(502f) Dramatically Increased Enzymatic Activity of Lysozyme and Myoglobin Adsorbed On the Internal Surface of SBA-15 Mesoporous Silica

When protein molecules are strongly adsorbed on a surface, their activity in catalyzing chemical conversions can be severely altered with respect to free protein molecules in solution, depending on the geometrical and chemical structure of the surface. Local surface curvature plays an important role. For example, on silica nanoparticles and carbon nanotubes, effects of convex surface curvature on protein stability and activity were noted [1,2]. If proteins are similar in size to the pore diameter of mesoporous materials, adsorption on their surface leads to confinement effects that could also alter their activity. Several researchers indeed noted significant, sometimes stabilizing, effects of concave surface curvature [3], although a systematic study requires accurate control over particle morphology and size, and pore morphology and diameter.

By using a series of monodisperse SBA-15 mesoporous silica particles with high surface area, and carefully controlled, unique pore diameter in the range of 5-11 nm, in which large amounts of proteins are strongly adsorbed from buffered solutions, effects of local curvature and protein-surface interactions could be studied in a consistent way. It was verified by multiple washes with buffered solution that the protein would not leach out of the particles. We focused on the activity and stability of two different proteins ? lysozyme and myoglobin ? after adsorption on SBA-15 with different pore sizes. Lysozyme is an ellipsoidal protein, with dimensions equal to 3nm × 3nm × 4.5nm and myoglobin is a globular protein, with dimensions equal to 3nm × 4nm × 5nm. Effects of the local pore curvature of SBA-15 on the protein structure of the adsorbed protein were studied by comparing the activity and stability of adsorbed and free enzyme, for two representative test reactions including substrates that could diffuse through the pores. The fluorescent and chromogenic products of the test reactions for each case were followed using spectrofluorometry and UV/VIS spectroscopy.

The specific activity of adsorbed lysozyme in pores with a diameter around 5.7 nm pore size was 3 times higher, and in pores of 10.5 nm diameter 2.5 times higher than that of free lysozyme in solution. For adsorbed myoglobin, the activity even increased by a factor of 9 in SBA-15 with pores of 5.7 nm diameter, and a factor of 6 when the pore size was 9 nm, again compared to the free protein. This suggests that the active conformation of each protein has been greatly stabilized by confinement on the concave pore surface of SBA-15. This stabilization is most pronounced in mesoporous SBA-15 with a pore size close to the dimensions of the protein, but leaving room for reactants and products to enter and leave the pores. Heat treatment of protein/SBA-15 samples also showed that the stability of the adsorbed protein was protected.

These results show that the activity and stability of proteins can be significantly enhanced by confining the protein in a mesoporous support with a pore size similar to the protein size. The concave, negative pore curvature of SBA-15 has a remarkable stabilizing effect, higher than that noted for protein adsorption on silica nanoparticles [1] or carbon nanotubes [2], which have a convex, positive surface curvature.

References:

[1]. A.A. Vertegel, R.W. Siegel and J.S. Dordick. Silica nanoparticles size influences the structure and enzymatic activity of adsorbed lysozyme. Langmuir 20, 6800-6807 (2004).

[2]. N.R. Palwai, D.E. Martyn, L.F.F. Neves, Y. Tan, D.E. Resasco and R.G. Harrison. Retention of biological activity and near-infrared absorbance upon adsorption of horseradish peroxidase on single-walled carbon nanotubes. Nanotechnology 18, 235601 (2007).

[3]. H.H.P. Yiu, and P.A. Wright. Enzymes supported on ordered mesoporous solids: a special case of an inorganic-organic hybrid. J. Mater. Chem. 15, 3690-3700 (2005).