【作者】 胡玉峰；

【导师】 任雪芹； 胡树文；

【作者基本信息】 中国农业大学 ， 环境工程， 2014， 博士

【摘要】 功能高分子材料学是研究功能高分子材料规律的一门学科,是高分子材料学领域发展最为迅速,并与化学、物理、生物等紧密联系的一门学科。按照性质和功能可以划分为以下几类,即化学高分子分离材料、高分子膜材、活性高分子材料(光活性、电活性、生物活性高分子材料)、高分子液晶材料、以及高分子智能材料几种。本论文着重研究高分子分离材料,并制备了四种新型功能高分子材料,并对其在生物目标物(磷酸化蛋白质、环境微生物以及蛋白质结晶)的应用进行探索,主要研究结果如下：(1)在水相中制备得到一种新型以聚乙烯醇为原料的固定钛离子亲和色谱材料(Ti4+-IMAC),采用扫描电子显微镜和红外光谱对材料的合成过程进行追踪表征。通过优化上样缓冲液的组成以及pH值提升材料从样品中富集磷酸化多肽的能力。研究发现,最佳上样缓冲液体系为邻苯二甲酸氢钾-盐酸缓冲液(pH2.5)。最后将优化后的体系应用在盐胁迫条件下的农大108玉米叶片组织磷酸化蛋白质研究中,首次发现多个与盐胁迫相关的磷酸化蛋白质,这些研究为更好地认识植物抵抗外界盐胁迫的机理提供了证据。(2)采用沉淀聚合的方法合成了一种新型的分子印迹聚合物(MIPs),该聚合物以苯基磷酸作为模板,钛离子固定乙二醇甲基丙烯酸磷酸酯作为功能单体,聚乙二醇丙烯酸酯和N,N-亚甲基双丙烯酰胺作为交联剂,去离子水作为致孔剂。首先通过苯基磷酸再吸附试验考察材料对模板分子的吸附能力,之后通过引入干扰物苯甲酸,验证材料对模板分子的选择性。结果表明,材料对苯基磷酸的印迹因子高达2.04,而对苯甲酸的仅有0.24,展现了优异的选择性和特异性。此外,通过对比发现,该MIPs对酪氨酸磷酸化多肽的吸附量和选择性均高于商业化的二氧化钛。最后,该MIPs被成功用于从复杂样品中选择性地分离酪氨酸磷酸化多肽。(3)采用聚乙二醇、蓖麻油和六亚甲基二异氰酸酯作为原料,以两种模式甲烷氧化菌作为模板分子,合成了新型的甲烷氧化菌印迹薄膜材料(PU-MIF),通过实验证明其具有良好的生物相容性、选择性以及低吸水性,可以被应用在甲烷氧化菌的筛选研究中。(4)将传统分子印迹技术和沉淀剂相结合,合成新型固定沉淀剂分子的印迹聚合物(piMIPs)。在五种标准蛋白质的结晶实验中,piMIPs可以快速的促进高质量晶体的产生,而且也可以促进其它成核剂难以诱导结晶出现的蛋白质(过氧化氢酶)产生晶体。研究结果表明,当MIPs上固定的沉淀剂种类和溶液里游离的沉淀剂种类一致时,piMIPs在促进蛋白质结晶中的表现最佳。通过这种方法,结构未知的脆性X智力缺陷蛋白质片段被成功结晶,并且在其结构中首次发现全新的KH-KHO结构域和分子间二硫键。更多还原

【Abstract】 Functional polymer materials science is the fastest developed areas of polymer materials, which integrated the disciplines of chemistry, physics, and biology and so on. According to the nature and functions, functional polymer materials could be divided into the following branches, namely, separation polymer materials, polymer membranes, the active polymer materials (photoactive, electro-active and bioactive polymer), polymer liquid crystal materials, and smart polymer materials. The present work focuses on the separation polymer materials, and four types of functional polymer materials were prepared and applied in plant phosphorylation proteomics, protein crystallization and environmental microbiology respectively. The main findings were as follows:(1) A novel immobilized titanium ion affinity chromatography material (Ti4+-IMAC) was prepared in the aqueous environment using polyvinyl alcohol as raw material. SEM and IR spectroscopy were used to monitor the synthesis process. The selectivity for phosphopeptide enrichment from sample was improved by optimizing the pH and components of the loading buffer, and the optimal buffer system was found to be potassium hydrogen phthalate/HCl (pH2.5) solution. The final optimized protocol was adapted to salt-stressed maize leaves of Nongda108for phosphoproteome analysis, and numerous phosphorylated proteins/peptides related with salt stress were found for the first time. Thses research would provide solid evidence for comprehensive understanding of the mechanism of plant response to external salinity stress.(2) Molecularly imprinted polymer (MIPs) for tyrosine-phosphorylated peptides was synthesized by precipitation polymerization method using phenyl phosphate (PPA) as a template, Ti4+-immobilized ethylene glycol methacrylate phosphate as functional monomer, poly(ethylene glycol) diacrylate and N,N-methylene bisacrylamide as crosslinker, deionized water as porogen. The performance of MIPs was firstly evaluated by rebinding PPA, and then a competition experiment was conducted to assess the selectivity and specificity for PPA when mixed with benzoic acid, a structural analogue. The imprinting factor of the MIPs was up to2.04, compared with just0.24for benzoic acid, suggesting this kind of MIPs held superior selectivity and specificity. The selectivity and capacity of the MIPs for pTyr peptides from a mixture of peptides were considerably higher than that of commercial TiO2. Finally, MIPs were used for pTyr enrichment from a complex sample containing pTyr peptide and tryptic digestion of β-casein, where it demonstrated a clear preference for the pTyr peptide over ones containing phosphorylated serine.(3) Molecularly imprinted film based on polyurethane (PU-MIF) was obtained by the reaction among polyethylene glycol, castor oil and hexamethylene diisocyanate using methanotroph bacteria as template. PU-MIF was firstly characterized by biocompatibility test, selectivity and water retention capacity. Finally, PU-MIF was applied in the cell sorting of methanotroph bacteria.(4) Precipitant-immobilized molecularly imprinted polymers (piMIPs) were obtained by the combination of traditional molecularly imprinted technique and precipitating agent. For five model proteins, piMIPs facilitated better and faster crystals formation compared to conventional nucleants. piMIPs also can grow crystals which were missed using other nucleants, such as catalase. And results demonstrated if the type of immobilized precipitant was consistent with free one in solution, cognate piMIPs performed best in producing larger crystals and higher diffraction resolution. By this method, high-quality single crystal of a highly flexible and unsolved segment of Fragile X Mental Retardation Protein, whose absence causes the most common inherited mental retardation in human, was obtained without changing its structure. A novel KH domain-KHO and an intermolecular disulfide bond were identified for the first time.更多还原