Biocomposites of Enzymes and Covalent Organic Frameworks: A Novel Family of Heterogenous Biocatalysis

Abstract

Enzymatic catalysis is a green alternative to chemical catalysis, with high activity and selectivity toward the target products in very mild reaction conditions. However, the three-dimensional active structure of an enzyme is very fragile and highly sensitive to external variables such as temperature, pH, and chemical stressors, severely limiting the application range of natural enzymes. A viable solution is to immobilize enzymes within solid porous matrices. Among the most recently developed porous materials are covalent organic frameworks (COFs). They hold great potential as enzyme carriers, as they are nontoxic, light, and highly porous crystalline polymers. Unlike metal-organic frameworks, COFs do not carry a risk of any potential metal-ion leakage, and they offer long-term water/chemical stability. COFs exhibit larger surface areas and variety in their structural/chemical design compared to other conventional supports, like silica or polymers. In this Review, we offer for the first time a comprehensive overview of all the synthetic methods created so far for biocomposites of enzymes and COFs, together with an in-depth discussion of their design principles. We then focus on the critical synthetic parameters that may affect the chemistry of the resultant biocomposites, which find applications in biocatalysis, photoenzymatic catalysis, biosensing, chiral resolution, and stimuli-responsive release. The review ends by discussing the challenges and future opportunities related to the immobilization methods as well as the practical applications. We hope that this Review might guide researchers in developing more advanced encapsulation strategies to boost the enzymatic performance in real-world applications.

Keywords: Catalysis; Chiral Resolution; Covalent Organic Frameworks; Enzyme Immobilization; Immobilization Mechanisms; Sensing.

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Some covalent bonds for building COFs and schematic representation of imine-linked COF formation by reversible reaction.

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Timeline of remarkable progress for the formation of enzyme@COF biocomposites.

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Schematic diagram of post-synthetic immobilization for the synthesis of enzyme@COF biocomposites.

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Synthesis of enzyme@COF biocomposites via physical adsorption. (a) Proposed mechanism of the synthesis of hollow COF-DhaTab and a schematic representation of trypsin adsorption in COF-DhaTab. Reproduced with permission from ref . Copyright 2015, Springer Nature. (b) Schematic representation of the encapsulation of large-sized enzymes within TDCOFs. Reproduced with permission from ref . Copyright 2022, American Chemical Society. (c) Illustration of the immobilization of lipase within the modulator-mediated 3D COF HFPTP-TAE. Reproduced with permission from ref . Copyright 2023, American Chemical Society. (d) (i) Graphic view of lipase PS and porous materials used for lipase PS immobilization. (ii) Enzyme uptake comparison of various biocomposites. (iii) Catalytic kinetics of lipase embedded in different matrices. Reproduced with permission from ref . Copyright 2018, American Chemical Society. (e) (i) Synthetic scheme of COF-ETTA-EDDA and a graphic view of the AA-stacking mode of a dual-pore structure. (ii) Catalytic performance comparison of free lipase PS and lipase PS embedded in different COF skeletons. Reproduced with permission from ref . Copyright 2019, Wiley-VCH.

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(a) Schematic illustration of the synthesis of COFETTA-TPAL and the encapsulation of both GOD and MP-11 within the dual pores. Reproduced with permission from ref . Copyright 2019, Elsevier. (b) Schematic representation of the hierarchical TpAzo-COF foam synthesis using and in situ CO₂ effervescence technique, and a graphic view of enzyme immobilization. Reproduced with permission from ref . Copyright 2023, Royal Society of Chemistry.

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Reaction schemes showing the strategies to covalently immobilize enzymes in COFs. EDC = 1-ethyl-3-(3-(dimethyl­amino)­propyl)­carbo­diimide, NHS = N-hydroxy­succin­imide, E = enzyme.

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Schematic illustration of EDC and EDC/NHS-mediated coupling protocol. Sulfo-NHS = N-hydroxysulfo­succinimide sodium salt.

