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

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Department of Chemistry, Lancaster University, Lancaster LA1 4YB, United Kingdom
WestCHEM, Department of Pure & Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, United Kingdom
§ Department of Chemistry, University of Basel, CH-4056 Basel, Switzerland
Cite this: J. Phys. Chem. C 2016, 120, 50, 28717–28726
Publication Date (Web):November 25, 2016
Copyright © 2016 American Chemical Society

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    Abstract Image

    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.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.6b11575.

    • SDS-PAGE of uniformly 15N-labeled hCA II-S50C-C206S before and after subsequent purification steps; deconvoluted ESI-MS spectra of uniformly 15N-labeled hCA II-S50C-C206S after TCEP reduction; plot of the rate of product formation at increasing concentration of hCA II; one-dimensional 29Si CPMAS spectra of silica, epoxy–silica, and hCA II immobilized on epoxy–silica; one-dimensional 15N CPMAS NMR spectra of [U-15N]/hCA II and [15N Leu]/hCA II before and after immobilization on epoxy–silica (PDF)

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    Cited By

    This article is cited by 14 publications.

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    8. Juan M. Bolivar, Bernd Nidetzky. On the relationship between structure and catalytic effectiveness in solid surface-immobilized enzymes: Advances in methodology and the quest for a single-molecule perspective. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2020, 1868 (2) , 140333.
    9. Linda Cerofolini, Enrico Ravera, Marco Fragai, Claudio Luchinat. NMR of Immobilized Enzymes. 2020, 363-383.
    10. Jose M. Guisan, Fernando López-Gallego, Juan M. Bolivar, Javier Rocha-Martín, Gloria Fernandez-Lorente. One-Point Covalent Immobilization of Enzymes on Glyoxyl Agarose with Minimal Physico-Chemical Modification: Immobilized “Native Enzymes”. 2020, 83-92.
    11. Daniel Joss, Roché M. Walliser, Kaspar Zimmermann, Daniel Häussinger. Conformationally locked lanthanide chelating tags for convenient pseudocontact shift protein nuclear magnetic resonance spectroscopy. Journal of Biomolecular NMR 2018, 72 (1-2) , 29-38.
    12. Makoto Yoshimoto, Peter Walde. Immobilized carbonic anhydrase: preparation, characteristics and biotechnological applications. World Journal of Microbiology and Biotechnology 2018, 34 (10)
    13. L. Dettori, F. Vibert, Y. Guiavarc'h, S. Delaunay, C. Humeau, J.L. Blin, I. Chevalot. N-α-acylation of lysine catalyzed by immobilized aminoacylases from Streptomyces ambofaciens in aqueous medium. Microporous and Mesoporous Materials 2018, 267 , 24-34.
    14. Sabu Varghese, Peter J. Halling, Daniel Häussinger, Stephen Wimperis. Two-dimensional 1H and 1H-detected NMR study of a heterogeneous biocatalyst using fast MAS at high magnetic fields. Solid State Nuclear Magnetic Resonance 2018, 92 , 7-11.

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