Preparation And Application Of Porous Chitosan-Silica Composite Material

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-SiO₂) 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 Cu²⁺ with CTS-SiO₂ (Cu-CTS-SiO₂). Effects of CS and PEG content in coating solution on the surface morphology and protein adsorption capacity of Cu-CTS-SiO₂ were investigated. The CTS-SiO₂ 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-SiO₂ possessed functional surface, low non-specific interaction with BSA, stability in both alkali and acid solution.The application of CTS-SiO₂ in enzyme immobilization was investigated. CTS-SiO₂ 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-SiO₂ in affinity chromatography was investigated. BSA adsorption to Cu-CTS-SiO₂ 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-SiO₂ could significantly affect the rate of adsorption toward BSA. Adsorptive isotherm of BSA on Cu-CTS-SiO₂ could be fitted to the Langmuir isotherm equation. A crude BSA sample was purified on the IMAC column packed with Cu-CTS-SiO₂. When CTS-SiO₂ 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 Cu²⁺. 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.