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Synthesis of enzyme@COF biocomposites via covalent attachment. (a) Schematic illustration of the covalent attachment of biomolecules within COFs and insights into the covalent strategy. Reproduced with permission from ref . Copyright 2018, Wiley-VCH. (b) Schematic representation of the synthetic route to H-COF-OMe and the procedure for covalent attachment of laccase. Reproduced with permission from ref . Copyright 2021, Elsevier. (c) Covalent immobilization of Cyt c into the confined channels of COFs. Reproduced with permission from ref . Copyright 2022, Wiley-VCH.

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Schematic illustration of glutaraldehyde-mediated covalent coupling.

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Schematic illustration of enzyme immobilization via multipoint covalent attachment on epoxy-activated COFs.

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Schematic diagram of the formation of PPF-2 and the strategies for CALB immobilization. Reproduced with permission from ref . Copyright 2019, Wiley-VCH.

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Schematic illustration of template-guided and in situ encapsulation strategies for the synthesis of enzyme@COF biocomposites.

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Template-guided methods for the fabrication of enzyme@COF biocomposites. (a) Schematic representation of the synthetic route to an enzyme@COF-42-B biocomposite using a sacrificial templating strategy. Reproduced with permission from ref . Copyright 2020, American Chemical Society. (b) Formation of porous COF microcapsules with a cross-linked shell (CALB@COF-MCs-SH) based on Pickering emulsion. Reproduced with permission from ref . Copyright 2023, Wiley-VCH.

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Synthesis of enzyme@COF biocomposites via various in situ immobilization strategies. (a) Synthesis diagram and formation mechanism of trypsin@COF-LZU1 capsules. Reproduced with permission from ref . Copyright 2022, American Chemical Society. (b) Schematic illustration of the in situ assembly process of lipase@NKCOF-98. Reproduced with permission from ref . Copyright 2022, Wiley-VCH. (c) Schematic depicting a one-pot synthesis of HRP-incorporated COFs. Reproduced with permission from ref . Copyright 2022, Wiley-VCH. (d) (i) Schematic showing in situ encapsulation of PVP-modified enzyme within COFs. (ii) Structure of PVP and a schematic showing its attraction of COF monomers. Reproduced with permission from ref . Copyright 2022, Elsevier.

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(a) Schematic representation of the mechanochemistry-induced assembly of enzyme@COFs. Reproduced with permission from ref . Copyright 2022, Elsevier. (b) Schematic diagram of a pre-protection strategy to immobilize enzyme into COFs via a self-repairing and crystallization process. Reproduced with permission from ref . Copyright 2023, American Chemical Society. (c) Diagram of the in situ encapsulation process of enzyme@COF-TAPT-TFB. Reproduced with permission from ref . Copyright 2023, American Chemical Society. (d) Schematic diagram of ionic liquid-mediated dynamic polymerization for one-step formation of highly crystalline enzyme@COFs. Reproduced with permission from ref Copyright 2024, Wiley-VCH.

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Schematic illustration of enzyme immobilization within a monopore-structured COF or a hierarchical COF and a graphical view of the effects on substrate diffusion.

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(a) (i) Structural formulas of NKCOF-98 and its analogs. (ii) Hydrophobic angles and relative activity of various lipase@COFs systems for the hydrolysis of 4-nitrophenyl acetate (p-NPA). Reproduced with permission from ref . Copyright 2022, Wiley-VCH. (b) (i) Graphical view of the structure of the COF materials. (ii) EPR spectra of all spin-labeled sites after association with COF-OMe, COF-OH, and COF-ONa, respectively; digits represent the surface spin sites of labeled T4L. (iii) Comparison of EPR spectra with theoretical models. Reproduced with permission from ref . Copyright 2019, Elsevier.

