Covalent Organic Networks for in situ Entrapment of Enzymes with Superior Robustness and Durability

Herein, a crystalline CONs-based EnNBHs is constructed under mild conditions through an in situ entrapment strategy. The promoting effect of enzymes on the nucleation, growth, and crystallization of CONs is systematically investigated. The crystalline CONs serve as a shielding layer to protect enzymes from being denatured under inhospitable environments, thus enhancing the robustness and durability of enzymes. Meanwhile, the hierarchically porous CONs accelerate the diffusion of substrates to entrapped enzymes, finally enhancing the enzyme activity. • Covalent Organic Networks (CONs) is reported for in situ enzyme entrapment. • CONs offer a powerful platform to construct enzyme-based nanobiohybrids (EnNBHs). • Enzymes accelerate the growth of CONs 2-fold faster than that without enzymes. • CONs-based EnNBHs retain over 70% of initial activity under 60 o C incubation for120 min. • CONs-based EnNBHs exhibit over 80% of the original activity after 12 reaction cycles. Enzyme-based nanobiohybrids (EnNBHs) are an emerging biocatalyst family that can manufacture industrial products such as fuels and chemicals in a green and low-carbon manner. Designing high-performance EnNBHs could confer enzymes with superior robustness and durability, while current strategies confront grand challenges. Indeed, the reticular chemistry materials, especially metal-organic frameworks (MOFs), covalent organic frameworks (COFs), hydrogen-bond organic frameworks (HOFs), are several good candidates for constructing EnNBHs. While, MOFs and HOFs are constructed by coordination bond and hydrogen bond, respectively, which may be destroyed by acid or organic solvents, thus causing structural degradation and loss of protection to enzymes. The use of acetic acid (6 mol L -1 ) and organic solvents is conventional conditions for the synthesis of COFs, which may be inapplicable for de novo constructing EnNBHs. Herein, a facile and versatile in situ entrapment strategy is developed to entrap a series of enzymes in crystalline imine-based covalent organic networks (CONs) under mild conditions. Notably, the growth rate of CONs induced by glucose oxidase (GOx) is increased by 2 folds than that of CONs in the absence of GOx. Moreover, the crystalline CONs could create confinement environment and safeguard the hosted enzymes from being denatured under unfavorable conditions. Given moderate crystallinity of CONs, short-range ordered micro/meso-porous structures are generated. Compared with GOx@ZIFs, the larger transport channels for reactants/products in GOx@CONs result in 1.73-fold enhancement in the catalytic efficiency. The crystalline CONs could also serve as a cell-mimic nanoreactor for multienzyme catalysis, demonstrating the potential applications in biomanufacturing, bioimaging, biosensing and so on.