Q Sepharose FF分离藻红蛋白的工艺研究

Study on Separation of Phycoerythrin by Q-Sepharose Fast Flow

[目的]建立Q Sepharose FF分离藻红蛋白的工艺方法并验证其放大可行性。[方法]通过对洗脱条件、上样体积以及洗脱流速的优化,确定最佳分离工艺条件;在确定的工艺条件下,将上样体积与柱体积等比例放大。[结果]Q Sepharose FF分离藻红蛋白的最佳工艺条件为:将30 ml坛紫菜提取液加载到8 ml Q Sepharose FF柱上,以1 ml/min的流速使用添加0-0.10-0.35-1.00 mol/L NaCl的NaH2PO4-Na2HPO4缓冲溶液(pH 6.0)分步洗脱,收集0.35 mol/L NaCl溶液洗脱时的色谱峰。在此条件下R-藻红蛋白的回收率和纯度系数(A565/A280)分别为44.3和1.15。按照该工艺,将75 ml藻红蛋白提取液加载到20 ml Q Sepharose FF柱上进行分离,发现分离效果没有发生明显变化。[结论]使用Q Sepharose FF介质能够分离坛紫菜提取液中的藻红蛋白且该工艺易于放大。

[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verify its feasibility.[Method] Elution gradient,loading volume and flow rate were optimized to determine the optimal separation condition,under which the scale up process was verified.[Result] The optimal condition for separation of phycoerythrin by using Q Sepharose Fast Flow resin was investigated： 30 ml of laver extract was loaded to a Q Sepharose Fast Flow column with a bed volume of 8 ml;subsequently,the column was eluted under a stepwise gradient elution of 0-0.10-0.35-1.00 mol/L NaCl solution（pH 6.0） at a constant flow rate of 1 ml/min;the elution peak under 0.35 mol/L NaCl solution was collected,and the recovery rate and purity coefficient（A565/A280） of phycoerythrin were determined as 44.3 and 1.15,respectively.Based on the established process,75 ml of phycoerythrin extract was loaded to a Q Sepharose Fast Flow column with bed volume of 20 ml for separation,while no significant variation was observed in separation result.[Conclusion] Phycoerythrin can be well separated from laver extract by Q Sepharose Fast Flow resin and the process is feasible for scale-up.