染料亲和色谱复性蛋白质新方法的研究及其应用

发布时间：2018-03-23 03:14

本文选题：壳聚糖　切入点：硅胶　出处：《青岛科技大学》2010年硕士论文　论文类型：学位论文

【摘要】： 本论文分三部分,第一部分,基于大孔生物材料的染料亲和色谱,发展了一种新型的蛋白质色谱复性新技术;第二部分,基于溶胶-凝胶法,通过氨水溶液简单处理制备了球形硅胶支载的固定化金属亲和吸附剂;第三部分,基于溶胶-凝胶法,通过氢氧化钠溶液简单处理制备了球形硅胶支载的生物吸附剂。 1、基于大孔生物材料的染料亲和色谱(DLAC),发展了一种新型的蛋白质色谱复性新技术。采用具有表面大孔结构的壳聚糖-硅基(CS-silica)材料作为DLAC的基质材料。利用基质中的氨基,共价偶联亲和染料配基活性蓝(CBF)得到DLAC吸附剂。模型蛋白质过氧化氢酶经6 mol/L尿素变性后,DLAC可迅速去除变性剂,并通过CBF-CS-silica的吸附成功实现过氧化氢酶的复性。利用荧光光谱和过氧化氢酶活性研究考察了变性过程并对色谱洗脱步骤进行优化。与常规吸附复性法相比,可在蛋白质浓度提高20倍的条件下进行DLAC复性。 2、提出了制备新型有机-无机复合基质的有效途径。以球形硅胶作为支载核、以壳聚糖有机-无机杂化层作为壳,基于溶胶-凝胶反应,通过氨水简单处理后制备了该吸附剂。在基质上固定金属离子后,即得到吸附蛋白质的固定化金属亲和吸附剂。扫描电镜表征表明,经氨水溶液简单处理后,基质具有稳定的多孔表面。X-衍射分析表明,有机-无机杂化和氨水处理过程可破坏壳聚糖的结晶区,从而提高活性氨基的可利用度。采用同步热重-微量热分析进一步对制备的基质进行了表征。在基质表面固定作为特异性亲和配体Cu2+后,制备了新型固定化金属亲和吸附剂。以BSA为模型蛋白,通过吸附实验考察了该吸附剂的吸附性能。该吸附剂对BSA吸附速度快、吸附容量大。提出的方法及制备的基质在生化分析中具有潜在的应用前景。 3、提出了制备新型有机-无机生物吸附剂的有效途径。以球形硅胶作为支载核、以壳聚糖有机-无机杂化层作为壳,基于通过溶胶-凝胶反应,经氢氧化钠溶液简单处理后制备了该生物吸附剂。有机-无机杂化层通过壳聚糖与前驱体γ-环氧丙氧丙基三甲氧基硅烷(GPTMS)溶胶-凝胶化过程共价固定到硅胶表面。CS上的氨基与GPTMS上的环氧基团交联后,克服了其酸溶性缺点。通过氢氧化钠溶液简单处理后发生的湿法相转移,使制备的复合生物吸附剂具有粗糙的表面。制备的生物吸附试剂可以用来处理电镀废水。
[Abstract]:This thesis is divided into three parts. In the first part, a new protein chromatography renaturation technique based on macroporous biomaterial dye affinity chromatography is developed, the second part is based on sol-gel method. The immobilized metal affinity adsorbent supported by spherical silica gel was prepared by the simple treatment of ammonia solution. The third part, based on the sol-gel method, the spherical silica supported biological adsorbent was prepared by the simple treatment of sodium hydroxide solution. 1. A novel protein chromatographic renaturation technique was developed based on dye affinity chromatography (DLA) of macroporous biomaterials. Chitosan / Si-based CS-silicon material with surface macroporous structure was used as the matrix material of DLAC. The model protein catalase was denatured by 6 mol/L urea, and the denaturant was removed rapidly. The renaturation of catalase was successfully achieved by the adsorption of CBF-CS-silica. The denaturation process was investigated by fluorescence spectrum and catalase activity. The chromatographic elution procedure was optimized. DLAC renaturation could be carried out under the condition of increasing protein concentration by 20 times. 2. An effective way to prepare a novel organic-inorganic composite matrix was proposed. The spherical silica gel was used as the supporting nucleus, the chitosan organic-inorganic hybrid layer was used as the shell, and the sol-gel reaction was carried out. The adsorbent was prepared by simple treatment of ammonia water. After immobilized metal ions were fixed on the substrate, the immobilized metal affinity adsorbent of adsorbed protein was obtained. The SEM characterization showed that the adsorbent was simply treated with ammonia solution. The matrix has a stable porous surface. X- diffraction analysis shows that organic-inorganic hybrid and ammonia treatment can destroy the crystalline region of chitosan. In order to improve the availability of active amino groups, the prepared matrix was further characterized by simultaneous thermogravimetry and microcalorimetry. After the substrate surface was fixed as a specific affinity ligand Cu2, A new immobilized metal affinity adsorbent was prepared. The adsorption performance of the immobilized metal affinity adsorbent was investigated by adsorption experiments using BSA as the model protein. The proposed method and the prepared matrix have potential application in biochemical analysis. 3. An effective way to prepare new organic-inorganic biological adsorbents was proposed. The spherical silica gel was used as the supporting nucleus, and the chitosan organic-inorganic hybrid layer was used as the shell, based on the sol-gel reaction. The biological adsorbent was prepared by simple treatment of sodium hydroxide solution. The organic-inorganic hybrid layer was covalently immobilized on the surface of silica gel through the sol-gelation process between chitosan and the precursor 纬 -epichloropropyltrimethoxysilane (GPTMS). When the amino group on CS is crosslinked with the epoxy group on GPTMS, Through the wet phase transfer after simple treatment of sodium hydroxide solution, the composite biological adsorbent has rough surface. The prepared biosorption reagent can be used to treat electroplating wastewater.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：Q51-3;R341

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