辛巴蓝F3GA-羟丙基淀粉的制备及其对蛋白质的亲和分离特性

Synthetisis of Cibacorn Blue F3GA-hydroxypropyl Starch and its Application in Affinity Separation of Protein

为了开发新型的淀粉基生物分离材料,制备了适用于亲和双水相萃取技术的辛巴蓝F3GA-羟丙基淀粉,并考察了其对蛋白的亲和分离特性。将三嗪类染料辛巴蓝F3GA分别偶联于不同取代度的羟丙基淀粉上,对其反应时间、温度以及pH进行考察,根据亲和配基密度,得到制备亲和分离材料的优化工艺条件为:反应温度60℃,反应时间6 h,pH 9.5。进一步考察了亲水性基团、亲和配基对羟丙基淀粉亲和材料/聚乙二醇双水相体系液液平衡的影响,表明,亲和配基的引入对系统的相平衡影响很小,系统结线都基本重合,斜率变化很小;随着羟丙基淀粉取代度的增大,双水相呈相区域向右移。牛血清白蛋白被分配到羟丙基淀粉亲和材料相中,当亲和配基密度达到1.25μmol/g时,分配效率从30%提高到80%以上。

In order to develop a novel starch based biological separation material, the F3GA-hydroxypropyl starch for the aqueous two-phase extraction system was prepared and its affinity separation characteristics for protein were investigated. Triazine dyes were respectively coupled with hydroxypropyl starch with different degrees of substitution. The effects of affmity ligand density, reaction time, temperature and pH on the degrees of substitution were observed and the optimal conditions of the preparation of affinity separation material were obtained. The affinity separation material was prepared at 60 ℃ for 6 hours at the pH value of 9.5. Furthermore, the effects of the hydrophilic group and affinity ligand on liquid-liquid equilibrium of hydroxypropyl starch affinity material/PEG aqueous two-phase system were studied. The results indicated that the effect of affinity ligand on phase equilibrium of system was slight and the slope change of the basically overlapping tie lines was also small and the area of aqueous two-phase was right-shift with increasing the degree of substitution of hydroxypropyl starch. Additionally, the distribution and affinity efficiency of bovine serum albumin （BSA） in this aqueous two-phase system were also observed. Most of the BSA was distributed to the phase of hydroxypropyl starch affinity materials, as well as the affmity efficiency was improved from 30% to more than 80% with the affinity ligand density of 1.25 μmol/g.