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Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes

Nature Protocols volume 18, pages 1641–1657 (2023)Cite this article

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Abstract

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1–24 h.

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Fig. 1: Two examples of isocyanide-based multi-component reactions.
Fig. 2: Carrier-bound covalent immobilization of enzymes by using ICMRs.
Fig. 3: Cross-linking of enzymes by using ICMRs.
Fig. 4: A simple activity assay for lipases.
Fig. 5: ABTS oxidation by laccase.
Fig. 6: Immobilization of RML on multi-wall carbon nanotubes.
Fig. 7: The preparation of CLEs of TLL.
Fig. 8: The formation of CLEs of RML.

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Data availability

The main data supporting the findings of this study are present in the paper. All other data supporting the findings of this study are available from the corresponding author upon request. No datasets or custom code was generated in this study.

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Acknowledgements

This work was financially supported by National Institute of Genetic Engineering and Biotechnology of Iran (grants 672 and 587), by a grant from PhosAgro/UNESCO/IUPAC partnership program (4500283719-A1) and by a grant from the Iran National Science Foundation (INSF) (95830819).

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Author notes

  1. These authors contributed equally: Mehdi Mohammadi, Mansour Shahedi.

Authors and Affiliations

  1. Bioprocess Engineering Department, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

    Mehdi Mohammadi & Faezeh Ahrari

  2. Department of Pure Chemistry, Faculty of Chemistry, Shahid Beheshti University, G.C., Tehran, Iran

    Mansour Shahedi, Faezeh Ahrari, Mostafa Mostafavi & Zohreh Habibi

  3. Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

    Maryam Yousefi

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Contributions

M. Mohammadi and M.Y. conceived the project. M. Mohammadi and M.S. wrote the manuscript with support from M.Y., Z.H., M. Mostafavi and F.A. F.A. performed the experiments. M. Mohammadi, Z.H., M.Y. and M.S. analyzed the data. M. Mohammadi and M.Y. supervised the project. M. Mostafavi provided the figures.

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Correspondence to Mehdi Mohammadi or Maryam Yousefi.

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Nature Protocols thanks Rénato Froidevaux and J. C. S. dos Santos for their contribution to the peer review of this work.

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Key references using this protocol

Mohammadi, M. et al. RSC Adv. 5, 32698–32705 (2015): https://doi.org/10.1039/C5RA03299G

Mohammadi, M. et al. RSC Adv. 6, 52838–52849 (2016): https://doi.org/10.1039/C6RA11284F

Ashjari, M. et al. Chem. Commun. 56, 9683–9686 (2020): https://doi.org/10.1039/D0CC03429K

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Mohammadi, M., Shahedi, M., Ahrari, F. et al. Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes. Nat Protoc 18, 1641–1657 (2023). https://doi.org/10.1038/s41596-023-00812-z

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