Biomimetic transmembrane transport of multi-enzymes to fabricate heterogeneous bioreactors with photoenzymatic reduction of CO_2

Domain: chemistry
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
The cascade of bio-/chemo-catalysis with quantitative integration and optimal activity is vital for catalytic conversion, but highly challenging. Here, we innovate a co-immobilization strategy by mimicking cellular transmembrane transport using ionic covalent organic framework capsules (iCOFcap) to co-encapsulate multi-enzymes and chemocatalysts. Thanks to the swellable polyethylene glycol and the adjustable charge of iCOFcap shells, multi-enzymes can be adsorbed onto the iCOFcap shells and then transported stepwise into the inner cavity of iCOFcap by adjusting electrostatic interaction between the enzymes and iCOFcap. Thus, the iCOFcap enables both transmembrane transport multi-enzymes and one-pot pre-encapsulation of chemocatalysts to construct various cascade catalysts, enabling quantitative integration and nearly complete retention of enzyme activity. The formed heterogeneous cascade catalysts show excellent comprehensive performance for CO2 reduction with good reusability. Thus, this study presents a co-encapsulation strategy for bio-/chemo-catalysis, providing an energy-efficient alternative for biotechnological applications.

Introduction
Green biomanufacturing with low energy consumption, high efficiency, and sustainability is a major transformation of manufacturing mode1. Enzymes, as the “core chip” of green biomanufacturing, are natural catalysts with good regio-, stereo-, and chemoselectivity, which offer great potential in various fields2,3. Actually, the practical biomanufacturing process usually involves several catalytic steps and may require the cascade of multiple biocatalysts (enzymes) and/or chemocatalysts4–7, e.g., the cascade of formate dehydrogenase (FDH) and glucose dehydrogenase (GDH) to achieve CO2 reduction8, the cascade of palladium and human carbonic anhydrase II to produce chiral alcohols9. However, the stable synergy of multiple enzymes under industrial conditions is challenging, complicated, and inefficient. In addition, for chemo-/bio- hybrid catalysis, the cascade systems usually possess low catalytic efficiency due to the unmatchable catalytic conditions for fragile enzymes and robust chemocatalysts. Co-immobilization of these catalysts on solid supports can be considered a convenient and effective way to address these limitations. Currently, reported enzyme carriers (e.g., sol–gel matrices, inorganic materials, adsorbent resin, and organic nanoparticles) mostly exhibit amorphous or nonporous structures, which unavoidably face issues with the mass transfer efficiency and the range of catalytic substrates. Their immobilization strategies, such as adsorption10,11, covalent bonding12, and in situ13, still have some limitations14,15. For instance, it is challenging to co-immobilize bio- and chemo-catalysts in controllable and precise proportions and locations, and to cascade these catalysts with excellent mass transfer performance for increasing catalytic performance. Moreover, unfavorable conditions (e.g., reactive agents, organic solvents, and high temperatures) during the immobilization process often led to reduced enzymatic activity. Thus, the development of biomanufacturing is in dire need of immobilization technology for constructing cascade catalyst systems, especially for a cascade of bio- and chemo-catalysts.

In nature, cells can achieve collaboration of multi-step intracellular reactions16,17 by the transmembrane transport of biomacromolecules (e.g., lipids, proteins) by endocytosis. Inspired by nature, the cascade immobilization of biomacromolecules can be achieved by mimicking transmembrane transport. Currently, flexible organic polymers are commonly used to mimic a phospholipid bilayer to simulate transmembrane transport of cells to achieve targeted delivery systems of biomacromolecules, which are usually used in living organisms18. However, the use of the transmembrane transport strategy for building the cascade immobilization platform and catalysis in vitro has not yet been reported due to the limitation of carrier stability. Covalent organic frameworks (COFs), a class of crystalline porous polymers, have emerged as ideal candidates for cascade immobilization because of their distinct advantages19, such as good stability, well-defined structures, high surface areas, and facile functionality. However, COFs are challenging to mimic transmembrane transport to immobilize enzymes due to their pore size and structural rigidity limitations. To address these limitations, hybridizing COFs with swellable polymers can be a feasible approach. The biocompatible polyethyleneglycol (PEG) polymer is widely used in diverse biomedical and biotechnological applications. PEG polymers exhibit swelling behavior in aqueous solutions, enabling them to imbibe biological fluids20,21, which have drawn considerable attention as carriers for transmembrane transport22. In previous reports23, our team pioneered a class of COFs, polymer-covalent-organic-frameworks (polyCOF), which uses linear PEGylate polymers as COF building blocks. PolyCOFs can combine the porosity, structural versatility, and chemical stability of COFs with PEG’s good mechanical properties and swelling behavior. Moreover, the presence of PEG polymers can promote the liquid–liquid phase separation (LLPS) to form stable droplets24,25, hence benefiting the formation of COF capsules. To promote the transmembrane transport of enzymes from the outer environment to the inner capsule, ionic COFs can be designed to adsorb enzymes by electrostatic interactions and transport enzymes by charge reversal via altering buffer pH.

Herein, we introduce a PEG-driven LLPS synthesis approach to produce ionic COF capsules (iCOFcap) that can achieve co-immobilization of multi-enzymes by mimicking cell transmembrane transport (Fig. 1). Due to the swellable PEG in the COF capsules, iCOFcap can swell upon adsorbing a buffer solution, thereby promoting the loading of enzymes into the COF shells by adjusting the electrostatic interaction between the enzymes and ionic COFs. Then, enzymes can be transported into the inner COF capsule cavity by altering the buffer pH, thereby changing the charge of the COF capsule shells (Fig. 1a). To construct cascade biocatalysts, chemocatalysts (e.g., electron mediator (Cp*Rh(bpy)H2O) and cofactors (NAD+)) can be pre-encapsulated by the COF capsules via a one-pot method and then transport multiple enzymes (e.g., FDH and GDH) stepwise into the COF capsule. The formed iCOFcap biocatalysts offer a feasible route for efficient photo- and multi-enzyme catalysis of CO2 reduction (Fig. 1b). Therefore, this study innovates an enzyme immobilization strategy and provides a versatile platform for multi-enzyme immobilization.

Results
Preparation and characterization of iCOFcap
To demonstrate the proof of concept, we designed and prepared a class of cationic COF capsules, iCOFcap-1, via condensation polymerization of BMTH (2,5-bis(2-methoxyethoxy)terephthalohydrazide), HPTMB (2-(2,5-di(hydrazinecarbonyl)phenoxy)-N,N,N-trimethylethan-1-aminium and), TB (1,3,5-triformylbenzene), and a PEGylated polymer additive, DTH-600 (M =~17800 g/mol)23, in an emulsion formed by water and chloroform with scandium trifluoromethanesulfonate (Sc(OTf)3) as a catalyst (Fig. 2a, Supplementary Figs. 1–3, and Supplementary Methods 1–4). The ratio of DTH-600:BMTH:HPTMB:TB was systematically studied (0:21:9:20; 1:20:9:20; 3:18:9:20; 5:16:9:20; 10:11:9:20) and profoundly impacted the reaction outcome (Supplementary Methods 5 and 6). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the morphology and particle size distribution of the iCOFcap-1 products. As shown in Fig. 2b and Supplementary Figs. 4 and 5, adding the PEGylated DTH-600 afforded dispersed high-quality iCOFcap-1 with hollow spherical morphology and smooth shell surfaces. It was consistent with our previous work in which PEGylated polymer additives improved the mechanical strength, processability, and flexibility of COF membranes23. The structure of iCOFcap-1 was identified by powder X-ray diffraction (PXRD). Among the ratios studied, 0:21:9:20; 1:20:9:20; 3:18:9:20; 5:16:9:20; 10:11:9:20 matched the characteristic PXRD peaks of COF-4223,26 (2θ=3.4°, 7.0°, and 26.9°, Fig. 2c). N2 sorption data of iCOFcap-1 (0:21:9:20; 1:20:9:20; 3:18:9:20; 5:16:9:20; 10:11:9:20) at 77 K revealed BET surface areas of 649 m2/g, 558 m2/g, 370 m2/g, 271 m2/g, and 41 m2/g, respectively (Fig. 2d). Pore size distribution, as calculated using the density functional theory (DFT) method, revealed intrinsic pores of 2.2 nm (COF-42), as well as a new pore of 1.2 nm, ascribed to the cross-linking effect of linear polymers (DTH-600) in iCOFcap-1 (Supplementary Fig. 6). It was found that PEGylated DTH-600 served as a droplet stabilizer and participated in the formation of iCOFcap-1. Fourier transform infrared spectra (FT-IR) showed the stretching modes at 2867 cm−1 in iCOFcap-1, which we attribute to PEGylated DTH-600 (Supplementary Fig. 7). FT-IR showed an increment of stretching modes at 1020 cm−1 in iCOFcap-1, attributed to HPTMB monomer (Fig. 2e). Increasing the amount of DTH-600 or HPTMB monomer decreased their crystallinity and surface area because these bulky monomers could block the COF pores (Supplementary Figs. 6 and 8). It should be noted that adding HPTMB could dramatically change the charge of the COF capsule. The zeta potentials of neutral COF capsules without HPTMB were negative in PB buffer solution (pH 7.4), while iCOFcap-1 was positively charged with the increase of positive monomer (Fig. 2f). Similarly, an anionic COF capsule, iCOFcap-2, was fabricated using SDHS (sodium 2-(2,5-di(hydrazinecarbonyl)phenoxy)ethane-1-sulfonate) instead of HPTMB (Fig. 2a). The iCOFcap-2 (0:21:9:20; 1:20:9:20; 3:18:9:20; 5:16:9:20; 10:11:9:20) was illustrated as detailed in SI, the morphology and particle size distribution being studied by SEM (Supplementary Fig. 9) and TEM (Supplementary Fig. 10), BET surface areas by N2 sorption (Supplementary Figs. 11 and 12), crystallinity by PXRD (Supplementary Fig. 13). The zeta potential measurements revealed that iCOFcap-2 was negative due to the anionic SDHS monomer (Supplementary Fig. 14). Meanwhile, similar to iCOFcap-1, their porosity and crystallinity gradually decreased with the increase of SDHS monomer (Supplementary Fig. 15).

Preparation and characterization of enzyme◎iCOFcap-1
Lipase (Aspergillus niger lipase) was selected as a model enzyme to study enzyme encapsulation ability by iCOFcap-1 (Lipase◎iCOFcap-1) using the stepwise transmembrane transport method. It has been reported that the materials formed by PEG could imbibe a large number of biological fluids by swelling experiments22. The swelling ratio of iCOFcap-1 is a critical parameter for the enzyme entrapment capability. The PEGylated DTH-n was varied (n=400, 600, 800, representing the molecular weight of the PEG connector) and profoundly impacted the reaction outcome. Similar to DTH-600, The DTH-400 and DTH-800 monomers also formed iCOFcap-1 with good quality, verified by SEM (Supplementary Fig. 16), TEM (Supplementary Fig. 17), N2 sorption data (Supplementary Fig. 18), and PXRD patterns (Supplementary Fig. 19). The dynamic light scattering (DLS) results revealed that the swelling ratio of iCOFcap-1 (taking DTH-n:DTH:HPTMB:TB=5:16:9:20 as a representative) could be adjusted from 22.5 ± 2.8% to 47.9 ± 4.1% when changing the length of PEG connector, i.e., changing DTH-400 to DTH-800 (Fig. 3c and Supplementary Fig. 20). Meanwhile, the loading amount of lipase increased from 420 ± 20 mg g−1 to 620 ± 30 mg g−1 when changing DTH-400 to DTH-800, where the proportion of fixed lipase and lipase in the solution was 38 ± 5 to 52 ± 4% and 62 ± 5 to 48 ± 4%, respectively. The afforded lipase◎iCOFcap-1 showed good activities compared to the free enzymes (Supplementary Fig. 21 and Supplementary Method 7). Moreover, we also found that increasing the cationic charge (i.e., amount of cationic HPTMB monomer) of iCOFcap-1 could increase the loading speed and number of enzymes (Fig. 3d). The iCOFcap-1 maintained good crystallinity after transporting lipase (Supplementary Fig. 22). The pore size distribution results revealed no significant changes in the pore size of iCOFcap-1 prepared using the stepwise method before and after enzyme immobilization. This observation indicates that the enzymes are encapsulated within the COF capsule cavity rather than within the pore channels (Supplementary Fig. 23). The consistency of this phenomenon was further confirmed through comparative studies using a one-pot enzyme immobilization approach, where similarly unaltered pore sizes were observed post-immobilization (Supplementary Fig. 24). Meanwhile, there was no significant enzyme leakage for lipase◎iCOFcap-1 in pH=5.0/7.0 (Supplementary Figs. 25 and 26 and Supplementary Method 8). Overall, iCOFcap-1 with DTH-600 possessed a better swelling ratio, enzyme activity, loading amount, and COF structure. Thus, we selected 5:16:9:20 (DTH-600:DTH:HPTMB:TB) to produce enzyme◎iCOFcap-1 for the following studies.