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(a) Hydrolysis of aspirin methyl ester (AME). (i) Conversion rates of free lipase (1), lipase@NKCOF-98 (2), lipase@NKCOF-99 (3), lipase@NKCOF-100 (4), lipase@MCM-41 (5), and lipase@ZIF-8 (6). (ii) Recyclability of lipase@NKCOF-98 and -99 for AME hydrolysis reactions. (iii) Conversion rates of various hydrolysis substrates. Reproduced with permission from ref . Copyright 2022, Wiley-VCH. (b) (i) Schematic illustration of the one-pot hydrolysis of carboxymethylcellulose (CMC) to glucose. (ii) Comparison of CMC conversion efficiency under various catalyst combinations. (iii) Retained conversion efficiency of the one-pot glucose synthesis during the recycling process. Reproduced with permission from ref . Copyright 2023, Royal Society of Chemistry. (c) (i) Esterification reaction of 1-hexanol and hexanoic acid catalyzed by CALB@COF-MCs-SH. (ii) Conversion yields of hexyl hexanoate catalyzed by CALB@COF-MCs-SH, CALB@COF-MCs, free CALB, COF-MCs, and COF-MCs-SH. (iii) Specific activities of free CALB, CALB@COF-MCs, and CALB@COF-MCs-SH within the first 10 min. Reproduced with permission from ref . Copyright 2023, Wiley-VCH.

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(a) (i) Control experiments on the photoenzymatic asymmetric catalysis of 2-phenylindole catalyzed by WGL@NKCOF-118­(Zn) using visible light irradiation. (ii) Catalytic tolerance of WGL@NKCOF-118­(Zn) to various 2-phenylindole derivatives. Reproduced with permission from ref . Copyright 2022, American Chemical Society. (b) (i) Schematic diagram of the synthesis of COPF-I1/2 and COF-V1/2 with similar topologies but different linkage units. (ii) Comparison of the specific activity of photoenzymatic reduction of CO₂. Reproduced with permission from ref . Copyright 2024, American Chemical Society.

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(a) (i) Differential pulse voltammetry (DPV) response curves of the proposed ratiometric electrochemical biosensor (0–3 mM). (ii) Selectivity of the proposed biosensor. Reproduced with permission from ref . Copyright 2019, Elsevier. (b) (i) Amperometric i–t curves for (a–g) 0, 2.5 × 10², 2.5 × 10³, 2.5 × 10⁴, 2.5 × 10⁶, and 2.5 × 10⁷ particles/μL. (ii) Clinical sample analyses using this proposed method (***, p < 0.001). Reproduced with permission from ref . Copyright 2023, Elsevier. (c) (i) Colorimetric response to different amounts of glucose; inset photo indicates the color changes at different glucose concentrations. (ii) Selectivity and anti-interference ability of the COF_HD-GOx-based biosensor. Reproduced with permission from ref . Copyright 2021, Royal Society of Chemistry.

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(a) Chiral catalysis of R,S-1-phenethanol. (i) Conversion rates of free lipase, lipase@NKCOF-99, and lipase@COF-98 in12 h. (ii) HPLC results of NJCOF-99 in different catalytic windows. (iii) Conversion rates toward various chiral resolution substrates. Reproduced with permission from ref . Copyright 2022, Wiley-VCH. (b) Schematic illustration of lysozyme⊂COF 1-based CSPs for chiral resolution and enantiomeric separation chromatograms of various racemates, including dl-threonine, dl-leucine, dl-tryptonphan, Ofloxacin, Metoprolol, and chlorpheniramine. Reproduced with permission from ref . Copyright 2018, Wiley-VCH.

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(a) Schematic illustration of the FITC-PEG-COF-based glucose and pH dual-responsive insulin delivery release system. Reproduced with permission from ref . Copyright 2021, Wiley-VCH. (b) Schematic diagram of the reversible protonation of the imine bond and illustration of the adsorption and release of lysozyme uptake. Reproduced with permission from ref . Copyright 2021, Elsevier.