阴离子交换层析法纯化聚唾液酸工艺

以大肠杆菌发酵所产聚唾液酸粗品(含蛋白质、核酸、无机盐和色素等杂质)为纯化对象，通过静态吸附和梯度洗脱实验选择了分离效果较好的阴离子交换介质Q-琼脂糖凝胶FF，对聚唾液酸的纯化工艺条件进行优化. 得到最佳洗脱条件为：洗脱液为pH 7.2的NaCl-0.02 mol/L磷酸钠缓冲液体系，流速0.8 mL/min，洗脱线性梯度方程为CNaCl=0.0017VEluent (CNaCl为NaCl浓度，VEluent为洗脱液体积)，层析柱体积46 mL时最大进样量4.0 mL. 该条件下聚唾液酸回收率在86.0%以上，纯化后样品中的蛋白质含量从1.9%降低至0.04%，纯度在98%以上. 紫外吸收光谱和高效凝胶过滤色谱分析表明，聚唾液酸产品组分均一，重均分子量为303 kDa.

Purification of Polysialic Acid by Anion Ion Exchange Chromatography

A crude product of polysialic acid produced by Escherichia coli was subjected to anion ion exchange chromatography for further refining. Based on the preliminary results of static adsorption and gradient elution, a Q-Sepharose FF was chosen for the chromatographic matrix. The ion exchange chromatography conditions for polysialic acid purification were optimized. The results showed that the optimal elution conditions for polysialic acid were as follows: pH 7.2 sodium chloride-0.02 mol/L sodium phosphate buffer, linear elution at CNaCl=0.0017VEluent, elution rate 0.8 mL/min, and maximum injection volume 4.0 mL in 46 mL column. The purity of polysialic acid product was achieved to 98%, the protein content was decreased from 1.9% to 0.04% and the recovery rate of polysialic acid was more than 86.0%. Analytical results of UV spectrometry and high performance gel filtration chromatography showed that the final polysialic acid product was homogeneous and had average molecular weight of 303 kDa.