The behaviors of enzymes loading and transporting into the capsule cavity were investigated by super-resolution imaging system (SIM) analysis (Supplementary Method 9). Figure 3a, b showed that lipase was first adsorbed by the shell of iCOFcap-1, then gradually transported into the capsule cavity by altering buffer pH. The capsular COF materials align structurally and functionally with the transmembrane dynamics of lamellar membranes, enabling analogous transport mechanisms. The iCOFcap-1 achieved 670 mg g−1 protein loading, declining to 600 mg g−1 with transporting 3 h and 560 mg g−1 with transporting 6 h, with ~16% outer-layer proteins released externally and ~84% inner-layer transported inward (Supplementary Fig. 27a). Thus, the reduction in total loading capacity serves as an indicator to track the progress of translocation. The transport process reached 65% completion within 3 h and achieved near completion (>95%) by 6 h, as evidenced by the nearly undetectable protein levels in the external solution (Supplementary Fig. 27b and Supplementary Method 10). Meanwhile, the confocal laser scanning microscope (CLSM) images of the neutral COF capsule (5:25:0:20) without adding ionic monomers showed that lipase could be loaded into the COF capsule shell but could not be transported into the capsule cavity under the same treatments (Supplementary Fig. 28). This phenomenon could be ascribed to the lack of electrostatic interaction between the negatively charged lipase and neutral COF capsule (5:25:0:20). Thus, lipase was difficult to release into the neutral COF capsule. We further proved the conclusion using iCOF film (a uniform and thickened version of iCOFcap-1 capsule wall) prepared via an interfacial polymerization method using identical monomers as iCOFcap-1 (Supplementary Methods 11 and 12). As shown in Fig. 3e, Supplementary Figs. 29–32 and Supplementary Method 13, the CLSM measurement revealed that the enzyme permeability of iCOF film (DTH-600:DTH:HPTMB:TB=5:16:9:20) was much higher than that of neutral COF film (DTH-600:DTH:HPTMB:TB=5:25:0:20). Zeta potential can quantify the electrostatic interactions between enzymes and carriers, thus enabling the prediction of protein affinity to the different carriers. The zeta potentials illustrated that iCOFcap-1 was positive in PB buffer solution, lipase with a negative charge, and the surface zeta potentials of the capsule decreased to 3.64 ± 0.7 mV after loading lipase and then increased gradually to 10.53 ± 0.9 mV with transporting into the capsule cavity (Supplementary Fig. 33a). Thus, zeta potentials can be used as an indicator to judge whether the enzyme has been transported into iCOFcap-1. As shown in Supplementary Fig. 33b, the Zeta potential analysis confirmed that the enzyme exhibited a negative charge at a pH of 7.0. Meanwhile, the COF capsule surface maintained a positive zeta potential, which drove successful enzyme adsorption onto the COF shell. Upon pH adjustment to 5.0, the protein underwent charge reversal to positive and has the same charge as the iCOFcap-1, causing the enzyme to break away from the COF shell and release it into the COF cavity. The reason the enzyme can be released is possibly because it is an amphoteric electrolyte. Furthermore, transporting HRP by iCOFcap-2 was illustrated as detailed in SI (Supplementary Method 14 and 15), with the swelling ratio being studied by DLS (Supplementary Fig. 34a), crystallinity by PXRD (Supplementary Fig. 34b), and the transporting process by CLSM (Supplementary Fig. 35). Meanwhile, there was no significant enzyme leakage for HRP◎iCOFcap-2 in pH=7.0/9.0 (Supplementary Figs. 36 and 37).

Cascade biocatalysis of the iCOFcap-1 bioreactor
Conversion of CO2 to formic acid was chosen as a representative reaction to evaluate the performance iCOFcap bioreactor (Fig. 4a). iCOFcap-1 was thereby used to construct a photoenzyme cascade reactor. To construct such a cascade biocatalyst, electron mediators (Cp*Rh(bpy)H2O) and NAD+) were first pre-encapsulated into the COF cap to generate RhCp*&NAD+◎iCOFcap-1 via a one-pot synthesis method. Then, FDH was transported into RhCp*&NAD+◎iCOFcap-1 using the stepwise transmembrane transport method (Fig. 4a and Supplementary Methods 16 and 17). To construct an optimal catalyst, the loading amount of Cp*Rh(bpy)H2O was first investigated. In a typical one-pot synthesis procedure, Cp*Rh(bpy)H2O was mixed with COF monomers (DTH-600, DTH, HPTMB, and TB) to form RhCp*◎iCOFcap-1 in one pot. Thereupon, RhCp* and NAD+ were co-immobilized into iCOFcap-1 to form RhCp*&NAD+◎iCOFcap-1 by a one-pot method. The XPS images supported the presence of NAD+ in RhCp*&NAD+◎iCOFcap-1 (Fig. 4c). The PXRD measurement showed that RhCp*◎iCOFcap-1 maintained the crystalline structure of iCOFcap-1 (Supplementary Fig. 38). Meanwhile, the BET surface area of RhCp*◎iCOFcap-1 had no significant decrease compared with that of iCOFcap-1 (Supplementary Fig. 39). As shown in Fig. 4b, Cp*Rh(bpy)H2O had indeed been encapsulated in iCOFcap-1 by TEM images and energy-dispersive spectroscopy (EDS) elemental mappings. X-ray photoelectron spectroscopy (XPS) can further probe the chemical composition of the photocatalysts (Supplementary Fig. 40). The existence of the Rh element in RhCp*◎iCOFcap-1 also indicated the successful incorporation of Rh into the capsule. A series of photocatalysis experiments was performed to optimize the visible-light-driven regeneration activity of NADH by the RhCp*◎iCOFcap-1. As shown in Fig. 4d, the wavelength-dependent evolution of NADH was also performed to gain the apparent quantum yield (AQY). The AQY of RhCp*◎iCOFcap-1 was higher than that of iCOFcap-1 under each wavelength, which was well-matched with the results of DRS. We adjusted the Cp*Rh(H2O) content from 2.2 to 11.2 wt% to investigate the influence of content on the photo-regeneration of NADH (Fig. 4e and Supplementary Methods 18 and 19). RhCp*◎iCOFcap-1 (the loading amount of Cp*Rh(H2O) was 9.9 wt%) was significantly effective for NADH photoregeneration, accumulating up to 78 ± 3% yield within 15 min, about 2.3 times higher than that of iCOFcap-1. Then, FDH was transported into RhCp*&NAD+◎iCOFcap-1 by the stepwise method. From the CLSM results (Supplementary Fig. 41), FITC-tagged FDH can be uniformly and firmly transported inside RhCp*&NAD+◎iCOFcap-1 using the stepwise method. For FDH, a load amount of 280 ± 10 mg g−1 afforded optimal activities of 93 ± 1% vs the respective free enzymes (Supplementary Fig. 42), where the proportion of fixed enzymes and enzymes in the solution was 70 ± 2% and 30 ± 2%, respectively. Before irradiation, the mixed system was placed in the dark for 1 h to ensure CO2 was fully dissolved in the system. After system optimization, FDH&RhCp*&NAD+◎iCOFcap-1 was able to generate 1.61 mmol h−1 g−1 formic acid from CO2 under visible light irradiation (Fig. 4f and Supplementary Fig. 43), almost 2 times higher than the iCOFcap-1 (one-pot integration of FDH, RhCp* and NAD+). Meanwhile, the product yield of FDH&RhCp*&NAD+◎iCOFcap-1 is significantly higher than that of corresponding free systems (Supplementary Fig. 44). FDH&RhCp*&NAD+◎iCOFcap-1 was ineffective in the absence of FDH or NAD+ (Supplementary Fig. 45). Due to the low NADH regeneration ability of iCOFcap-1, FDH&NAD+◎iCOFcap-1 had a low yield rate of formic acid. Importantly, this heterogeneous catalyst could be easily reused and achieved a productivity of >84% after ten cycles (Supplementary Fig. 46).

In practical applications, multiple enzymes are often required to cooperate with each other for advanced functions. However, it is difficult to quantify and regulate the rational proportion of multiple enzymes to boost catalytic performance using traditional enzyme immobilization strategies. Our strategy can achieve step-by-step enzyme transport to resolve limitations of quantification and regulation. To prove the concept of multi-enzyme cascade biocatalysis in COF capsules, FDH was first encapsulated in NAD+◎iCOFcap-1 capsule and then GDH (Fig. 5a). For FDH and GDH, load amounts were 280 mg g−1 and 200 mg g−1, respectively, which afforded optimal activities of 92% and 95% vs the respective free enzymes (Fig. 5c). By contrast, we also used the one-pot method to encapsulate FDH or GDH together into iCOFcap-1, and the loading amounts were 120 mg g−1 and 100 mg g−1, respectively (Supplementary Figs. 47–49 and Supplementary Methods 20–22). Obviously, the cascade catalysts fabricated by the transmembrane transport method showed much higher enzyme loading amount (>2.5 times or >2.0 times) and enzyme activity (>25% or >20%) compared with those using the one-pot encapsulating method. It should be noted that the one-pot method had some limitations, e.g., the COF crystallinity gradually decreased, and the morphology worsened with the increase of the enzyme amount, possibly due to the influence of the functional groups of enzymes (e.g., amino groups, carboxyl groups). To test for cascade reactions in iCOFcap-1, we encapsulated fluorescent-labeled FDH and GDH into iCOFcap-1. The CLSM images further verified the uniform dispersion of FDH and GDH in iCOFcap-1 capsules (Fig. 5b). The iCOFcap-1 retained good crystallinity after transporting FDH and GDH (Supplementary Fig. 50). The ratios of FDH:GDH in iCOFcap-1 were systematically studied (e.g., 1.0:0.5, 1.0:1.0, 1.0:2.0), revealing that FDH:GDH (1.0:1.0) showed the best performance (Fig. 5d). The product yield of FDH&GDH&NAD+◎iCOFcap-1 is significantly higher than that of corresponding free multi-enzyme systems (Supplementary Fig. 51). The product yield of FDH&GDH&NAD+◎iCOFcap-1 is significantly higher than that of FDH&NADH◎iCOFcap-1, which demonstrated the significance of the coenzyme regeneration system for continuous catalysis (Supplementary Fig. 52). Meanwhile, the loading amount of FDH+GDH by stepwise co-immobilization was higher than that by simultaneous co-immobilization, and the product yield of FDH+GDH by stepwise co-immobilization was significantly higher than that by simultaneous co-immobilization (Supplementary Fig. 53). This heterogeneous catalyst had good reusability and achieved a good productivity of >80% after ten cycles (Supplementary Fig. 54 and Supplementary Method 23).

