High-Resolution Structural Characterization of a Heterogeneous Biocatalyst Using Solid-State NMR

Solid-state magic-angle spinning (MAS) NMR spectroscopy was employed to investigate structural detail in the enzyme human carbonic anhydrase II (hCA II) in uniformly N-15 and selectively (N-15 leucine) enriched states, covalently immobilized on epoxy-functionalized silica. The immobilized hCA II retained 71% of its specific enzymatic activity when compared to the free enzyme in solution. On the basis of the one- and two-dimensional H-1, C-13, N-15, and Si-29 MAS NMR. spectra, chemical shift assignments could be obtained from the silica support, covalent linker, and immobilized enzyme. The successful covalent immobilization of the enzyme on epoxy silica was confirmed by the appearance of signals from the aromatic and carbonyl groups in the immobilized enzyme in addition to signals from the modified support. Most notably, our MAS NMR results suggest that the covalent immobilization of the hCA II on epoxy silica does not significantly affect the structural integrity of the protein.