超支化固定金属离子亲和膜的制备及其吸附性能研究

发布时间：2018-03-31 02:02

本文选题：再生纤维素膜　切入点：固定化金属离子亲和膜　出处：《西北大学》2015年硕士论文

【摘要】：固定金属亲和膜色谱具有可以在较短时间内处理大量的实际样品,低压降,容易扩大化生产等优点,因此在蛋白等生物大分子的分离纯化方面有潜在应用价值。但是与传统的以微球装柱的柱色谱相比,膜色谱比表面积较低,因而对蛋白的吸附容量较低,限制其分离效率的提高。为提高蛋白的吸附量,本文采用超支化方法对膜进行表面改性,通过在表面引入更多的功能基团,增加吸附位点来提高蛋白的吸附量,制备了一种对于金属离子和蛋白都有较高吸附量的超支化固定金属离子亲和膜。同时研究了该改性膜对蛋白的吸附性能,并将其用于含组氨酸标签蛋白的分离纯化。首先通过两步反应在膜表面引入带有氨基的宫能团,接下来将丙烯酸甲酯和乙二胺逐步加入,通过迈克加成反应和酰胺化反应在膜表面形成超支化结构,最后依次与环氧氯丙烷和亚氨基二乙酸反应,再螯合金属离子制备了超支化固定金属离子亲和膜。用FTIR和XPS表征了膜表面的化学组成。溶菌酶被作为模型蛋白来探究蛋白的吸附性能,该改性膜对于溶菌酶的最大吸附量为162 mg/cm3,高于文献报道值。同时探究了溶液pH,离子强度,疏水作用和吸附时间对吸附量的影响。将该改性膜用于蛋清中溶菌酶的纯化,回收率可达到94.3%,比未超支化的固定金属离子亲和膜的回收率提高了50%。最后用本文制备的改性膜从大肠杆菌粗提液中分离和富集含六聚组氨酸标签的天蚕抗菌肽B-人表皮生长因子(CB-EGF)融合蛋白,获得的蛋白纯度较高。所有这些结果证明本文合成的超支化固化金属离子亲和膜在His-tagged蛋白的分离纯化方面有潜在的应用价值。
[Abstract]:Stationary metal affinity membrane chromatography has the advantages of being able to deal with a large number of actual samples in a relatively short time, falling at low pressure, easy to expand production, etc. Therefore, it has potential application value in the separation and purification of biological macromolecules such as protein, but compared with the traditional column chromatography with microspheres, the specific surface area of membrane chromatography is lower, so the adsorption capacity of protein is lower. In order to improve the adsorption capacity of protein, the hyperbranched method was used to modify the surface of the membrane. By introducing more functional groups on the surface and increasing the adsorption sites, the adsorption capacity of the protein was increased. A hyperbranched fixed metal ion affinity membrane with high adsorption capacity for both metal ions and proteins was prepared. It was used for the separation and purification of histidine labelled proteins. Firstly, a hysteric group with amino groups was introduced on the membrane surface through two steps reaction, and then methyl acrylate and ethylenediamine were added step by step. The hyperbranched structure was formed on the membrane surface by Michael addition reaction and amidation reaction, and then reacted with epichlorohydrin and iminodiacetic acid. The hyperbranched immobilized metal ion affinity membrane was prepared by chelating metal ions. The chemical composition of the membrane surface was characterized by FTIR and XPS. Lysozyme was used as a model protein to investigate the adsorption properties of the membrane. The maximum adsorption capacity of the modified membrane for lysozyme was 162 mg / cm 3, which was higher than that reported in the literature. The effects of pH, ionic strength, hydrophobic action and adsorption time on the adsorption capacity of the modified membrane were investigated. The modified membrane was used to purify lysozyme from egg white. The recovery rate was 94. 3%, which was 50% higher than that of unhyperbranched immobilized metal ion affinity membrane. Finally, the modified membrane prepared in this paper was used to separate and enrich the antimicrobial peptide B- of Bombyx mori containing Hexahistidine label from the crude extract of Escherichia coli. Human epidermal growth factor CB-EGF fusion protein, All these results show that the hyperbranched solidified metal ion affinity membrane synthesized in this paper has potential application value in the separation and purification of His-tagged protein.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TQ051.893

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