A key feature of this co-immobilization strategy is that it can combine the one-pot pre-encapsulation method with stepwise transmembrane transport method to immobilize multiple enzymes and chemocatalysts into one capsule, especially for the systems in which different catalyst moieties have different optimal immobilization conditions (e.g., chemocatalyst’s best condition is in organic solvents and can be pre-encapsulated by the one-pot method, while enzyme’s best condition is in buffer solution and can be immobilized via the stepwise transmembrane transport method). In a word, the integration of the one-pot pre-encapsulation method and stepwise transmembrane transport method can help enzymes and chemocatalysts maintain their optimal activity. To demonstrate the effect of immobilization methods on catalysis efficiency, we synthesized FDH&GDH&NAD+◎iCOFcap-1 by different formulations. Formulation 1: Co-encapsulated FDH and NAD+ by the one-pot method and GDH by the stepwise method. Formulation 2: Co-encapsulated GDH and NAD+ by the one-pot method and FDH by the stepwise method. Formulation 3: Co-encapsulated FDH, GDH, and NAD+ by the one-pot method. Formulation 4: encapsulated NAD+ by the one-pot method and GDH and FDH by the stepwise method (Fig. 6a). As shown in Fig. 6b, the performance of FDH&GDH&NAD+◎iCOFcap-1 (Formulation 4) surpassed all other formulations. Notably, the comprehensive performance of FDH&GDH&NAD+◎iCOFcap-1 and FDH&RhCp*&NAD+◎iCOFcap-1 (HCOOH yield: 2.05 and 1.61 mmol h−1 g−1, cycle time: 100 and 100 h, relative yield: 84% and 80%, respectively) surpassed all reported heterogeneous platforms with photoenzyme or multi-enzyme cascade systems (Fig. 6c and Supplementary Table 1).

In summary, we innovate a multi-enzyme and chemocatalyst co-immobilization strategy by mimicking the cell transmembrane transport using ionic COF capsules (iCOFcap). A series of iCOFcap could be fabricated using PEGylated linear polymers and positive/negative monomers as structural building blocks promoted by the PEG-driven LLPS effect. The iCOFcap can efficiently load enzymes by swelling and then transport them into the capsule cavity by charge reversal. This stepwise enzyme-transporting process was visualized in situ by SIM analysis. A systematic investigation revealed that the capacity and rate of enzyme transport could be adjusted by altering the content of PEGylated polymer or ionic monomers. Notably, due to the mild operation conditions, enzymes could achieve nearly complete retention of enzyme activity. Meanwhile, multiple enzymes can be transported into iCOFcap-1 with controllable proportions. Combining this stepwise transmembrane transport method with the one-pot pre-encapsulation method could afford various types of cascade catalysts of multiple enzymes and chemocatalysts with different formulations. For instance, the iCOFcap can co-immobilize Cp*Rh(bpy)H2O and NAD+ via the one-pot method and then transport FDH into the capsule cavity to build a photoenzymatic system for CO2 reduction. Being heterogeneous catalysts, FDH&RhCp*&NAD+◎iCOFcap-1 was able to generate 1.61 mmol h−1 g−1 formic acid from CO2 under visible light irradiation and had facile recycling and recovery, and productivity of >84% after ten cycles. Overall, our multi-enzyme and photoenzymatic cascade catalytic system showed good comprehensive performance for CO2 conversion, superior to all reported heterogeneous catalysts using the same enzymes. This study innovates an immobilization strategy and provides an energy-efficient alternative for biotechnology applications.

Methods
Synthesis of iCOFcap-1/2 with different ratios of monomer
The DTH-400/600/800:BMTH:HPTMB/SDHS (0:21:9; 1:20:9; 3:18:9; 5:16:9; 10:11:9, 0.03 mmol) and water/chloroform mixture (V/V=1.0/7.5 mL/mL) were added into a 50 mL bottle and stirred at >600 rpm for 10 min. Then, TB (3.3 mg, 0.02 mmol) was ultrasonically dissolved in 1.0 mL chloroform and 2.0 mL acetonitrile. The TB solution and Sc(OTf)3 aqueous solution (70 μL, 1.0 g/mL) were added to the DTH-400/600/800:BMTH:HPTMB/SDHS mixture by drops and stirred at room temperature for 50 min. At the end of the reaction, the product was collected by centrifugation at 6000×g for 10 min, washed three times with methanol, and the faint yellow solid was obtained by filtration.

Synthesis of lipase◎iCOFcap-1
The freeze-dried iCOFcap-1 (5 mg) was added to the lipase solution (6.0 mg/mL, PB buffer, 50 mM, pH=7.0) for 6 h. And then, iCOFcap-1 loading lipase was added into PB buffer (50 mM, pH=5.0) for 6 h. The product was collected by centrifugation at 6000×g for 10 min, washed three times with DI water, and freeze-dried to isolate the solid. After the encapsulation and washing process, the supernatant of each operation was collected, and the concentration of protein was quantified by following the standard protocol of the Bradford assay, before which the calibration curve of lipase was fitted. The loading efficiency (LE%) and the loading capacity (LC, g·g−1) were calculated as follows:
LE% = (total enzyme − free enzyme in supernatant) / total enzyme × 100% (1)
LC = (quantified amount of enzyme loaded into materials) / (weight of materials) (2)

Synthesis of RhCp*&NAD+◎iCOFcap-1
The DTH-600:BMTH:HPTMB (5:16:9, 0.03 mmol), Cp*Rh(bpy)H2O (9.9 mg), NAD+ (5.0 mg) and water/chloroform mixture (V/V=1.0/7.5 mL/mL) were added into a 50 mL bottle and stirred at >600 rpm for 10 min. Then, TB (3.3 mg, 0.02 mmol) was ultrasonically dissolved in 1.0 mL chloroform and 2.0 mL acetonitrile. The TB solution and Sc(OTf)3 aqueous solution (70 μL, 1.0 g/mL) were added to the mixture by drops and stirred at room temperature for 50 min. At the end of the reaction, the solid was precipitated with 15 mL of methanol, and the faint yellow solid was obtained by filtration.

Synthesis of FDH&RhCp*&NAD+◎iCOFcap-1
The freeze-dried RhCp*&NAD+◎iCOFcap-1 (5 mg) was added to 2.0 mL FDH solution (1.0 mg/mL, PB buffer, 50 mM, pH=7.0) for 3 h. And then, iCOFcap-1 loading FDH was added into 2.0 mL PB buffer (50 mM, pH=5.0) for 6 h. The product was collected by centrifugation at 6000×g for 10 min, washed three times with DI water, and freeze-dried to isolate the solid. The supernatants were detected at different time points using the Bradford method and quantified by the standard curve of BSA.

Synthesis of FDH&GDH&NAD+◎iCOFcap-1
The freeze-dried NAD+◎iCOFcap-1 (5 mg) was added to the FDH solution (1.0 mg/mL, PB buffer, 50 mM, pH=7.0) for 3 h. And then, iCOFcap-1 loading FDH was added into the PB buffer (50 mM, pH=5.0) for 6 h. The product was collected by centrifugation at 6000×g for 10 min, washed three times with DI water, and freeze-dried to isolate the solid. The supernatants were detected at different time points using the Bradford method and quantified by the standard curve of BSA.

The freeze-dried FDH&NAD+◎iCOFcap-1 (5 mg) was added to the GDH solution (1.0 mg/mL, PB buffer, 50 mM, pH=7.0) for 3 h. And then, iCOFcap-1 loading GDH was added into the PB buffer (50 mM, pH=5.0) for 6 h. The product was collected by centrifugation at 6000×g for 10 min, washed three times with DI water, and freeze-dried to isolate the solid. The supernatants were detected at different time points with the Bradford method and quantified by the standard curve of BSA.

Photobiocatalytic conversion of CO2 into formic acid
Photobiocatalytic conversion of CO2 was conducted under 1 atm CO2 and 30 °C in a quartz reactor under visible light. The light could pass through the quartz window and reach the reaction solution. The reaction solution contains 5 mg integrated photocatalysts (containing NAD+ (1.0 mg), RhCp* (0.5 mg), and FDH (1.4 mg)), 5 wt% TEOA in 50 mL PB buffer (10 mM, pH 7.0) using a solar simulator (300 W Xe lamp, PLS-SXE300) outputting stable solar flux at 1.0 kW/m2 (1 sun). The supernatants were detected at different time points by ion chromatography (Thermo Fisher, ICS-1100).

Conversion of CO2 into formic acid by a multi-enzyme cascade
Multi-enzyme catalyzing conversion of CO2 was conducted under 1 atm CO2 and 30 °C in a quartz reactor. The reaction solution contains 5 mg integrated catalysts, 5 wt% TEOA in 50 mL PB buffer (10 mM, pH 7.0). The supernatants were detected at different time points by ion chromatography (Thermo Fisher, ICS-1100).

