Abstract
Enzymes are natural catalysts with high catalytic activity, substrate specificity and selectivity. Their widespread utilization in industrial applications is limited by their sensitivity to harsh reaction conditions and difficulties relating to their removal and re-use after the reaction is complete. These limitations can be addressed by immobilizing the enzymes in solid porous supports. Covalent organic frameworks (COFs) are ideal candidate carriers because of their good biocompatibility, long-term water stability and large surface area. In post-synthetic immobilization, the enzyme is added to an existing COF; this has had limited success because of enzyme leaching and pore blockage by enzymes that are too large. Direct-immobilization methods—building the COF around the enzyme—allow tailored incorporation of proteins of any size and result in materials with lower levels of leaching and better mass transport of reactants and products. This protocol describes direct-immobilization methods that can be used to fabricate enzyme@COF (@ = engulfing) biocomposites with rationally programmed structures and functions. If COF construction requires harsh reaction conditions, the enzyme can be protected by using a removable metal-organic framework. Alternatively, a direct in situ approach, in which the enzyme and the COF monomers assemble under very mild conditions, can be used. Examples of both approaches are described: enzyme@COF-42-B/43-B capsules (enzymes including catalase, glucose oxidase, etc.) with ZIF-90 or ZPF-2 as protectors, and lipase@NKCOF-98/99 via in situ direct-immobilization methods (synthesis timing: 30–100 min). Example assays for physical and functional characterization of the COF and enzyme@COF materials are also described.
Key points
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Covalent organic frameworks (COFs) protect enzymes from the harsh reaction conditions required for organic synthetic chemistry. If there is no enzyme leaching and pore blockage, enzyme removal and reuse are possible.
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Direct in situ assembly of enzyme@COFs works for COFs that form under very mild reaction conditions. In cases in which harsher conditions are required, a sacrificial metal-organic framework can be constructed to protect the enzyme.
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Data availability
The main data discussed in this protocol are available within the figures and the Supplementary Information. Additional data that support the findings of this study can be obtained from the corresponding author on request.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (22022808).
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Q.Z. and Y.Z. contributed equally. All authors contributed to the development of the protocol and the design of the experiments.
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Key references using this protocol
Li, M. et al. J. Am. Chem. Soc. 142, 6675–6681 (2020): https://doi.org/10.1021/jacs.0c00285
Zheng, Y. et al. Angew. Chem. Int. Ed. Engl. 61, e202208744 (2022): https://doi.org/10.1002/anie.202208744
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Supplementary Figs. 1–22 and Table 1
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Zhu, Q., Zheng, Y., Zhang, Z. et al. Enzyme immobilization on covalent organic framework supports. Nat Protoc 18, 3080–3125 (2023). https://doi.org/10.1038/s41596-023-00868-x
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DOI: https://doi.org/10.1038/s41596-023-00868-x
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