摘要
壳聚糖(Chitosan，CS)是来源丰富的环境可再生资源，具有良好的生物相容性。CS分子中丰富的羟基和氨基使其易于衍生化。将CS用作蛋白质分离纯化和酶固定化基质材料具有很好的应用前景。本论文建立了多孔性壳聚糖-硅基复合材料的制备方法，并将所得材料用于生物分离和酶的固定化。
论文概述了蛋白质色谱分离模式，总结了蛋白质色谱和酶固定化所需基质材料的特性及分类。认为固相材料对蛋白质的非特异性吸附、蛋白质在固相表面的构象稳定性、蛋白质分离与制备的速度及材料的功能化是开发新型生物色谱及酶固定化基质材料的关键。
论文将基于CS的有机-无机复合材料分为无机材料负载CS型和原位复合型两大类，综述了CS与硅胶、玻璃微珠、氧化铝、量子点、金、碳纳米管、Fe_3O_4、羟基磷灰石等无机材料的复合方法和应用，并总结了制备多孔CS材料的致孔方法。
论文将CS负载在高比表面积、高机械强度的硅胶表面，以环氧氯丙烷为交联剂，聚乙二醇(PEG)为致孔剂，建立了新型多孔性CS-硅胶复合基质(CTS-SiO_2)的制备方法。研究了涂覆液中CS含量、致孔剂的分子量及含量对复合材料形貌和性能的影响。结果表明，涂覆液中CS含量为2％；致孔剂为PEG 20000，在涂覆液中含量为10％；涂覆液与硅胶质量比为2∶1；交联剂浓度为1mol L~(-1)时，所得复合材料具有表面大孔结构。CTS-SiO_2吸附Cu~(2+)后，制备了固定化金属亲和吸附剂(Cu-CTS-SiO_2)。以牛血清白蛋白(BSA)为模型蛋白，研究了CTS-SiO_2对BSA的非特异性吸附以及Cu-CTS-SiO_2对BSA的特异性吸附性能。研究发现，CTS-SiO_2对BSA具有低的非特异性吸附，具有大孔结构的Cu-CTS-SiO_2具有较高的BSA吸附容量。考察了CTS-SiO_2的酸碱稳定性等性能。发现CTS-SiO_2具有功能性表面和良好的酸碱稳定性。
论文考察了CTS-SiO_2在酶固定化中的应用。将CTS-SiO_2经环氧化、重氮化或醛基化三种不同的衍生化方法活化后，用于共价固定胰蛋白酶。采用生物信息学手段，推测了各种固定化方法中酶与活化微珠的多位点或单位点结合模式。实验结果表明，CTS-SiO_2易于衍生化，不同固定化方法对酶固定化率和稳定性有显著影响。与游离酶相比，固定化胰蛋白酶的热稳定性、pH稳定性、长期保存稳定性均显著提高。环氧化活化法制备的固定化胰蛋白酶与微珠的多位点结合导致酶刚性提高，并表现出最好的稳定性和重复利用性。
论文考察了CTS-SiO_2在固定化金属亲和色谱、生物配基亲和色谱中的应用。复合材料吸附Cu~(2+)后，研究了所得固定化金属亲和色谱填料(Cu-CTS-SiO_2)对BSA的吸附性能。结果表明，缓冲液酸度影响Cu-CTS-SiO_2对BSA的吸附。在pH 6.0时，Cu-CTS-SiO_2对BSA的吸附达到最大。Cu-CTS-SiO_2具有快的BSA吸附速度，吸附等温线符合Langmuir吸附模式。Cu-CTS-SiO_2可用于粗BSA样品的亲和色谱纯化。将CTS-SiO_2经环氧基团活化后，共价偶联胰蛋白酶，制备的生物配基亲和色谱填料可用于蛋清中胰蛋白酶抑制剂的亲和纯化。
论文以HAc为反应介质，以γ-缩水甘油基丙基三甲氧基硅氧烷为无机源，通过PEG印迹的溶胶-凝胶过程，将CS的共价交联与无机网络的引入一步完成，建立了尼龙膜支载CS／硅基多孔性复合膜的制备方法。研究了涂膜液中PEG分子量和含量对所得复合膜材料形貌的影响。当涂膜液中CS含量为2％，致孔剂PEG 20000的含量为15％时，所得复合膜具有等级式孔结构：除具备大孔结构外，还具有与大孔互相贯穿的孔结构。推测了等级式孔结构形成机理，研究了复合膜的热稳定性、对BSA的非特异性吸附等性能。结果表明，复合膜材料具有高的热稳定性和低的BSA非特异性吸附。具有等级式孔结构的膜材料结合Cu~(2+)后，对BSA具有较高的吸附容量。
As a functional biopolymer, chitosan (CS) remains a focus of study in recent years. A set of unique characteristics including hydrophilicity, remarkable biocompatibility and high concentration of amino groups, guides its applications in bioseparation and enzyme immobilization. In this thesis, CS-based porous organic-inorganic composite materials were prepared and their applications in enzyme immobilization and affinity chromatography were evaluated.