Statistics and reproducibility
No statistical methods were used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessments. All experiments were performed at least three times with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper presents a novel ionic COF capsule (iCOFcap) synthesized via polyethylene glycol-driven liquid-liquid phase separation (LLPS), simulating cellular transmembrane transport and establishing a platform for immobilizing chemical catalysts and enzyme cascades. The work demonstrates significant innovation and thorough research. However, some aspects require further clarification and stronger theoretical support. Below are some suggestions for improvement:\n1. Is the change in pore size sufficient evidence for DTH-600’s role in structural formation? Could it simply be adsorbed on the surface or between layers?\n2. The claim of \"monodispersity\" needs stronger evidence. Could additional TEM images showing dispersed materials in multiple fields of view be provided?\n3. Is the iCOFcap precipitate in membrane form? Please clarify the methods for measuring enzyme permeability and preparing iCOF and COF membranes. Since iCOF and COF membranes are multilayered, how does stepwise transmembrane transport occur on monodisperse materials? This requires further explanation.\n4. Could a SIM or LSCM video clip showing enzyme transmembrane transport into the carrier after charge reversal be provided?\n5. Please provide BET data and analysis after enzyme immobilization. Comparing the surface area and the pore size before and after immobilization using the one-pot method and the stepwise method could help clarify enzyme loading sites.\n6. How are chemical catalysts encapsulated into the carrier via the one-pot method? Relevant literature should be cited to support this.\n7. Lines 216-219: Does iCOFcap-1 function as a nanozyme? A clearer conclusion is needed.\n8. Lines 220-221: Should the XPS characterization of NAD+ in RhCp*&NAD +◎iCOFcap-1 be placed after Figure (b)?\n9. The enzyme activity assay is not clearly described. Please include potential reasons for enzyme activity loss.\n10. The staining method for Figure 4(b) requires clarification.\n11. Does the encapsulated enzyme leak when the pH of the reaction solution or environment changes?\n12. Is washing required during the synthesis of iCOFcap-1? If so, please include this step to ensure experimental rigor.", "reviewer_2": "The study conducted by Zhang, Chen, and co-workers focuses on the development of materials, specifically ionic capsules of covalent organic frameworks (iCOFs), designed to encapsulate enzymes and chemo-catalysts. The research primarily investigates the immobilization of formate dehydrogenase (FDH), which is employed as a biocatalyst for the reduction of CO2 to formic acid. The innovative aspect of this study lies in the encapsulation strategy of the enzyme. Unlike conventional methodologies, which typically employ a one-pot encapsulation approach, the material in this study is synthesized in a manner that mimics transmembrane transport. This ensures that the enzymes are encapsulated at a later stage, thereby preserving high enzymatic activity. Various monomers were tested to optimize capsule composition, aiming to maintain efficient enzymatic performance and a comparison between one pot and stepwise encapsulation wads made. In addition to FDH immobilization, the study also reports the encapsulation of RhCp within the structure, which was used as a photocatalyst for the regeneration of the NADH cofactor required for CO2 reduction. Alternatively, GDH was also employed as a strategy for cofactor regeneration.\nAs noted in the Supporting Information (SI), numerous studies in literature have already reported the co-immobilization of FDH and GDH for CO2 reduction to formic acid. Nevertheless, the immobilization methodology presented is indeed innovative and the data analysis is highly detailed, comprehensive, and compelling. Beyond this, several aspects require further clarification to allow for a conclusive evaluation.\n- Figure S19 refers to the leakage of lipase protein, but the methodology used to assess this parameter is not mentioned. Could further details be provided?\n- In lines 224–225 and 251–252, the residual activity of immobilized enzymes is compared to their free counterparts. How was this evaluation performed?\n- The co-immobilization of FDH and GDH within the same capsule is conducted in separate steps. Has the possibility of preparing a single starting solution containing both FDH and GDH been considered, allowing for their simultaneous immobilization? Would this approach affect the reaction yield compared to sequential encapsulation?\n- Could you clarify the rationale behind selecting DHT-600 over DHT-400 and DHT-800? The study states that DHT-600 provides superior properties in terms of relative activity, enzyme loading, and COF structure. However, Figure S16(a) indicates that DHT-800 exhibits the highest enzyme loading. Maybe it would be better to say that the choice primarily based on achieving a balance among swelling ratio, loading amount, COF structure, and enzyme activity?\n- The title refers to a \"boosted\" CO2 reduction. Is this truly the case? A comparison with the reaction yield using the free enzyme is missing, making it difficult to substantiate the claim that immobilization enhances reaction performance. It would be beneficial to report these values to enable a direct comparison. Additionally, it would be advisable to evaluate the reaction yield in a system containing NADH without any regeneration process. This would serve as a control, allowing for a better understanding of the maximum conversion achievable under the conditions studied.\nOverall, the study presents a significant advancement in enzyme immobilization strategies. However, addressing these points would strengthen the validity of the findings and further substantiate the claims made in the article.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The manuscript by Zhu et al. describes a co-encapsulation strategy of two enzymes, formate dehydrogenase (FDH), for CO2 reduction to formate, and glucose dehydrogenase (GDH) for recycling NADH, in ionic covalent organic framework capsules (named iCOFcap). By introducing polyethylene glycol (PEG) polymers and adjusting the charge of COF capsule shells they achieve the stepwise transport of the two proteins inside the capsule by what is named “transmembrane transport”. The iCOFcap were previously loaded with pre-encapsulated NAD+ (cofactor for FDH) and Cp*Rh(bpy)H2O (to reform NADH) and used for photocatalytic CO2 reduction.\nThe whole concept of enzyme integration into COFcap capsules lacks novelty as it was recently reported by the same team for the reduction of N2 using co-encapsulatation of GluDH, GCDH, ω-TA, Au NPs and NAD+, in a slightly different set up (DOI: 10.1038/s41467-025-56214-0). Furthermore, I found that the experimental work is not properly described and there is lack of adequate controls.\nSeveral points need to be addressed:\n1. How is the enzyme loading amount (Fig. 2d) and loading efficiency into the capsules quantified? This is not described.\nWe are shown SIM images of isolated capsules, but no information on the efficiency of the process. What relative proportion of the FDH enzyme (and lipase) is incorporated and how much remains in solution? How much of the total activity is left in solution after preparing the enzyme-loaded capsules? Apparently, possible enzyme left in solution is not separated from the loaded capsules, and can thus contribute to the reaction.\n2. What is the enzyme permeability (Fig. 2e)? How is this determined? Also how is leakage (Fig. S19) quantified? This is not described.\n3. Line 166/167: The pH was not altered over 6h. Should rephrase.\n4. The reason for the physical process of enzyme incorporation inside the capsules should be better described (what is called “transmembrane transport”)\n5. Line 224-225: How are the 93% activity versus the free enzyme quantified? How is the activity quantified in the capsules? This is not described and these results are not shown.\n6. What are the reaction volumes used for the synthesis of FDH&RhCp*&NAD+@iCOFcap-1?\n7. What is the light source and intensity used in the photocatalytic assays with the FDH&RhCp*&NAD+@iCOFcap-1 systems? Also not described.\n8. What does it mean that 5 mg integrated photocatalysts are present in the solution?? This is vague. Should describe the amount of each component. How much enzyme is present?\n9. Figure 3: The results of formate production by the FDH&RhCp*&NAD+@iCOFcap-1 need be compared with the system when the enzyme and co-catalysts are free in solution, so the effect of enzyme encapsulation can be properly assessed. This is a critical control that is missing.\n10. Is there a meaning in the use of two colours for the graphs in figs 2e, 3f, 4c and 4d?\n11. For the experiments in Fig. 5b, why is there no effect for the transport of the isolated enzymes (formulations 2 and 3), when this was observed in the previous studies?\n12. Figs S31 and S32 – give reaction conditions, such as volume, amount of catalysts, …\n13. Fig. S33 – describe what recycling experiment means\n14. In the comparison with literature values the authors are omitting systems studying metal-dependent Fdhs where much higher photocatalytic activities have been reported (eg. up to 600 mmol.gh-1.g-1 of formate, DOI: 10.1002/anie.201814419). These should be included.\nOverall, I do not see a reason to justify publishing this study in a broad journal like Nature Communications. It should go to a specialized journal.\nOther points:\n- The title of the manuscript is not accurate, as the chemocatalysts were not integrated through transmembrane transport, but by direct initial incorporation\n- The sentence “In nature, cells can achieve precision catalysis by confinement effect and collaboration of multi-step intracellular reactions by the transmembrane transport of biomacromolecules (e.g., lipids, proteins) by endocytosis.” is misleading and simply not correct! While the first part is true (confinement effect in intracellular catalysis), this catalysis does not require “transmembrane transport of biomacromolecules” or endocytosis. Should be corrected."}, "author_response": "Response: We appreciate the positive support of the reviewer. Per the suggestion of reviewers, we have made modifications and improvements to the overall content of the manuscript.\nQ1. Is the change in pore size sufficient evidence for DTH-600's role in structural formation? Could it simply be adsorbed on the surface or between layers?\nResponse: We thank the reviewer for the suggestion. Due to the cross-linking effect of DTH-600 in iCOFcap-1, the pores of ∼2.2 nm gradually disappeared, and new pores centered at ∼1.2 nm appeared (Figure S3). This result is consistent with our previous work (ACS Cent. Sci. 2019, 5, 1352). If DTH-600 is only adsorbed on the surface sites or interlayer positions of iCOFcap-1, the pore sizes will not change. Per the suggestion, we have added the FT-IR data in the revised manuscript to prove the DTH-600 monomer existing in the iCOFcap-1 (Figure S4). The band at ~2867 cm-1 can be attributed to the presence of PEG moieties.\nNankai University\nFigure S3. Pore size distribution of iCOFcap-1 (DTH-600) with different ratios calculated from the N sorption data using the DFT method.\nQ2. The claim of \"monodispersity\" needs stronger evidence. Could additional TEM images showing dispersed materials in multiple fields of view be provided?\nResponse: We thank the reviewer for the suggestion. Per the suggestion, to avoid misunderstanding, we have changed the term “monodispersity” to “dispersed” in Line 108. We\nNankai University\nhave added TEM images to the manuscript to illustrate the dispersed capsules (Figure S2). Furthermore, the SEM images also demonstrated the dispersibility of the material (Figure S6).\nFigure S2. TEM images of iCOFcap-1 (DTH-600:BMTH:HPTMB:TB = 5:16:9:20) with different monomer ratios, Scale bar: 500 nm.\nFigure S6. SEM images of iCOFcap-2 (DTH-600) with different monomers, Scale bar: 500\nnm.\nQ3. Is the iCOFcap precipitate in membrane form?\nResponse: We thank the reviewer for the comment. The iCOFcap forms a dispersed hollow COF capsule rather than a membrane form.\nQ4. Please clarify the methods for measuring enzyme permeability and preparing iCOF and COF membranes.\nResponse: We thank the reviewer for the suggestion. The preparing iCOF and COF membranes have been described in SI (page S6). Per the suggestion, we have added the methods for measuring enzyme permeability in SI (page S7). The measuring enzyme permeability iCOF and COF membranes is as follows: “The enzymatic permeability of iCOF and COF membranes\nNankai University\nwas quantified through confocal laser scanning microscopy (CLSM) analysis of enzyme infiltration depth, combined with membrane thickness measurements. And the enzymatic permeability of iCOF and COF films was calculated using the following equation:\nEnzyme infiltration depth\nEnzyme Permeability % = × 100 %\nMembrane thickness\nQ5. Since iCOF and COF membranes are multilayered, how does stepwise transmembrane transport occur on monodisperse materials? This requires further explanation.\nResponse: We thank the reviewer for the suggestion. The COF material facilitates enzyme transport across the COF shells through a unique mechanism. Due to the swellable PEG monomer in the COF capsules, iCOFcap can swell upon adsorbing a buffer solution, thereby promoting the loading of enzymes into the COF shells by adjusting the electrostatic interaction between enzymes and ionic COFs. Then, enzymes can be transported into the inner COF capsule cavity by altering the buffer pH. This explanation was emphasized in the revised manuscript.\nQ6. Could a SIM or LSCM video clip showing enzyme transmembrane transport into the carrier after charge reversal be provided?