Spatial Distribution of Enzymes Immobilized in Mesoporous Silicas for Biocatalysis

Abstract

Mesoporous silica (MPS) particles are promising materials for the immobilization of a variety of enzymes due to the possibility of tailoring their morphology and surface properties. A better understanding of the spatial distribution of enzymes immobilized in these materials is important for improved biocatalytic applications. In this work three types of MPS materials are compared for the immobilization of alcohol dehydrogenase (ADH): two ordered mesoporous silica type SBA-15 particles with 6.8 and 10 nm pore diameters and one mesostructured cellular foam (MCF) with a pore diameter of 26.8 nm and window diameter of 10.7 nm. Nitrogen sorption analysis and transmission electron microscopy (TEM) using immunogold staining (IGS) are used to study the immobilized amount and spatial distribution of ADH in the MPS. In the MCF the mesopore volume occupied by the enzymes measured by nitrogen sorption analysis agrees well with the calculated pore filling, which indicates that the amount of enzyme bound to the external surface is small as supported by TEM imaging showing an essentially even distribution in the MCF particles at least up to 2000 nm from the particle surface. In the case of SBA-15 with 6.8 nm pore size the ADH can enter the pores even though the pore size distribution is smaller than the hydrodynamic size of the protein, but the enzymes only penetrate to about 500 nm from the particle surface. With 10 nm pores the ADH penetrates further into SBA-15 but is still not evenly distributed. MCF can accommodate the largest amount of enzyme per gram of particles and still has a substantial fraction of its mesopore volume available, showing that MCF is a good host for the immobilization of large enzymes such as ADH.

Mesoporous Silica Particles as Drug Delivery Systems—The State of the Art in Loading Methods and the Recent Progress in Analytical Techniques for Monitoring These Processes

“…In the following studies, the technique was improved by the silver enhancement procedure, allowing the increase in the size of ultrasmall gold nanoparticles (GNPs), which enables the direct imaging of the absorbed conjugates [151,152]. Visualization and quantification of enzyme immobilized in MSNs have been facilitated by the combination of nitrogen sorption analysis and IGS-TEM, allowing direct imaging of guest distribution inside the particles [153]. An optical microscope uses visible light and a system of lenses to generate magnified images of the sample.…”

Direct Imaging of Protein Clusters in Metal–Organic Frameworks

“…Yet, SIM can only obtain a spatial resolution of ∼120 nm by mathematical deconvolution of interference patterns . Stochastic optical reconstruction microscopy (STORM) can achieve single molecule localization with a spatial resolution of 10–20 nm , and has assisted in revealing bionano-interactions and structure–functional relationships. − However, in order to apply STORM to investigate protein distribution patterns inside P@MOFs, fluorescent signals of internal molecules need to be well acquired and a data analysis method needs to be developed. Cluster analysis provides a possible solution for analyzing spatial coordinates generated by STORM to evaluate complex organizational patterns of proteins within the P@MOFs.…”

Fabrication of a Floatable Micron-Sized Enzyme Device Using Diatom Frustules

“…The use of porous materials is another prevalent approach. − Porous materials have larger surface areas than spheres or rods. The number of attached enzyme molecules can be dramatically increased because of the large surface area of porous materials.…”