Activity and Stability of Nanoconfined Alpha-Amylase in Mesoporous Silica

Mesoporous silica particles (MSPs) have been studiedfor theirpotential therapeutic uses in controlling obesity and diabetes. Previousstudies have shown that the level of digestion of starch by & alpha;-amylaseis considerably reduced in the presence of MSPs, and it has been shownto be caused by the adsorption of & alpha;-amylase by MSPs. In thisstudy, we tested a hypothesis of enzymatic deactivation and measuredthe activity of & alpha;-amylase together with MSPs (SBA-15) usingcomparably small CNP-G3 (2-chloro-4-nitrophenyl alpha-d-maltotrioside)as a substrate. We showed that pore-incorporated & alpha;-amylase wasactive and displayed higher activity and stability compared to amylasein solution (the control). We attribute this to physical effects:the coadsorption of CNP-G3 on the MSPs and the relatively snug fitof the amylase in the pores. Biosorption in this article refers tothe process of removal or adsorption of & alpha;-amylase from its solutionphase into the same solution dispersed in, or adsorbed on, the MSPs.Large quantities of & alpha;-amylase were biosorbed (about 21% w/w)on the MSPs, and high values of the maximum reaction rate (V (max)) and the Michaelis-Menten constant(K (M)) were observed for the enzyme kinetics.These findings show that the reduced enzymatic activity for & alpha;-amylaseon MSP observed here and in earlier studies was related to the largeprobe (starch) being too large to adsorb in the pores, and potatostarch has indeed a hydrodynamic diameter much larger than the poresizes of MSPs. Further insights into the interactions and environmentsof the & alpha;-amylase inside the MSPs were provided by H-1 fast magic-angle spinning (MAS) nuclear magnetic resonance (NMR)and C-13/N-15 dynamic nuclear polarization MASNMR experiments. It could be concluded that the overall fold and solvationof the & alpha;-amylase inside the MSPs were nearly identical to thosein solution.