\nResponse: We thank the reviewer for the suggestion. The confocal laser scanning microscope (LCSM, LSM 800 with Airyscan) and Super-resolution imaging system (SIM, Elyra 7) in enzyme localization have a limited spatial resolution (typically ~120 nm of XY resolution and ~300 nm Z resolution), making it difficult to distinguish whether proteins are embedded in the capsule shell or distributed within the internal cavity. This resolution barrier prevents the precise localization of proteins at the submicron scale.\nSIM can realize the processing function of single-molecule fluorescence localization microscope images and calculate the position of fluorescent molecules according to the intensity of the light spot, which relies on post-acquisition computational processing (e.g., Z-stack reconstruction or intensity profiling) to generate static 2D/3D maps of protein distribution. Although this method provides finer spatial details, it captures only fixed-timepoint snapshots and cannot dynamically visualize real-time protein transport (e.g., movement between the shell and cavity over hours) or generate a continuous “video” of spatial redistribution over time.\nNankai University\nTo resolve the ambiguity in protein localization and indirectly infer transport dynamics, we implemented the following strategy: we performed 3D SIM imaging at multiple time points (e.g., t = 0, loading 3 hours, transporting 3 and 6 hours) during the interaction between proteins and capsules (Figure 2a and b). This allowed us to reconstruct the 3D spatial distribution of proteins at each stage and observe progressive shifts in their localization. A gradual increase in cavity-localized protein fluorescence across time points supports load or transport mechanisms.\nQ7. Please provide BET data and analysis after enzyme immobilization. Comparing the surface area and the pore size before and after immobilization using the one-pot method and the stepwise method could help clarify enzyme loading sites.\nResponse: We thank the reviewer for the suggestion. As shown in Figure S20, BET data and analysis of the iCOFcap-1 using the stepwise method before and after enzyme immobilization revealed no significant changes in pore size. This observation demonstrates that the enzymes are encapsulated within the COF capsule cavity rather than adsorbed in the pore channels. The consistency of this phenomenon was further confirmed through comparative studies using a one-pot enzyme immobilization approach, where similarly unaltered pore sizes were observed post-immobilization. Per the suggestion, we have added the BET data of the iCOFcap-1 using the one-pot method before and after enzyme immobilization in the SI (Figure S21).\nFigure S20. N sorption isotherms (a) and pore size distribution (b) of iCOFcap-1 and Lipase\n2\n◎iCOFcap-1 using stepwise methods.\nNankai University\nFigure S21. N sorption isotherms (a) and pore size distribution (b) of iCOFcap-1 and Lipase\n2\n◎iCOFcap-1 using one-pot methods.\nQ8. How are chemical catalysts encapsulated into the carrier via the one-pot method? Relevant literature should be cited to support this.\nResponse: We thank the reviewer for the suggestion. The chemical catalysts (Cp*Rh(bpy)H O)) 2 were pre-synthesized and then added with DTH-600, BMTH, HPTMB and TB to form RhCp* ◎iCOFcap-1. Relevant literature is as follows: (1) The article (Appl. Catal. B: Environ. 2020, 272, 119035) reported Au nanocrystals are incorporated into COFs using O/W emulsion technique via the one-pot method. (2) The article (J. Am. Chem. Soc. 2022, 144, 4168–4177) reported Cp*Rh(bpy)H O are incorporated into thylakoid membrane-inspired capsule (TMC) 2 via the one-pot method. These papers have been added in the reference part.\nQ9. Lines 216-219: Does iCOFcap-1 function as a nanozyme? A clearer conclusion is needed.\nResponse: We thank the reviewer for the suggestion. No, iCOFcap-1 did not function as a nanozyme due to a lack of catalytic sites, while the RhCp*◎iCOFcap-1 composite demonstrates intrinsic nanozyme functionality due to the existence of Rh moieties, enabling efficient coenzyme regeneration.\nQ10. Lines 220-221: Should the XPS characterization of NAD+ in RhCp*&NAD +◎ iCOFcap-1 be placed after Figure (b)?\nNankai University\nResponse: We thank the reviewer for the suggestion. Per the suggestion, we have changed the position of the XPS data in the main text (Figure 3).\nQ11. The enzyme activity assay is not clearly described. Please include potential reasons for enzyme activity loss.\nResponse: We thank the reviewer for the suggestion. Per the suggestion, we have added detailed testing methods for enzyme activity in SI (page S7-S8). Compared to free enzymes, a slight decrease in enzymatic activity (only 6-7%) has been observed after immobilization. We think that the catalytic process of the enzyme after immobilization increases the process of substrate diffusion into the capsule’s interior, thereby resulting in a marginal decline in enzymatic activity.\nQ12. The staining method for Figure 4(b) requires clarification.\nResponse: We thank the reviewer for the suggestion. The staining method has been documented in SI. Per the suggestion, we have incorporated relevant descriptions of the staining protocol into the main text (Lines 282-283): “To test for cascade reactions in iCOFcap-1, we encapsulated fluorescent labeled FDH and GDH into iCOFcap-1.”.\nQ13. Does the encapsulated enzyme leak when the pH of the reaction solution or environment changes?\nResponse: We thank the reviewer for the suggestion. Enzyme leakage rates in buffer solutions have been systematically quantified, with no significant enzyme leakage (Figure S22). Per the suggestion, we have expanded the analysis to include leakage measurements of lipase◎ iCOFcap-1 under pH = 5.0/7.0 (Figures S22 and S23) and HRP◎iCOFcap-2 under pH = 7.0/9.0 conditions (Figures S32 and S33). The expanded experimental data consistently demonstrate negligible enzyme leakage across all tested environments.\nNankai University\nFigure S22. The protein leakage of lipase◎iCOFcap-1 in pH = 7.0.\n100\n80\n)\n%\n(\ne\nm 60\ny\nz\nn\ne\nf\ne\no 40\ng\na\nk\na\ne\nL 20\n0\n0 50 100 150 200 250 300 350 400\nTime (min)\nFigure S23. The protein leakage of lipase◎iCOFcap-1 in pH = 5.0.\nNankai University\n100\n80\n)\n%\n(\ne\nm 60\ny\nz\nn\ne\nf\ne\no 40\ng\na\nk\na\ne\nL 20\n0\n0 50 100 150 200 250 300 350 400\nTime (min)\nFigure S32. The protein leakage of HRP◎iCOFcap-2 in pH = 7.0.\n100\n80\n)\n%\n(\ne\nm 60\ny\nz\nn\ne\nf\ne\no 40\ng\na\nk\na\ne\nL 20\n0\n0 50 100 150 200 250 300 350 400\nTime (min)\nFigure S33. The protein leakage of HRP◎iCOFcap-2 in pH = 9.0.\nQ14. Is washing required during the synthesis of iCOFcap-1? If so, please include this step to ensure experimental rigor.\nResponse: We thank the reviewer for the suggestion. Yes, the synthesis of iCOFcap-1 requires purification through washing steps, as detailed in the revised protocol. Per the suggestion, we have described the synthesis process of iCOFcap-1 in further detail in the main text (Lines 360-361).\nNankai University\nReviewer 2:\nThe study conducted by Zhang, Chen, and co-workers focuses on the development of materials, specifically ionic capsules of covalent organic frameworks (iCOFs), designed to encapsulate enzymes and chemo-catalysts. The research primarily investigates the immobilization of formate dehydrogenase (FDH), which is employed as a biocatalyst for the reduction of CO2 to formic acid. The innovative aspect of this study lies in the encapsulation strategy of the enzyme. Unlike conventional methodologies, which typically employ a one-pot encapsulation approach, the material in this study is synthesized in a manner that mimics transmembrane transport. This ensures that the enzymes are encapsulated at a later stage, thereby preserving high enzymatic activity. Various monomers were tested to optimize capsule composition, aiming to maintain efficient enzymatic performance and a comparison between one pot and stepwise encapsulation wads made. In addition to FDH immobilization, the study also reports the encapsulation of RhCp within the structure, which was used as a photocatalyst for the regeneration of the NADH cofactor required for CO2 reduction. Alternatively, GDH was also employed as a strategy for cofactor regeneration.\nAs noted in the Supporting Information (SI), numerous studies in literature have already reported the co-immobilization of FDH and GDH for CO2 reduction to formic acid. Nevertheless, the immobilization methodology presented is indeed innovative and the data analysis is highly detailed, comprehensive, and compelling. Beyond this, several aspects require further clarification to allow for a conclusive evaluation.\nOverall, the study presents a significant advancement in enzyme immobilization strategies. However, addressing these points would strengthen the validity of the findings and further substantiate the claims made in the article.\nResponse: We appreciate the positive support of the reviewer. Per the suggestion of reviewers, we have made modifications and improvements to the overall content of the manuscript.\nQ1. Figure S19 refers to the leakage of lipase protein, but the methodology used to assess this\nNankai University\nparameter is not mentioned. Could further details be provided?\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have added the test methodology of enzyme leakage in SI (page S8): “In the leaching study, after washing 3 times with deionized water quickly, the Lipase◎iCOFcap-1 and HRP◎iCOFcap-2 complexes were immersed into fresh PB buffer, and the supernatants were measured at different time intervals with the replacement of buffer after each test. And the leakage of Lipase◎iCOFcap-1 and HRP◎iCOFcap-2 was calculated using the following equation:\nleaked enzyme\nLeakage % = × 100 %”\nImmobilized enzyme\nQ2. In lines 224–225 and 251–252, the residual activity of immobilized enzymes is compared to their free counterparts. How was this evaluation performed?\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have described the measurement of enzyme activity in further detail in the SI (page S7): “ The free FDH or FDH◎iCOFcap-1 containing the same amount of FDH was added to the 3 mL solution containing PB buffer (50 mM, pH=7.0), 0.16 mM NAD+, and 5 mM sodium formate. The increase in absorbance change at 340 nm was recorded in a UV-Vis spectrophotometer with a constant stirring device and temperature controller. The activities were then determined by UV-Vis using the same method as free enzyme.\nImmobilized FDH\nRelative activity % = × 100 %\nFree FDH\nThe free GDH or GDH◎iCOFcap-1 containing the same amount of GDH was added to the 3 mL solution containing PB buffer (50 mM, pH=7.0), 0.16 mM NAD+, and 5 mM glucose. The increase in absorbance change at 340 nm was recorded in a UV-Vis spectrophotometer with a constant stirring device and temperature controller. The activities were then determined by UV-Vis using the same method as free enzyme.\nImmobilized GDH\nRelative activity % = × 100 %”\nFree GDH\nQ3. The co-immobilization of FDH and GDH within the same capsule is conducted in separate steps. Has the possibility of preparing a single starting solution containing both FDH and GDH\nNankai University\nbeen considered, allowing for their simultaneous immobilization? Would this approach affect the reaction yield compared to sequential encapsulation?\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have achieved simultaneous co-immobilization of FDH and GDH using iCOFcap-1, and tested the catalytic performance of the composite (Figure S49). The catalytic yield of co-immobilization of FDH and GDH remains lower compared to sequentially immobilized enzymes. This performance gap is primarily attributed to the reduced enzyme loading capacity in the simultaneous immobilization process (FDH+GDH, 320 mg g-1 vs. 480 mg g-1 in the stepwise process). Furthermore, the co-immobilization approach introduces critical limitations: the inability to precisely determine individual enzyme ratios (FDH:GDH quantification uncertainty) means that controlled enzyme ratios are hard to achieve, making it difficult to optimize cascade catalysis.\nFigure S49. The loading amount and the yield rate of HCOOH catalyzed by\nFDH&GDH&NAD+◎iCOFcap-1 with FDH and GDH stepwise and FDH and GDH co-immobilization.\nQ4. Could you clarify the rationale behind selecting DHT-600 over DHT-400 and DHT-800? The study states that DHT-600 provides superior properties in terms of relative activity, enzyme loading, and COF structure. However, Figure S16(a) indicates that DHT-800 exhibits the highest enzyme loading. Maybe it would be better to say that the choice primarily based on achieving a balance among swelling ratio, loading amount, COF structure, and enzyme activity?\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have\nNankai University\nredescribed the reasons for selecting DTH-600 over DTH-400 and DTH-800 as the balance among swelling ratio, loading amount, COF structure, and enzyme activity in main text (Lines 166-167). Although DTH-800 demonstrates good loading capacity, it was excluded from selection due to low crystallinity characteristics that compromise structural stability and substrate transport rates.\nQ5. The title refers to a \"boosted\" CO2 reduction. Is this truly the case?\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, to avod misunderstanding, we have changed the title as “Biomimetic Transmembrane Transport of Multi-Enzymes to Fabricate Heterogeneous Bioreactor with Photoenzymatic Reduction of CO ”\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we added the reaction yield using the free enzyme in SI (Figure S40 and S47). The product yield of FDH&RhCp*&NAD+◎iCOFcap-1 and FDH&GDH&NAD+◎iCOFcap-1 is significantly higher than that of corresponding free systems.\n-1\n)\ng\nmaterial 2.0\n-1\nh\nM 1.5\nm\n(\nH\nO\nO\nC 1.0\nH\nof\ne\nat\nr\nd 0.5\nel\nyi\ne\nh\nT\n0.0\nFDH&RhCp*&NAD+◎iCOFcap -1 corresponding free catalysts\nFigure S40. The yield of HCOOH catalyzed by FDH&RhCp*&NAD+◎iCOFcap-1 and the\nNankai University\ncorresponding free systems.