The type of chromatography for protein separation was described. The characteristics for matrices used for enzyme immobilization and bioseparation were summarized. The matrices usually used were also listed. The keys for the development of new matrices inculding non-specific interaction with protein, stability of protein on solid matrix, speed of bioseparation and functionality of materials, were described.
The preparation and application of CS-based organic-inorganic composite materials were reviewed. The methods for the preparation of porous CS materials were also summarized。
The preparation of new CS-silica porous organic-inorganic composite matrix (CTS-SiO_2) was prepared by coating cross-linked CS on silica. Porous structure could be formed on the coated layer using polyethylene glycol (PEG) as porogen. A new immobilized metal affinity chromatography (IMAC) matrix was prepared by coordinating Cu~(2+) with CTS-SiO_2 (Cu-CTS-SiO_2). Effects of CS and PEG content in coating solution on the surface morphology and protein adsorption capacity of Cu-CTS-SiO_2 were investigated. The CTS-SiO_2 prepared using 2% CS and 10% PEG 20000 in coating solution, 1 mol L~(-1) epichlorohydrin as cross-linking reagent, possessed macroporous surface. The non-specific protein adsorption was investigated using BSA as model protein. Results showed that CTS-SiO_2 possessed functional surface, low non-specific interaction with BSA, stability in both alkali and acid solution.
The application of CTS-SiO_2 in enzyme immobilization was investigated. CTS-SiO_2 was easily activated with the functional group of epoxy, diazo and aldehyde respectively. Trypsin was covalently immobilized on the three activated matrices. The characteristics of the immobilized trypsin were investigated in comparison with free enzyme. The results were explained and discussed by analyzing the protein structure such as surface exposure rate of amino acid residues related to enzyme coupling. Results showed that trypsin immobilized via all employed methods could tolerate relatively tough environmental conditions, such as high temperature and wide pH range. The immobilized trypsin was obviously stable in long time storage. The best results were obtained when trypsin was immobilized via epoxy activation, which might be ascribed to the multi-point attachment (MPA) between enzyme and the support.
The application of CTS-SiO_2 in affinity chromatography was investigated. BSA adsorption to Cu-CTS-SiO_2 was conducted for elucidating the optimal pH, adsorption kinetics and adsorption isotherm. The adsorption of BSA reached the maximum at pH 6.0. The macroporous morphology of Cu-CTS-SiO_2 could significantly affect the rate of adsorption toward BSA. Adsorptive isotherm of BSA on Cu-CTS-SiO_2 could be fitted to the Langmuir isotherm equation. A crude BSA sample was purified on the IMAC column packed with Cu-CTS-SiO_2. When CTS-SiO_2 was activated epoxy groups, trypsin was covalently coupled as a bio-ligand for capturing typsin inhibitor (TIs). The separation of TIs from egg white by column packing with trypsin-immobilized bead could be achieved.
The preparation of porous CS/silica composite membrane supported by microfiltration nylon membrane was developed. Gamma-glycidoxypropyltrimethoxysilane (GPTMS) was used as an inorganic source as well as crosslinking reagent. Polyethylene glycol (PEG) with different molecular weight and content was used as porogen for morphology control. Results showed that the molecular weight and content of PEG had remarkable effect on the surface morphology of the conposite membrane. A special porous surface with 3-D hierarchical structure-in-structure pore fashion was obtained when content of PEG 20000 was controlled at 15%. The CS component in the composite materials could directly coordinate with Cu~(2+). The immobilized metal affinity membrane could effectively adsorb BSA. As expected, the affinity membrane imprinted with 15% PEG 20000 had remarkably high copper ion binding and BSA adsorption capacity, which might result from its large surface area, high ligand density and suitable interconnected 3-D hierarchical porous surface.
引文
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