.09 by crystallization. The remained ammonium sulfate was removed by chromatography on a Sephadex G-25 column pre-equilibrated with the same buffer. The results showed that the purity of R-PE reached 2.97. Then the R-PE was further purified through hydroxylapatite （HA） column and the purity of R-PE reached up to 4.98. There was a maximum absorption peak at 565 nm, and a fluorescence emission peak at 578 nm. SDS-PAGE for R-PE showed that there were two subunits, α subunit and β subunit, with 17.0 kD and 19.0 kD of molecular weights respectively.