Enzyme immobilization in mesoporous silica

University dissertation from Chalmers University of Technology

Abstract: Enzymes are highly effective and versatile biological catalysts, with a high chemo-, stereo- and regioselectivity while operating under mild conditions, such as physiological temperature and pH and atmospheric pressure. Therefore, enzymes are a sustainable alternative to conventional catalysts in organic synthesis processes. They are also used in detergent formulations, in biosensors for analytical and diagnostic purposes, and in biofuel cells. However, the use of enzymes in their native form often results in high costs, low operational stability and difficulties in recovery and reuse. By immobilization of enzymes to a solid support these limitations can be minimized. Mesoporous silica materials are a promising support for enzyme immobilization, offering a large surface area, a narrow pore size distribution and a high stability. Immobilization of enzymes in mesoporous silica has become a popular research topic, but despite the many techniques available, there is still a need for controlled strategies in order to adapt the support to the specific enzyme. It is crucial to better understand how the enzyme is affected by the microenvironment inside the pores and to gain more knowledge about the immobilization process. A major part of this work has been focused on finding suitable mesoporous silica particles customized for an optimal performance of each individual type of enzyme investigated. Enzymes of different character were immobilized in mesoporous silica particles with varied pore size, particle size and particle morphology through physical adsorption. The influence of these parameters on the loading capacity, catalytic activity and reusability of the immobilized enzymes was evaluated. A method to study the microenvironment inside mesoporous silica particles by covalently attaching a pH-probe (SNARF1) to proteins was developed. The results indicate that the immobilized proteins experience an environment inside the pores which is closer to neutral compared to the bulk solution. This approach can be used to characterize the pore environment without perturbing the properties of the material. The immobilization process has been studied directly with quartz crystal microbalance with dissipation monitoring (QCM-D) by attaching silica particles to the sensor. We have demonstrated that QCM-D is a simple and robust measuring technique for real time study of enzyme immobilization into mesoporous silica particles and that it is a useful complement to conventional indirect measuring techniques. With QCM-D we were also able to follow the co-immobilization of glucose oxidase (GOD) and horseradish peroxidase (HRP), which was done by immobilization of GOD in mesoporous silica particles followed by adsorption of HRP covalently linked to a polycationic dendronized polymer. A cascade reaction was confirmed with enzymatic activity analysis.

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