High-resolution structural characterization of a heterogeneous biocatalyst using solid-state NMR

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Varghese, Sabu and Halling, Peter J. and Haüssinger, Daniel and Wimperis, Stephen (2016) High-resolution structural characterization of a heterogeneous biocatalyst using solid-state NMR. Journal of Physical Chemistry C, 120 (50). pp. 28717-28726. ISSN 1932-7447 (https://doi.org/10.1021/acs.jpcc.6b11575)

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Abstract

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 15N and selectively (15N 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 1H, 13C, 15N, and 29Si 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.

ORCID iDs

Varghese, Sabu, Halling, Peter J. ORCID logoORCID: https://orcid.org/0000-0001-5077-4088, Haüssinger, Daniel and Wimperis, Stephen;
CORE (COnnecting REpositories)