\n-1\n)\ng\nmaterial 2.5\n-1 h 2.0\nM\nm\n(\nH 1.5\nO\nO\nC\nH\nof 1.0\ne\nat\nr\nd\nel\n0.5\nyi\ne\nh\nT\n0.0\nFDH&GDH&NAD+◎iCOFcap-1 corresponding free catalysts\nFigure S47. The yield of HCOOH catalyzed by FDH&GDH&NAD+◎iCOFcap-1 and the\ncorresponding free systems.\nQ7. Additionally, it would be advisable to evaluate the reaction yield in a system containing NADH without any regeneration process. This would serve as a control, allowing for a better understanding of the maximum conversion achievable under the conditions studied.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have evaluated the reaction yield in iCOFcap-1 containing NADH without any regeneration process in SI (Figure S48). The product yield of FDH&GDH&NAD+◎iCOFcap-1 is significantly higher than that of FDH&NADH◎iCOFcap-1, which demonstrated the significance of the coenzyme regeneration system for continuous catalysis.\n-1\n)\ng\nmaterial 2.5\n-1 h 2.0\nM\nm\n(\nH 1.5\nO\nO\nC\nH\nof 1.0\ne\nat\nr\nd\nel\n0.5\nyi\ne\nh\nT\n0.0\nFDH&GDH&NAD+◎iCOFcap-1 FDH&NADH◎iCOFcap-1\nFigure S48. The yield of HCOOH catalyzed by FDH&GDH&NAD+◎iCOFcap-1 and\nNankai University\nFDH&NADH◎iCOFcap-1.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe manuscript by Zhu et al. describes a co-encapsulation strategy of two enzymes, formate dehydrogenase (FDH), for CO2 reduction to formate, and glucose dehydrogenase (GDH) for recycling NADH, in ionic covalent organic framework capsules (named iCOFcap). By introducing polyethylene glycol (PEG) polymers and adjusting the charge of COF capsule shells they achieve the stepwise transport of the two proteins inside the capsule by what is named “transmembrane transport”. The iCOFcap were previously loaded with pre-encapsulated NAD+ (cofactor for FDH) and Cp*Rh(bpy)H2O (to reform NADH) and used for photocatalytic CO2 reduction.\nThe whole concept of enzyme integration into COFcap capsules lacks novelty as it was recently reported by the same team for the reduction of N2 using co-encapsulatation of GluDH, GCDH, ω-TA, Au NPs and NAD+, in a slightly different set up (DOI: 10.1038/s41467-025-56214-0). Furthermore, I found that the experimental work is not properly described and there is lack of adequate controls.\nResponse: We appreciate the reviewer's comments. The high novelty of this work was supported by reviewer 1 and 2, who stated “the study presents a significant advancement in enzyme immobilization strategies”, “the immobilization methodology presented is indeed innovative”, “The work demonstrates significant innovation and thorough research”. The\nNankai University\nmulti-enzyme integration method reported in this work is completely different from our previous immobilization methodology (DOI: 10.1038/s41467-025-56214-0). Here are the\nreasons.\n(1) For this new enzyme immobilization method, we pre-synthesized the COF capsules and then stepwise transported multi-enzymes into the inner cavity of the COF capsules. By contrast, our previous work employed a one-pot encapsulation method, in which the multi-enzymes were co-encapsulated during the synthesis process of COF capsules.\n(2) The mechanisms to encapsulate enzymes are completely different. In this work, we designed and fabricated ionic COF capsules that can efficiently load enzymes by swelling effect of the PEG moieties and then transport them into the capsule cavity through charge reversal. By contrast, our previous work reported an emulsion templating method that promotes liquid-liquid phase separation (LLPS) to form stable droplets, resulting in 300– 400 nm COFcaps suitable for the one-pot integration of multiple enzymes.\n(3) In this work, we designed and fabricated new ionic COF capsules as enzyme immobilization matrixes, which were completely different from the neutral COF capsule matrixes in our previous works.\n(4) In this work, we directly compare the catalytic yields of enzymes immobilized using our novel stepwise transmembrane transport method versus our previous one-pot encapsulation method. The results showed that our new approach (92 ± 3%) yielded nearly twice the catalytic yield of the one-pot encapsulation method (67 ± 4%).\nIn summary, the enzyme immobilization methods, immobilization mechanisms, immobilized matrices, and catalysis applications are completely different. Thus, the previous work should not harm the novelty of this work.\nQ1. How is the enzyme loading amount (Fig. 2d) and loading efficiency into the capsules quantified? This is not described.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have added the calculation formulas and detailed steps for the enzyme loading amount and loading efficiency into the capsules in the revised main text (Lines 371-372).\nQ2. We are shown SIM images of isolated capsules, but no information on the efficiency of the process.\nNankai University\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have added the efficiency for loading and transporting enzymes in SI (Figure S24). Freshly synthesized COF capsules exhibited a protein loading capacity of 670 mg g-1. The total protein loading decreased to 600 mg g-1 (10.4% reduction) with transporting 3 hours. A further reduction to 560 mg g-1 (16.4% reduction) was observed with transporting 6 hours. A fraction of proteins (~16%) loading in the outer layer of COF capsule shell diffused out of the COF capsules into the surrounding solution, and that in the inner layer of COF capsule shell diffused into the COF capsules.\nThus, the reduction in total loading capacity serves as an indicator to track the translocation progress. The transport process reached 65% completion within 3 hours and achieved near completion (>95%) by 6 hours, as evidenced by the nearly undetectable protein levels in the external solution. The transporting efficiency of lipase◎iCOFcap-1 was calculated using the following equation:\nInitial loading amount-Loading amount at different time\nTransporting efficiency % = × 100 %\nInitial loading−Final loading amount\nFigure S24. (a) The loading amount and (b) transporting efficiency of lipase◎iCOFcap-2 after loading lipase for 3 hours, transporting lipase for 3 hours, and transporting lipase for 6 hours.\nQ3. What relative proportion of the FDH enzyme (and lipase) is incorporated and how much remains in solution?\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we introduced the proportion of fixed FDH (70 ± 2%) and lipase (38 ± 5% to 52 ± 4%) and the proportion of FDH (30 ± 2 %) and lipase (62 ± 5% to 48 ± 4%) in the solution in\nNankai University\nthe revised main text (Lines 154-155 and 246-247).\nQ4. How much of the total activity is left in solution after preparing the enzyme-loaded capsules?\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have tested the enzyme activity in the solution after preparing the enzyme-loaded capsules in the revised main text (Lines 248-249): “the residual free enzyme activity in the solution phase remains 97 ± 2%.”\nQ5. Apparently, possible enzyme left in solution is not separated from the loaded capsules, and can thus contribute to the reaction.\nResponse: We thank the reviewer for the suggestion and comment. Our standardized post-synthesis protocol involving three cycles of high-speed centrifugation (5500 rpm, 10 min) coupled with gradient washing effectively removes any unbound enzymes, ensuring that only the immobilized enzyme fraction contributes to catalytic activity. Per the suggestion, we added the corresponding content in the revised main text (Lines 366-373).\nQ6. What is the enzyme permeability (Fig. 2e)? How is this determined?\nResponse: We thank the reviewer for the suggestion. Per the suggestion, we added the corresponding content about the enzyme permeability in SI (page S7). Enzyme permeability refers to the percentage ratio of the enzyme’s penetration depth within a membrane to the total thickness of the membrane itself. The measuring enzyme permeability iCOF and COF membranes is as follow: The enzymatic permeability of iCOF and COF films was quantified through confocal laser scanning microscopy (CLSM) analysis of enzyme infiltration depth, combined with membrane thickness measurements. And the enzymatic permeability of iCOF and COF films was calculated using the following equation:\nEnzyme infiltration depth\nEnzyme Permeability % = × 100 %\nMembrane thickness\nQ7. Also how is leakage (Fig. S19) quantified? This is not described.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we\nNankai University\nadded the corresponding content about the enzyme leakage in SI (page S8): “In the leaching study, after washing 3 times with deionized water quickly, the Lipase◎iCOFcap-1 and HRP ◎iCOFcap-2 complexes were immersed into fresh PB buffer, and the supernatants were measured at different time intervals with the replacement of buffer after each test. And the leakage of Lipase◎iCOFcap-1 and HRP◎iCOFcap-2 was calculated using the following equation:\nleaked enzyme\nLeakage % = × 100 %”\nImmobilized enzyme\nQ8. Line 166/167: The pH was not altered over 6h. Should rephrase.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we changed the corresponding content in the revised main text (Lines 172-173).\nQ9. The reason for the physical process of enzyme incorporation inside the capsules should be better described (what is called “transmembrane transport”)\nResponse: We thank the reviewer for the suggestion and comment. The transmembrane transport mechanism of enzymes is governed by their amphoteric electrolyte nature. As shown in Figure S29b, Zeta potential analysis confirms that the enzyme exhibits a negative charge (- 8.44 mV) at pH = 7.0, while the COF capsule surface maintains a positive zeta potential (+11.5 mV). This electrostatic complementarity drives successful enzyme adsorption onto the COF shell. Upon pH adjustment to 5.0, the protein undergoes charge reversal to positive (+2.3 mV) and has the same charge as the iCOFcap-1, causing the enzyme to break away from the COF shell and release it into the COF cavity. Per the suggestion, we have added the corresponding content for further explaining the cause of enzyme incorporation inside the capsules in main text (Lines 197-202).\nQ10. Line 224-225: How are the 93% activity versus the free enzyme quantified? How is the activity quantified in the capsules? This is not described and these results are not shown.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have added the corresponding content for enzyme activity quantified in the capsules in SI (page S7):\nNankai University\n“The free FDH or FDH◎iCOFcap-1 containing the same amount of FDH was added to the 3 mL solution containing PB buffer (50 mM, pH=7.0), 0.16 mM NAD+, and 5 mM sodium formate. The increase in absorbance change at 340 nm was recorded in a UV-Vis spectrophotometer with a constant stirring device and temperature controller. The activities were then determined by UV-Vis using the same method as free enzyme.\nImmobilized FDH\nRelative activity % = × 100 %”\nFree FDH\nPer the suggestion, we have added the corresponding results for enzyme activity in SI (Figure S38):\n100\n)%\n80\n(\ny\nvit\ncti\n60\na\nH\nD\nF\ne 40\nh\nT\n20\nFigure S38. The FDH activity of free FDH and immobilized FDH (FDH&RhCp*&NAD+◎\niCOFcap-1).\nQ11. What are the reaction volumes used for the synthesis of\nFDH&RhCp*&NAD+@iCOFcap-1?\nResponse: We thank the reviewer for the suggestion and comment. The total reaction volumes used for the synthesis of FDH&RhCp*&NAD+◎iCOFcap-1 is 4.0 mL. Meanwhile, the total reaction volumes used for the synthesis of RhCp*&NAD+◎iCOFcap-1 is 11.5 mL. Per the suggestion, we have added the corresponding reaction volumes in the revised main text (Lines 383-385).\nQ12. What is the light source and intensity used in the photocatalytic assays with the\nNankai University\nFDH&RhCp*&NAD+@iCOFcap-1 systems? Also not described.\nResponse: We thank the reviewer for the suggestion and comment. The photocatalytic assays with the FDH&RhCp*&NAD+◎iCOFcap-1 systems reacted using a solar simulator (300 W Xe lamp, PLS-SXE300) outputting stable solar flux at 1.0 kW/m2 (1 sun). Per the suggestion, we have added the corresponding content for explaining the light source and intensity in the revised main text (Lines 399-401).\nQ13. What does it mean that 5 mg integrated photocatalysts are present in the solution?? This is vague. Should describe the amount of each component. How much enzyme is present?\nResponse: We thank the reviewer for the suggestion and comment. The integrated photocatalysts (5.0 mg) include NAD+ (2.5 mg), RhCp* (5.0 mg), and FDH (1.4 mg). Per the suggestion, we have added the corresponding content in the revised main text (Lines 399-400).\nQ14. Figure 3: The results of formate production by the FDH&RhCp*&NAD+@iCOFcap-1 need be compared with the system when the enzyme and co-catalysts are free in solution, so the effect of enzyme encapsulation can be properly assessed. This is a critical control that is missing.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we added the reaction yield using the free enzyme and co-catalysts in SI (Figure S40 and S47). The product yield of FDH&RhCp*&NAD+◎iCOFcap-1 and FDH&GDH&NAD+◎iCOFcap- 1 is significantly higher than that of corresponding free systems.\n-1\n)\ng\nmaterial 2.0\n-1\nh\nM 1.5\nm\n(\nH\nO\nO\nC 1.0\nH\nof\ne\nat\nr\nd 0.5\nel\nyi\ne\nh\nT\n0.0\nFDH&RhCp*&NAD+◎iCOFcap -1 corresponding free catalysts\nFigure S40. The yield of HCOOH catalyzed by FDH&RhCp*&NAD+◎iCOFcap-1 and the\nNankai University\ncorresponding free systems.\n-1\n)\ng\nmaterial 2.5\n-1 h 2.0\nM\nm\n(\nH 1.5\nO\nO\nC\nH\nof 1.0\ne\nat\nr\nd\nel\n0.5\nyi\ne\nh\nT\n0.0\nFDH&GDH&NAD+◎iCOFcap-1 corresponding free catalysts\nFigure S47. The yield of HCOOH catalyzed by FDH&GDH&NAD+◎iCOFcap-1 and the\ncorresponding free systems.\nQ15. Is there a meaning in the use of two colours for the graphs in figs 2e, 3f, 4c and 4d?\nResponse: We thank the reviewer for the suggestion and comment. The dual-color figures employed in the bar charts serve solely for visual distinction purposes, with no categorical or quantitative significance associated with the color selection. Per the suggestion, we have changed the corresponding colors for the graphs in Figs. 2e, 3f, 4c and 4d in main text.