Markedly Increased Activity of Lysozyme Adsorbed on SBA-15 Silica with Controlled Nanopore Diameter
With their high surface area and well-controlled pore morphology, nanostructured mesoporous materials are increasingly considered as supports for enzymes, for applications in industrial catalysis, membranes and sensing. While research has mainly focused on these applications, our fundamental understanding of the local effects of a support on an enzyme remains unclear. This, in turn, is important for the rational design of immobilized enzymes for targeted applications. Answering the question of how the local curvature of nanostructured materials influences enzyme activity is the objective of our research.
Here we report how the local curvature of mesoporous materials affects the diffusion and adsorption of lysozyme, and its catalytic activity, when it is immobilized on mesoporous supports. Lysozyme is a test molecule in our fundamental study, because it is almost spherical and many data have been reported in the literature. As mesoporous supports, we synthesized a series of SBA-15s, which are silicas with a well-defined, hexagonal array of parallel mesopores of the same pore diameter. The pore diameter of SBA-15s was varied between 5 and 12 nm by changing the synthesis conditions. Given the similar dimensions of the pores and lysozyme (approximately 3.0 X 3.0 X 4.5 nm3), confinement effects may occur. We measured the kinetics and the thermodynamics of lysozyme adsorption on this series of SBA-15 experimentally, and compared the results to model calculations, as a function of pore size and surface area.
The surface of mesoporous materials might change the conformation of the attached molecules, and their activity. When lysozyme was immobilize on to the internal surface of SBA-15, whose local curvature changed with the pore diameter, we demonstrated a dramatic increase in enzyme activity, for experiments with a substrate (4-Methylumbelliferyl-N,N',N''-triacetyl-Beta-chitotrioside, MW=787.75 g/mol) small enough to enter the pores. However, the activity when using whole cells (Micrococcus lysodeikticus, D=0.8µm), which cannot enter the nanopores, dropped to almost zero, proving that the enzyme is indeed mostly adsorbed on the internal surface, and that it is this enzyme which is responsible for the activity increase in the first experiments. Results with external adsorption on nanoparticles and nanotubes by others [1, 2] had indicated a constant or dropped activity, rather than an increase. This suggests remarkable confinement effects in nanoporous media, induced by geometrical and/or chemical effects, which further studies on other supports and with different substrates will help to elucidate.
References:
[1] Alexey A. Vertegel, Richard W. Siegel, Jonathan S. Dordick; Silica Nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme; Langmuir 2004, 20, 6800-6807
[2] Hao-Min Ding, Lei Shao, Run-Jing Liu, Qing-Gui Xiao, Jian-Feng Chen; Silica nanotubes for lysozyme immobilization; J. Coll. Inter. Sci. 2005, 290(1), 102-106