High-Resolution Structural Characterization of a Heterogeneous Biocatalyst Using Solid-State NMR
View Author Information
† 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
*E-mail: [email protected]
*E-mail: [email protected]
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.
Cited By
This article is cited by 14 publications.
- Wenjie Yang, Weibin Liang, Luke A. O’Dell, Hamish D. Toop, Natasha Maddigan, Xingmo Zhang, Alena Kochubei, Christian J. Doonan, Yijiao Jiang, Jun Huang. Insights into the Interaction between Immobilized Biocatalysts and Metal–Organic Frameworks: A Case Study of PCN-333. JACS Au 2021, 1
(12)
, 2172-2181. https://doi.org/10.1021/jacsau.1c00226
- Riley M. Bednar, Thaddeus W. Golbek, Kelsey M. Kean, Wesley J. Brown, Subhashis Jana, Joe E. Baio, P. Andrew Karplus, Ryan A. Mehl. Immobilization of Proteins with Controlled Load and Orientation. ACS Applied Materials & Interfaces 2019, 11
(40)
, 36391-36398. https://doi.org/10.1021/acsami.9b12746
- Ranjana Das, Renuka Ranjan, Neeraj Sinha, Arvind M. Kayastha. Comparative Characterization of Peanut β-Amylase Immobilization onto Graphene Oxide and Graphene Oxide Carbon Nanotubes by Solid-State NMR. The Journal of Physical Chemistry C 2018, 122
(33)
, 19259-19265. https://doi.org/10.1021/acs.jpcc.8b06219
- Ajit Pradhan Cesar Ovalles Michael Moir . Characterization of Heavy Petroleum Fractions by NMR Techniques. 2018, 73-86. https://doi.org/10.1021/bk-2018-1282.ch004
- Linda Cerofolini, Stefano Giuntini, Alexandra Louka, Enrico Ravera, Marco Fragai, and Claudio Luchinat . High-Resolution Solid-State NMR Characterization of Ligand Binding to a Protein Immobilized in a Silica Matrix. The Journal of Physical Chemistry B 2017, 121
(34)
, 8094-8101. https://doi.org/10.1021/acs.jpcb.7b05679
- Fangfang Yang, Pierre Rousselot‐Pailley, Cendrine Nicoletti, A. Jalila Simaan, Bruno Faure, Elise Courvoisier‐Dezord, Agnès Amouric, Yolande Charmasson, Rénal Backov, Thierry Tron, Yasmina Mekmouche. Controlled Immobilization of a Palladium Complex/Laccase Hybrid into a Macrocellular Siliceous Host. ChemPlusChem 2023, 88
(5)
https://doi.org/10.1002/cplu.202300156
- Linda Cerofolini, Marco Fragai, Claudio Luchinat, Enrico Ravera. Orientation of immobilized antigens on common surfaces by a simple computational model: Exposition of SARS-CoV-2 Spike protein RBD epitopes. Biophysical Chemistry 2020, 265 , 106441. https://doi.org/10.1016/j.bpc.2020.106441
- 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. https://doi.org/10.1016/j.bbapap.2019.140333
- Linda Cerofolini, Enrico Ravera, Marco Fragai, Claudio Luchinat. NMR of Immobilized Enzymes. 2020, 363-383. https://doi.org/10.1007/978-1-0716-0215-7_24
- 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. https://doi.org/10.1007/978-1-0716-0215-7_4
- 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. https://doi.org/10.1007/s10858-018-0203-4
- Makoto Yoshimoto, Peter Walde. Immobilized carbonic anhydrase: preparation, characteristics and biotechnological applications. World Journal of Microbiology and Biotechnology 2018, 34
(10)
https://doi.org/10.1007/s11274-018-2536-2
- 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. https://doi.org/10.1016/j.micromeso.2018.03.018
- 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. https://doi.org/10.1016/j.ssnmr.2018.03.003