\nQ16. For the experiments in Fig. 5b, why is there no effect for the transport of the isolated enzymes (formulations 2 and 3), when this was observed in the previous studies?\nResponse: We thank the reviewer for the suggestion and comment. The superior catalytic performance of formulation 4 over formulations 2-3 stems from fundamental limitations of the one-pot co-immobilization approach, which reduces both enzymatic activity retention (FDH: 67 ± 4% in one-pot immobilization vs. 92 ± 3% in stepwise immobilization; GDH: 75 ± 3% in one-pot immobilization vs. 95 ± 2% in stepwise immobilization) and loading amount (FDH: 120 ± 8 mg g-1 in one-pot immobilization vs. 280 ± 15 mg g-1 in stepwise immobilization; GDH: 100 ± 9 mg g-1 in one-pot immobilization vs. 200 ± 10 mg g-1 in stepwise immobilization). The reason why there is no significant difference in the catalytic yields of formulations 2 and 3 and that of formulations 1 is that the synthesis of formic acid\nNankai University\nrequires the synergy of FDH and GDH. The decrease in the catalytic performance of either catalyst will lead to a decrease in the catalytic performance of the overall reactor.\nQ17. Figs S31 and S32 – give reaction conditions, such as volume, amount of catalysts,\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we added reaction conditions in the revised SI (page S26 and S27).\nQ18. Fig. S33 – describe what recycling experiment means\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we added a measurement method for recycling experiments in the SI (page S8): “The FDH&RhCp*&NAD+◎iCOFcap-1 is subjected to multiple reaction cycles under optimized conditions. After each cycle, the composite is recovered via centrifugation, washed thoroughly with buffer to remove residual substrates/products, and reused in a fresh reaction. The product yield is quantified after each cycle using ion chromatography. The relative yield (%) is calculated relative to the initial cycle to assess performance decay. And the relative yield of FDH&RhCp*&NAD+◎iCOFcap-1 was calculated using the following equation:\nCycle yield\nRelative yield % = × 100 %\nInitial yield\nThe significance of recycling experiments is as follows: (1) The composite’s stability during storage is evaluated to simulate real-world applications. (2) The composite exhibit reusability and reduce costs and waste in industrial biocatalysis compared to free enzymes (single-use). (3) The composite aligns with green chemistry principles by minimizing resource consumption. (4) The recycling experiments illustrates the robustness of the enzyme-COF binding strategy and the COF’s durability, which avoid potential issues like enzyme leaching, COF degradation, or active site blocking. (5) Good recyclability is critical for scalable processes in biofuel production and pharmaceutical synthesis.\nQ19. In the comparison with literature values the authors are omitting systems studying metal- dependent Fdhs where much higher photocatalytic activities have been reported (eg. up to 600\nNankai University\nmmol.gh-1.g-1 of formate, DOI: 10.1002/anie.201814419). These should be included.\nResponse: We thank the reviewer for the suggestion and comment. The comprehensive performance analysis in Figure 5c focuses on heterogeneous catalytic systems, evaluating three critical performance metrics: Initial catalytic yield, Operational durability, Recycled productivity yield (HCOOH yield: 2.05 mmol h-1 g material-1, cycle time: 100 hours, relative yield: 84%).\nWhile the referenced study (DOI: 10.1002/anie.201814419) demonstrates exceptional homogeneous catalytic performance, our systematic comparison reveals fundamental limitations of homogeneous systems, including non-recoverable catalyst consumption and cumulative cost inefficiency. Per the suggestion, we updated the reported heterogeneous platforms with photoenzyme or multi-enzyme cascade systems in Figure 5c and Table S1.\nOther points:\n- The title of the manuscript is not accurate, as the chemocatalysts were not integrated through transmembrane transport, but by direct initial incorporation\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have changed the title as “Biomimetic Transmembrane Transport of Multi-Enzymes to Fabricate Heterogeneous Bioreactor with Photoenzymatic Reduction of CO ”\n2\n- The sentence “In nature, cells can achieve precision catalysis by confinement effect and collaboration of multi-step intracellular reactions by the transmembrane transport of biomacromolecules (e.g., lipids, proteins) by endocytosis.” is misleading and simply not correct! While the first part is true (confinement effect in intracellular catalysis), this catalysis does not require “transmembrane transport of biomacromolecules” or endocytosis. Should be corrected.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we changed the corresponding content as in main text (Lines 56-57): “In nature, cells can achieve collaboration of multi-step intracellular reactions by the transmembrane transport of biomacromolecules (e.g., lipids, proteins) by endocytosis.”\nNankai University"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors put a lot of energy and effort into solving most of the previous problems, but there are still the following deficiencies:\n1.For question 3-4, the authors point out that iCOFcap-1 is a dispersed particle, and supplemented the preparation methods of COF films and iCOF films, but the response to question 5 is not pertinent. According to the preparation conditions of iCOF film and Fig.S28, can iCOF film be considered as a plane formed by a large number of iCOFcap-1? Therefore, in the penetration range of -240 μm to 270 μm, the lipase must pass through the diffusion path of ' outside the capsule-inside the capsule-outside the capsule '. Does it conflict with the low leakage rate of iCOFcap-1 immobilized enzyme? If the diffusion is carried out from the gap of iCOFcap-1 or COF membrane, it is impossible to verify the conjecture that ' the lack of electrostatic interaction between lipase and neutral COF makes it difficult to release into the capsule '. If iCOF film is not aggregated by a large number of iCOFcap-1, but a uniform and thickened version of iCOFcap-1 capsule wall, why can lipase penetrate the micron-level membrane for diffusion, but not leak from the nano-level shell? Please further clarify the experimental design, explanation and conclusion of this part to avoid misunderstanding and confusion.\n2.For question 7, does the additional Figure S21 mean that one-pot immobilization of the enzyme can also achieve the effect of enzyme encapsulation in the capsule (since the pore size remains unchanged, and the authors use this as one of the evidence of enzyme encapsulation)? Then the necessity of the membrane expansion transportation method will be challenged. In order to avoid this situation, the method of one-pot preparation of iCOFcap-1 immobilized enzyme, EDS-TEM characterization of one-pot immobilized enzyme, enzyme loading and activity of one-pot immobilized enzyme should be supplemented and explained according to the actual situation.\n3.In addition, the scale of Fig.S14 is suspected to be wrong, which is inconsistent with other pictures.\n4. It is suggested that the author can refer to more recent research progress on the topic of the practical biomanufacturing process and the introduction of COF, such as related papers published in International Journal of Biological Macromolecules Volume 283, Part 3, December 2024, Page 137856.\nI sincerely hope that the work will go further.", "reviewer_2": "I would recommend to accept and publish this revised manuscript.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have addressed several of the points raised and introduced important information that was missing.\nHowever, some answers are not clear.\nIn the answer to Q4 the authors state that “the residual free enzyme activity in the solution phase remains 97%.”. Does this mean that 97% of the activity remains in solution? This contradicts the previous answer (Q3) where the authors state that 30% of FDH remains in solution. These numbers do not add up. If 30% FDH remains in solution, then about the same level of activity should remain in solution.\nThe answer to Q19 also does not make sense. The catalytic system in reference DOI: 10.1002/anie.201814419 is also heterogeneous (FDH bound to TiO2 nanoparticles). In Supplementary Table S1 the wrong reference is cited for FDH-ITO and the value cited is also not correct. The correct reference (DOI: 10.1002/anie.201814419), has in Fig. 5 a production of 3 mM formate after 6h, which corresponds to 602 umol.h-1.g-1. This would be the meaningful comparison, using the same units."}, "author_response": "Response: We appreciate the positive support of the reviewer. Per the suggestion of reviewers, we have made modifications and improvements to the overall content of the manuscript.\nQ1. For question 3-4, the authors point out that iCOFcap-1 is a dispersed particle, and supplemented the preparation methods of COF films and iCOF films, but the response to question 5 is not pertinent. According to the preparation conditions of iCOF film and Fig.S28, can iCOF film be considered as a plane formed by a large number of iCOFcap-1? Therefore, in the penetration range of -240 μm to 270 μm, the lipase must pass through the diffusion path of ' outside the capsule-inside the capsule-outside the capsule '. Does it conflict with the low leakage rate of iCOFcap-1 immobilized enzyme? If the diffusion is carried out from the gap of iCOFcap-1 or COF membrane, it is impossible to verify the conjecture that ' the lack of electrostatic interaction between lipase and neutral COF makes it difficult to release into the capsule '. If iCOF film is not aggregated by a large number of iCOFcap-1, but a uniform and thickened version of iCOFcap-1 capsule wall, why can lipase penetrate the micron-level membrane for diffusion, but not leak from the nano-level shell? Please further clarify the experimental design, explanation and conclusion of this part to avoid misunderstanding and confusion.\nResponse: We thank the reviewer for the suggestion. Because we employed an interfacial polymerization approach, the SEM image showed that the iCOF film is not aggregated by a large number of iCOFcap-1, but instead exhibited a uniform and thickened version of the iCOFcap-1 capsule wall (Figure S29). Per the suggestion, we added the corresponding content and SEM image in the revised main text (Lines 187-189) and Figure S29. Compared to the COF film without ionic monomers, the iCOF film with ionic monomers exhibits significantly higher enzyme permeability. These results indicate that the enzyme penetrates the internal wall of the capsule, rather than merely adsorbing superficially on the outer wall of the capsule. By adjusting the pH of the buffer, enzymes near the outer layers of the iCOF capsule wall are released outwards into the buffer. In contrast, enzymes that have penetrated the inner layers of\nNankai University\nthe wall are released inwards into the cavity of the capsule. This differential release demonstrates that enzymes permeate more deeply into the iCOF shell structure, allowing a greater number of enzymes to be effectively fixed and retained inside the capsule.\nThe reason why lipase can be successfully loaded and fixed within the interior cavity of iCOFcap-1 but almost does not leak from the iCOFcap-1 is as follows: (1) Enzymes load into the shell of COF capsules during the shell swelling process (dry capusles are put into a solution environment). However, under a non-swelling environment (capsules are kept in solution without drying), the COF shell structure imposes kinetic barriers to enzyme movement. (2) After enzyme loading, the system reaches a state of equilibrium or near-equilibrium. There is no sustained concentration gradient or other driving force (swelling treatment) pushing the lipase molecules outwards through the capsule shell. Equilibrium dynamics trap the enzyme inside. Leakage would only occur if the shell were damaged or COF capsules were put in a swelling environment. The lipase molecules trapped inside the capsule cavity must overcome an energy barrier to reverse their path back out through the shell, which is kinetically slow.\nFigure S29. Cross-section SEM images for iCOF film.\nQ2. For question 7, does the additional Figure S21 mean that one-pot immobilization of the enzyme can also achieve the effect of enzyme encapsulation in the capsule (since the pore size remains unchanged, and the authors use this as one of the evidence of enzyme encapsulation)? Then the necessity of the membrane expansion transportation method will be challenged. In order to avoid this situation, the method of one-pot preparation of iCOFcap-1 immobilized enzyme, EDS-TEM characterization of one-pot immobilized enzyme, enzyme loading and activity of one-pot immobilized enzyme should be supplemented and explained according to\nNankai University\nthe actual situation.\nResponse: We thank the reviewer for the suggestion. We had tested the TEM, PXRD characterization, enzyme loading amount and activity of one-pot immobilized FDH and GDH in Figure 4c and S44-46. Per the suggestion, we have added the method of one-pot preparation of iCOFcap-1 immobilized enzyme in SI.\nMeanwhile, we directly compare the catalytic yields of enzymes immobilized using our novel stepwise transmembrane transport method versus our previous one-pot encapsulation method. The results showed that our new approach yielded nearly twice the catalytic yield of the one-pot encapsulation method in Figure 5b. This yield increase is primarily driven by improvements in both enzymatic activity (FDH: 67±4% in one-pot immobilization vs. 92± 3% in stepwise immobilization; GDH: 75±3% in one-pot immobilization vs. 95±2% in stepwise immobilization) and enzyme loading (FDH: 120±8 mg g-1 in one-pot immobilization vs. 280 ± 15 mg g-1 in stepwise immobilization; GDH: 100±9 mg g-1 in one-pot immobilization vs. 200±10 mg g-1 in stepwise immobilization) in Figure 4c and S43.