Architecture And Performance Of Bio-mimeticly Immobilized Enzyme On Mesoporous Silica

Enzyme immobilization has attracted continuous attention due to its widespread application in industrial organic synthesis, medical diagnosis, energy development and environmental engineering. To harmonize the conflict between activity retention and firm immobilization is a main challenge in enzyme immobilization. Mesoporous silicate (MPS) materials have been recognized as energetic hosts for protein adsorption due to their high surface area, tunable nano-sized pores, tailorable surface property, and good mechanical stability. High performance of mesoporous bioreactor by shrinking pore openings has been achieved in our precious research. However, there are still some key problems to be solved in enzyme immobilization with MPS such as determination of protein distribution, application in macrosubstrate and continuous process. This thesis aims at these problems above and the main innovative research findings and conclusions include:1. To make clear of the diffusion and distribution of enzyme in mesoporous materials, confocal laser scanning microscopy (CLSM) and X-ray diffraction (XRD) observations in combination with hindered diffusion simulation, intra-particle diffusion analysis, and apparent kinetics calculation have been carried out. The smallest and best pore sizes for PPL are revealed. The hindered diffusion simulation and CLSM observation indicates protein distribution seems more uniform in the larger pore. Besides pore diffusion, boundary resistance is revealed by the intra-particle diffusion model and the PPL amount adsorbed on the inner or external surface are further calculated. The PPL amount adsorbed in the pores, in accordant with XRD result, indicates the existence of confinement, which is further confirmed by kinetics calculation.2. A strategy based on pH-triggered release of enzyme from mesopores, lysis of bacterial cells and recovery has been presented to overcome the limitation of mesoporous materials-support enzyme in macrosubstrate. By controlling pH-sensitive interfacial interactions, lysozymes are effectively released from SBA-15, display full bacteriolytic activity and are almost completely recovered. CLSM and XRD observations give the direct evidence of the adsorption-readsorption recycle. The results have also demonstrated that substrate-enhanced enzyme desorption, privileged enzyme recovery, and the maintenance of secondary structure ensure the high activity and recycle efficiency.3. To achieve continuous manipulation of immobilized enzyme applied in catalysis, structured bio-catalysts are presented by synthesis of mesoporous silica in anodic aluminum oxide (AAO), which combines the advantage of mesoporous silica as host of enzyme and the membrane structure of AAO. The study shows that AAO-silica hybrid with 3D cubic, circular hexagonal or columnar hexagonal mesostructure is superior in enzyme uptake to AAO, and can be used in continuous mold.3D cubic mesostructure has highest permeability due to 3D connection and more pore opening. It also exhibit superiority in protein retention and activity expression. To avoid protein leaching in circular hexagonal mesostructure, a hierarchically-structured immobilized enzyme is prepared by further modification of AAO-silica hybrid. The immobilized enzyme demonstrates multi-functions of bio-membrane-like including size-selective permeability, bio-catalytic activity, continuous manipulation, and concurrent separation. Various enzymes can be encapsulated in the material with the protein elution inhibited and full bio-activity reserved. Excellent reusability has also been achieved.