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Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworks

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.

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.

References

  1. Thornberry, N. A. & Lazebink, Y. Caspases: enemies within. Science 281, 1312–1316 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Sheldon, R. A. & Woodley, J. M. Role of biocatalysis in sustainable chemistry. Chem. Rev. 118, 801–838 (2018).

    Article  CAS  PubMed  Google Scholar 

  4. Intasian, P. et al. Enzymes, in vivo biocatalysis, and metabolic engineering for enabling a circular economy and sustainability. Chem. Rev. 121, 10367–10451 (2021).

    Article  CAS  PubMed  Google Scholar 

  5. Devine, P. N. et al. Extending the application of biocatalysis to meet the challenges of drug development. Nat. Rev. Chem. 2, 409–421 (2018).

    Article  Google Scholar 

  6. Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Wong, L. S., Khan, F. & Micklefield, J. Selective covalent protein immobilization: strategies and applications. Chem. Rev. 109, 4025–4053 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Sheldon, R. A., Basso, A. & Brady, D. New frontiers in enzyme immobilisation: robust biocatalysts for a circular bio-based economy. Chem. Soc. Rev. 50, 5850–5862 (2021).

    Article  CAS  PubMed  Google Scholar 

  9. Huang, S., Chen, G. & Ouyang, G. Confining enzymes in porous organic frameworks: from synthetic strategy and characterization to healthcare applications. Chem. Soc. Rev. 51, 6824–6863 (2022).

    Article  CAS  PubMed  Google Scholar 

  10. Liang, W. et al. Metal–organic framework-based enzyme biocomposites. Chem. Rev. 121, 1077–1129 (2021).

    Article  CAS  PubMed  Google Scholar 

  11. Lian, X. et al. Enzyme–MOF (metal–organic framework) composites. Chem. Soc. Rev. 46, 3386–3401 (2017).

    Article  CAS  PubMed  Google Scholar 

  12. Wang, B., Lin, R.-B., Zhang, Z., Xiang, S. & Chen, B. Hydrogen-bonded organic frameworks as a tunable platform for functional materials. J. Am. Chem. Soc. 142, 14399–14416 (2020).

    Article  CAS  PubMed  Google Scholar 

  13. Li, P., Ryder, M. R. & Stoddart, J. F. Hydrogen-bonded organic frameworks: a rising class of porous molecular materials. Acc. Mater. Res. 1, 77–87 (2020).

    Article  CAS  Google Scholar 

  14. Song, X. et al. Design rules of hydrogen-bonded organic frameworks with high chemical and thermal stabilities. J. Am. Chem. Soc. 144, 10663–10687 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Lin, R.-B. & Chen, B. Hydrogen-bonded organic frameworks: chemistry and functions. Chem 8, 2114–2135 (2022).

    Article  CAS  Google Scholar 

  16. Liang, W. et al. Enzyme encapsulation in a porous hydrogen-bonded organic framework. J. Am. Chem. Soc. 141, 14298–14305 (2019).

    Article  CAS  PubMed  Google Scholar 

  17. Wied, P. et al. Combining a genetically engineered oxidase with hydrogen-bonded organic frameworks (HOFs) for highly efficient biocomposites. Angew. Chem. Int. Ed. 61, e202117345 (2022).

    Article  CAS  Google Scholar 

  18. Chen, G. et al. Protein-directed, hydrogen-bonded biohybrid framework. Chem 7, 2722–2742 (2021).

    Article  CAS  Google Scholar 

  19. Tang, Z. et al. A biocatalytic cascade in an ultrastable mesoporous hydrogen-bonded organic framework for point-of-care biosensing. Angew. Chem. Int. Ed. 60, 23608–23613 (2021).

    Article  CAS  Google Scholar 

  20. Tang, J. et al. In-situ encapsulation of protein into nanoscale hydrogen-bonded organic frameworks for intracellular biocatalysis. Angew. Chem. Int. Ed. 60, 22315–22321 (2021).

    Article  CAS  Google Scholar 

  21. Chen, G. et al. Hydrogen-bonded organic framework biomimetic entrapment allowing non-native biocatalytic activity in enzyme. Nat. Commun. 13, 4816 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Nelson, J. & Griffin, E. G. Adsorption of invertase. J. Am. Chem. Soc. 38, 1109–1115 (1916).

    Article  CAS  Google Scholar 

  23. Hartmann, M. & Kostrov, X. Immobilization of enzymes on porous silicas—benefits and challenges. Chem. Soc. Rev. 42, 6277–6289 (2013).

    Article  CAS  PubMed  Google Scholar 

  24. Pierre, A. C. The sol-gel encapsulation of enzymes. Biocatal. Biotransformation 22, 145–170 (2004).

    Article  CAS  Google Scholar 

  25. Furukawa, H., Cordova, K. E., O’Keeffe, M. & Yaghi, O. M. The chemistry and applications of metal-organic frameworks. Science 341, 1230444 (2013).

