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Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworks

Nature Protocols volume 18, pages 2032–2050 (2023)Cite this article

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Abstract

Enzymes are outstanding natural catalysts with exquisite 3D structures, initiating countless life-sustaining biotransformations in living systems. The flexible structure of an enzyme, however, is highly susceptible to non-physiological environments, which greatly limits its large-scale industrial applications. Seeking suitable supports to immobilize fragile enzymes is one of the most efficient routes to ameliorate the stability problem. This protocol imparts a new bottom-up strategy for enzyme encapsulation using a hydrogen-bonded organic framework (HOF-101). In short, the surface residues of the enzyme can trigger the nucleation of HOF-101 around its surface through the hydrogen-bonded biointerface. As a result, a series of enzymes with different surface chemistries are able to be encapsulated within a highly crystalline HOF-101 scaffold, which has long-range ordered mesochannels. The details of experimental procedures are described in this protocol, which involve the encapsulating method, characterizations of materials and biocatalytic performance tests. Compared with other immobilization methods, this enzyme-triggering HOF-101 encapsulation is easy to operate and affords higher loading efficiency. The formed HOF-101 scaffold has an unambiguous structure and well-arranged mesochannels, favoring mass transfer and understanding of the biocatalytic process. It takes ~13.5 h for successful synthesis of enzyme-encapsulated HOF-101, 3–4 d for characterizations of materials and ~4 h for the biocatalytic performance tests. In addition, no specific expertise is necessary for the preparation of this biocomposite, although the high-resolution imaging requires a low-electron-dose microscope technology. This protocol can provide a useful methodology to efficiently encapsulate enzymes and design biocatalytic HOF materials.

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Fig. 1: The molecular tecton of H4TBAPy.
Fig. 2: Schematic diagram of the in situ encapsulation method.
Fig. 3: The procedure for in situ growth of HOF-101 onto an enzyme.
Fig. 4: The Vacuum, High Tension and Aperture interface.
Fig. 5: The Autoloader interface.
Fig. 6: The enzyme-triggered in situ growth of HOF-101.
Fig. 7: PXRD and FT-IR characterization.
Fig. 8: Nitrogen isotherm characterization.
Fig. 9: The spatial distribution of RhB-enzyme (RhB-Cyt c) within HOF-101.
Fig. 10: The microstructure of enzyme (Cyt c)@HOF-101.
Fig. 11: The bioactivity and recyclability.

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Data availability

The main data discussed in this protocol are available within our primary research papers (refs. 18,21) and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We acknowledge financial support from projects of the National Natural Science Foundation of China (22174164, to G.C.; 22104159, to S.H.; 21904146, to G.C.).

Author information

Authors and Affiliations

  1. MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, China

    Guosheng Chen & Gangfeng Ouyang

  2. School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou, China

    Siming Huang

  3. Cryo-EM Center, Southern University of Science and Technology, Shenzhen, China

    Xiaomin Ma

  4. School of Chemical Engineering and Technology, Sun Yat-sen University, Zhuhai, China

    Rongwei He & Gangfeng Ouyang

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Contributions

G.C. designed the research. G.C. and G.O. directed the project. G.C. organized the in situ encapsulation and material characterizations. G.C. and S.H. organized the tables and the figures. X.M. organized the text about the cryo-EM characterization. R.H. participated in the discussion and helped with the data analysis. G.C. and G.O. wrote the manuscript.

Corresponding authors

Correspondence to Guosheng Chen or Gangfeng Ouyang.

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Nature Protocols thanks Kang Liang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Chen, G. et al. Chem 7, 2722–2742 (2021): https://doi.org/10.1016/j.chempr.2021.07.003

Chen, G. et al. Nat. Commun. 13, 4816 (2022): https://doi.org/10.1038/s41467-022-32454-2

Ye, N. et al. Anal. Chem. 93, 13981−13989 (2021): https://doi.org/10.1021/acs.analchem.1c03381

Extended data

Extended Data Fig. 1 In situ encapsulation of HRP within HOF-101.

Snapshots of HRP@HOF-101 sediment after assembly (a) and the dried product (b). c, Cryo-EM image of HRP@HOF-101. d, The nitrogen isotherms of standard HOF-101 and HRP@HOF-101, respectively. e, The catalytic kinetics of HRP, HRP@HOF-101 and HRP@ZIF-8. The HRP dosages in each group are the same (0.4 μg/ml). The H2O2 concentration is 0.1 mM. ΔAbs, change in absorbance. a–c and e adapted with permission from ref. 18, Elsevier.

Source data

Supplementary information

Supplementary Information

Supplementary Method 1 and Tables 1 and 2

Source data

Source Data Fig. 7

Unprocessed PXRD and FT-IR data

Source Data Fig. 8

Unprocessed N2 isotherm data for Cyt c@HOF-101

Source Data Extended Data Fig. 1d

Unprocessed N2 isotherm and bioactivity data for HRP@HOF-101

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Chen, G., Huang, S., Ma, X. et al. Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworks. Nat Protoc 18, 2032–2050 (2023). https://doi.org/10.1038/s41596-023-00828-5

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