壳聚糖锌离子固定化亲和层析填料的制备与性质

Preparation and Properties of Chitosan Immobilized Zinc Ion Affinity Chromatography Matrix

目的:制备了壳聚糖Zn2+固定化亲和层析填料,并对其性能进行了研究。方法:采用反相悬浮法制备了交联壳聚糖;再以环氧氯丙烷为活化剂,乙二胺为螯合配基,制备了固定化亲和层析填料;表征了其有效粒径以及均匀系数、含水量、失重率、氨基含量、骨架密度、堆积密度以及孔度值。从时间、加入ZnCl2的浓度、温度、pH方面对Zn2+固定化条件进行了优选,并确定了Zn2+的固定化量。含组氨酸标签的乙醛脱氢酶粗酶液,经硫酸铵盐析后,考察了壳聚糖Zn2+固定化亲和层析填料的亲和性能。结果:制备的填料有效粒径为105μm;均匀系数为1.46;含水量为58.03%;失重率为85.43%;氨基含量为9.20mmol/g;骨架密度为1.217 8g/ml;堆积密度为0.843 2g/ml;孔度值为36.40%。固定化Zn2+的最佳条件是:时间3 h、加入ZnCl2溶液浓度0.1mol/L、温度28℃、pH 5.5;且此条件下,亲和层析填料中Zn2+固定化量为3.35mmol/g。壳聚糖Zn2+固定化亲和层析填料对乙醛脱氢酶的亲和性能为4.14IU/g(干重)。结论:制备了壳聚糖Zn2+固定化亲和层析填料,可用于带有组氨酸标签重组蛋白的快速分离与纯化。

Purpose：Chitosan immobilized zinc ion affinity chromatography matrixes were prepared,and its properties were studied.Methods：Immobilized metal affinity chromatography（IMAC） matrixes were successfully prepared with cross-linked chitosan prepared by reverse phase suspension polymerization,and 3-Chloro-1,2-epoxypropane as activated agent,ethylenediamine as chelating ligand and Zn^2＋ as center ions.Its effective grain size and uniformity coefficient,moisture content,rate of lostmass,amino content,skeletaldensity,bulk density and porosity were characterized.Time,concentration of ZnCl2 solution,temperature,pH of Zn^2＋ immobilized conditions were optimized to determine the amount of Zn^2＋ immobilized.After purifying with salting out,aldehyde dehydrogenase containing a histidine-tag was used to examine the affinity of chitosan immobilized Zn^2＋affinity chromatography matrix.Results：the effective grain size,uniformity coefficient,moisture content,rate of lostmass,amino content,skeletaldensity,bulk density and porosity of the prepared matrix is 105μm,1.46,58.03%,85.43%,9.20 mmol/g,1.2178 g/ml,0.8432 g/ml and 36.40% respectively.The optimum conditions of Zn^2＋ immobilized were：time 3h,ZnCl2 concentration 0.1 mol / L,temperature 28℃,pH 5.5.And the amount of Zn^2＋ immobilized is 3.35 mmol/g under these optimum conditions.The affinity of chitosan immobilized zinc ion affinity chromatography matrix for aldehyde dehydrogenase is 4.14 IU/g（dry weight）.Conclusions： The high-efficiency chitosan immobilized zinc ion affinity chromatography matrixes were prepared,and it can be used for quickly separating and purifying the recombinant protein containing a His-tag.