    Article  PubMed  Google Scholar 

  26. Cote, A. P. et al. Porous, crystalline, covalent organic frameworks. Science 310, 1166–1170 (2005).

    Article  CAS  PubMed  Google Scholar 

  27. Lykourinou, V. et al. Immobilization of MP-11 into a mesoporous metal–organic framework, MP-11@mesoMOF: a new platform for enzymatic catalysis. J. Am. Chem. Soc. 133, 10382–10385 (2011).

    Article  CAS  PubMed  Google Scholar 

  28. Li, P. et al. Nanosizing a metal–organic framework enzyme carrier for accelerating nerve agent hydrolysis. ACS Nano 10, 9174–9182 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Li, P. et al. Hierarchically engineered mesoporous metal-organic frameworks toward cell-free immobilized enzyme systems. Chem 4, 1022–1034 (2018).

    Article  CAS  Google Scholar 

  30. Sun, Q. et al. Pore environment control and enhanced performance of enzymes infiltrated in covalent organic frameworks. J. Am. Chem. Soc. 140, 984–992 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Lyu, F., Zhang, Y., Zare, R. N., Ge, J. & Liu, Z. One-pot synthesis of protein-embedded metal−organic frameworks with enhanced biological activities. Nano Lett. 14, 5761–5765 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Liang, K. et al. Biomimetic mineralization of metal-organic frameworks as protective coatings for biomacromolecules. Nat. Commun. 6, 7240 (2015).

    Article  CAS  PubMed  Google Scholar 

  33. Li, M. et al. Fabricating covalent organic framework capsules with commodious microenvironment for enzymes. J. Am. Chem. Soc. 142, 6675–6681 (2020).

    Article  CAS  PubMed  Google Scholar 

  34. Zheng, Y. et al. Green and scalable fabrication of high-performance biocatalysts using covalent organic frameworks as enzyme carriers. Angew. Chem. Int. Ed. 61, e202208744 (2022).

    Article  CAS  Google Scholar 

  35. Gao, R. et al. Mechanochemistry-guided reticular assembly for stabilizing enzymes with covalent organic frameworks. Cell Rep. Phys. Sci. 3, 101153 (2022).

    Article  CAS  Google Scholar 

  36. Hu, C. et al. Defect-induced activity enhancement of enzyme-encapsulated metal-organic frameworks revealed in microfluidic gradient mixing synthesis. Sci. Adv. 6, eaax5785 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Liang, W. et al. Enhanced activity of enzymes encapsulated in hydrophilic metal–organic frameworks. J. Am. Chem. Soc. 141, 2348–2355 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Tong, L. et al. Atomically unveiling the structure-activity relationship of biomacromolecule-metal-organic frameworks symbiotic crystal. Nat. Commun. 13, 951 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wu, X. et al. Packaging and delivering enzymes by amorphous metal-organic frameworks. Nat. Commun. 10, 5165 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Feng, Y. et al. Defect engineering of enzyme-embedded metal-organic frameworks for smart cargo release. Chem. Eng. J. 439, 135736 (2022).

    Article  CAS  Google Scholar 

  41. Wu, X. et al. A versatile competitive coordination strategy for tailoring bioactive zeolitic imidazolate framework composites. Small 17, e2007586 (2021).

    Article  PubMed  Google Scholar 

  42. Huang, W. et al. Photodynamic hydrogen-bonded biohybrid framework: a photobiocatalytic cascade nanoreactor for accelerating diabetic wound therapy. JACS Au 2, 2048–2058 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ye, N. et al. Hydrogen-bonded biohybrid framework-derived highly specific nanozymes for biomarker sensing. Anal. Chem. 93, 13981–13989 (2021).

    Article  CAS  PubMed  Google Scholar 

  44. Wijesundara, Y. H. et al. Carrier gas triggered controlled biolistic delivery of DNA and protein therapeutics from metal–organic frameworks. Chem. Sci. 13, 13803–13814 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Chen, T.-T., Yi, J.-T., Zhao, Y.-Y. & Chu, X. Biomineralized metal-organic framework nanoparticles enable intracellular delivery and endo-lysosomal release of native active proteins. J. Am. Chem. Soc. 140, 9912–9920 (2018).

    Article  CAS  PubMed  Google Scholar 

  46. Maddigan, N. K. et al. Protein surface functionalisation as a general strategy for facilitating biomimetic mineralisation of ZIF-8. Chem. Sci. 9, 4217–4223 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Shen, B. et al. A single-molecule van der Waals compass. Nature 592, 541–544 (2021).

    Article  CAS  PubMed  Google Scholar 

  48. Lazić, I. et al. Single-particle cryo-EM structures from iDPC–STEM at near-atomic resolution. Nat. Methods 19, 1126–1136 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Gao, L. et al. Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat. Nanotechnol. 2, 577–583 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Wei, H. & Wang, E. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem. Soc. Rev. 42, 6060–6093 (2013).

    Article  CAS  PubMed  Google Scholar 

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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

Authors

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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Competing interests

The authors declare no competing interests.

Peer review

Peer review information

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. ac 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 (2023). https://doi.org/10.1038/s41596-023-00828-5

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