\nFigure 4. Multi-enzyme cascade bioreactor of iCOFcap-1. (a) The illustration of the synthesis of FDH&GDH&NAD+◎iCOFcap-1 and cascade biocatalysis of CO to formic acid.\n2\n(b) The CLSM images of FDH&GDH◎iCOFcap-1. Scale bar: 1 μm. (c) The loading amount and relative activity of FDH◎iCOFcap-1 (stepwise), FDH◎iCOFcap-1 (one-pot), GDH◎ iCOFcap-1 (stepwise), GDH◎iCOFcap-1 (one-pot). (d) The yield rate of HCOOH catalyzed\nNankai University\nby FDH&GDH&NAD+◎iCOFcap-1 with different ratios of FDH:GDH. The error bar of the sample refers to the standard deviation of three groups prepared at different times.\nFigure S44. The loading amount and loading efficiency of iCOFcap-1 immobilization FDH (a) and GDH (b) by the one-pot method.\nFigure S45. The PXRD patterns of iCOFcap-1 immobilizing FDH (a) and GDH (b) by one- pot method.\nNankai University\nFigure S46. The TEM images of iCOFcap-1 immobilizing FDH (a: 0.25 mg, b: 0.50 mg, c: 1.00 mg) and GDH (d: 0.25 mg, e: 0.50 mg, f: 1.00 mg) by one-pot method.\nFigure 5. Cascade performance of iCOFcap-1 bioreactors using different formulations.\n(a) The illustration of the synthesis of Formulation 1, Formulation 2, Formulation 3, and Formulation 4. (b) The yield rate of HCOOH catalyzed by Formulation 1, Formulation 2, Formulation 3, and Formulation 4. (c) The comprehensive performance of this work compared with reported heterogeneous platforms with photoenzyme cascade or multi-enzyme cascade. The error bar of the sample refers to the standard deviation of three groups prepared at different times.\nQ3. In addition, the scale of Fig.S14 is suspected to be wrong, which is inconsistent with other pictures.\nNankai University\nResponse: We thank the reviewer for the comment. Per the suggestion, we have changed the scale of Figure S14.\nQ4. It is suggested that the author can refer to more recent research progress on the topic of the practical biomanufacturing process and the introduction of COF, such as related papers published in International Journal of Biological Macromolecules Volume 283, Part 3, December 2024, Page 137856.\nResponse: We thank the reviewer for the suggestion. Per the suggestion, we have carefully studied the literature you recommended and cited the paper (International Journal of Biological Macromolecules Volume 283, Part 3, December 2024, Page 137856) in the revised main text.\nI sincerely hope that the work will go further.\nReviewer 2:\nI would recommend to accept and publish this revised manuscript.\nResponse: We appreciate the positive support of the reviewer.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed several of the points raised and introduced important information that was missing.\nHowever, some answers are not clear.\nNankai University\nResponse: We appreciate the positive support of the reviewer. Per the suggestion of reviewers, we have made modifications and improvements to the overall content of the manuscript.\nQ1. In the answer to Q4 the authors state that “ the residual free enzyme activity in the solution phase remains 97%.”. Does this mean that 97% of the activity remains in solution? This contradicts the previous answer (Q3) where the authors state that 30% of FDH remains in solution. These numbers do not add up. If 30% FDH remains in solution, then about the same level of activity should remain in solution.\nResponse: We thank the reviewer for the suggestion and comment. The 30% FDH mentioned in Q3 refers to the mass fraction of residual protein in the solution, representing 30% of the total protein mass initially added. In contrast, the 97% value in Q4 denotes the residual enzymatic activity of FDH remaining in the solution.\nQ2. The answer to Q19 also does not make sense. The catalytic system in reference DOI: 10.1002/anie.201814419 is also heterogeneous (FDH bound to TiO2 nanoparticles). In Supplementary Table S1 the wrong reference is cited for FDH-ITO and the value cited is also not correct. The correct reference (DOI: 10.1002/anie.201814419), has in Fig. 5 a production of 3 mM formate after 6h, which corresponds to 602 umol.h-1.g-1. This would be the meaningful comparison, using the same units.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have added the related reference and corrected production in Figure 5c and Table S1.\nNankai University"}, {"round": 3, "reviewer_comments": {"reviewer_1": "I would recommend to accept this revised manuscript.", "reviewer_4": "I still have a problem with the soundness of this research, because the paper continues to lack important details about the experimental methods. How is it possible that 97% of FDH activity remain in solution when 70% of the protein has been incorporated? This is not possible. If 97% of FDH activity remain in solution this means that only 3% of FDH has been incorporated. The authors should rely on activity, not on protein amount, as the protein may be contaminated. The purity is not assessed. 3% incorporation is very low efficiency and does not agree with the results for lipase or Fig. S44. Are protein contaminants being incorporated preferentially?\nI asked before (Q5) about how the enzyme in solution is separated from the capsules. The authors answered only about lipase and not about FDH. There is nothing in the paper saying that FDH in solution is separated from the capsules, so it may be contributing to the reaction. This is highly important, specially when 97% of activity remain in solution.\nMore details are still required for several figures in SI. What is being compared in Fig. S39? I mean how do the authors control the amount of protein in each case. The amount of protein in each case should be stated. The same for Fig. S41 and S48. What does it mean \"corresponding free catalysts\"? The conditions in comparison should be fully described."}, "author_response": "Response: We appreciate the positive support of the reviewer. Per the suggestion of reviewers, we have made modifications and improvements to the overall content of the manuscript.\nQ1. I still have a problem with the soundness of this research, because the paper continues to lack important details about the experimental methods. How is it possible that 97% of FDH activity remain in solution when 70% of the protein has been incorporated? This is not possible. If 97% of FDH activity remain in solution this means that only 3% of FDH has been incorporated. The authors should rely on activity, not on protein amount, as the protein may be contaminated. The purity is not assessed. 3% incorporation is very low efficiency and does not agree with the results for lipase or Fig. S44. Are protein contaminants being incorporated preferentially?\nResponse: We thank the reviewer for the suggestion and comment.\n(1) The relative loading proportion of the enzyme represents the proportion of enzyme immobilized onto the carrier relative to the total amount added. The calculation method of relative enzyme loading proportion is consistent with reported work (ACS Sustainable Chem. Eng. 2024, 12, 17566–17577; ACS Sustainable Chem. Eng. 2021, 9, 4054–4063), and the relative loading proportion of the enzyme was calculated using the following equation:\nquantified amount of enzyme loaded into materials\nRelative loading proportion of enzyme % = ×100 %\ntotal weight of enzyme\nThe proportion of fixed FDH (70%) indicates that the FDH immobilized on the material accounts for 70% of the total protein mass used, with an absolute immobilized mass of approximately 1.4 mg.\n(2) The relative enzyme activity quantifies the enzyme performance compared to free enzymes (typically its initial activity under optimal conditions), expressed as a percentage. It normalizes activity measurements to eliminate variables like enzyme concentration or purity—for example,\nNankai University\nif initial enzymes show 200 μmol/min activity and the enzyme in the solution for 6 h yields 194 μmol/min under identical conditions, the relative activity of the enzyme in the solution is 97% (Line 254). The 97% relative enzyme activity denotes that the 30% unimmobilized enzyme fraction retained 97% activity relative to the native enzyme benchmark. The calculation method of relative enzyme activity is consistent with reported work (ACS Sustainable Chem. Eng. 2021, 9, 4054–4063), and the relative enzyme activity was calculated using the following equation:\nImmobilized lipase\nRelative activity % = ×100 %\nFree lipase\nUsually, the immobilization process inherently reduces enzyme activity: (1) For unimmobilized enzymes in solution, prolonged ambient exposure induces time-dependent denaturation, depressing relative activity slightly lower than 100%. (2) For immobilized enzymes, synergistic effects of substrate diffusion barriers and extended room-temperature incubation collectively diminish catalytic rates, resulting in activity below 100%.\n(3) The loading efficiency indicates that the FDH/GDH immobilized on the material accounts for the total protein mass used in Figure S44. The calculation method of loading efficiency is consistent with reported work (Inorg. Chem. Front., 2025, 12, 2459-2467; ACS Sustainable Chem. Eng. 2021, 9, 4054–4063), and the loading efficiency (LE %) were calculated as follows:\ntotal enzyme-free enzyme in supernatant\nLE % = ×100 %\ntotal enzyme\nIn conclusion, enzyme activity defines practical utility, while enzyme loading merely measures immobilized quantity or proportion. Therefore, both the enzyme immobilization ratio and enzyme activity are jointly required to comprehensively evaluate the overall performance of immobilized enzymes.\nQ2. I asked before (Q5) about how the enzyme in solution is separated from the capsules. The authors answered only about lipase and not about FDH. There is nothing in the paper saying that FDH in solution is separated from the capsules, so it may be contributing to the reaction. This is highly important, specially when 97% of activity remain in solution.\nNankai University\nResponse: We thank the reviewer for the suggestion and comment. Our standardized post-synthesis protocol involving three cycles of high-speed centrifugation (5500 rpm, 10 min) coupled with gradient washing effectively removes any unbound FDH, ensuring that only the immobilized FDH fraction contributes to catalytic activity. Per the suggestion, we added the corresponding content in the revised main text.\nQ3. More details are still required for several figures in SI. What is being compared in Fig. S39? I mean how do the authors control the amount of protein in each case. The amount of protein in each case should be stated. The same for Fig. S41 and S48. What does it mean \"corresponding free catalysts\"? The conditions in comparison should be fully described.\nResponse: We thank the reviewer for the suggestion and comment. Per the suggestion, we have added the related condition details in Figure S39, S41 and S48. Since the protein loading per unit mass of the material is predetermined, the total protein input is regulated by controlling the amount of material added.\n100\n)%\n80\n(\ny\nvit\ncti\n60\na\nH\nD\nF\ne 40\nh\nT\n20\nFigure S39. The FDH activity of free FDH and immobilized FDH (FDH&RhCp*&NAD+◎\niCOFcap-1) with the same amount of FDH (0.5 mg).\nNankai University\n-1\n)\ng\nmaterial 2.0\n-1\nh\nM 1.5\nm\n(\nH\nO\nO\nC 1.0\nH\nof\ne\nat\nr\nd 0.5\nel\nyi\ne\nh\nT\n0.0\nFDH&RhCp*&NAD+◎iCOFcap -1 corresponding free catalysts\nFigure S41. The yield of HCOOH catalyzed by FDH&RhCp*&NAD+◎iCOFcap-1 and the\ncorresponding free systems (the same amount of free FDH, RhCp*, NAD+, and iCOFcap-1 in the reaction system).\n-1\n)\ng\nmaterial 2.5\n-1 h 2.0\nM\nm\n(\nH 1.5\nO\nO\nC\nH\nof 1.0\ne\nat\nr\nd\nel\n0.5\nyi\ne\nh\nT\n0.0\nFDH&GDH&NAD+◎iCOFcap-1 corresponding free catalysts\nFigure S48. The yield of HCOOH catalyzed by FDH&GDH&NAD+◎iCOFcap-1 and the\ncorresponding free systems (the same amount of free FDH, GDH, NAD+, and iCOFcap-1 in the reaction system).\nNankai University"}, {"round": 4, "reviewer_comments": {"reviewer_4": "The answer to question 1 describes several obvious things, but a proper justification for the findings is not given. The answers are not congruent with each other.\nIt is simply not possible that when 70% mass of FDH has been incorporated in the capsules, that 97% of activity remains in solution. The authors state that “The 97% relative enzyme activity denotes that the 30% unimmobilized enzyme fraction retained 97% activity relative to the native enzyme benchmark.” This is impossible. 30% of the enzyme in solution cannot present 97% of the initial enzyme activity present!!! It would mean that the enzymatic activity was going up with time in solution. The authors themselves describe that the activity goes down with time, as normal.\nThis just does not add up and suggests the authors are not doing the appropriate controls. This also does not agree with the results of Figure 39, where the same activity is observed for the same amount of free and immobilized enzyme."}, "author_response": "Response: We respectively disagree with the reviewer’s comment. The reviewer misunderstood the assessment of relative enzyme activity and biocatalytic performance, which are fundamental concepts widely accepted and routinely applied in high-impact publications, such as J. Am. Chem. Soc. 2025, 147, 28912; Nat. Commun. 2024, 15, 10861; Nat. Protoc. 2023, 18, 2032-2050; Nat. Protoc. 2023, 18, 3080-3125; Nat. Commun. 2022, 13, 4816; Angew. Chem. Int. Ed. 2022, 61, e202201378; J. Am. Chem. Soc. 2019, 141, 2348; J. Am. Chem. Soc. 2019, 141, 14298.\nAccording to the literature, the definition of relative enzyme activity: The relative enzyme activity quantifies the immobilized/un-immobilized enzyme performance compared to the initial free enzyme performance (defined as 100%).\n(1) For the un-immobilized enzyme in the solution:\nThe initial activity of the free enzyme before any treatments (total enzyme amount containing both immobilized and un-immobilized enzymes = 2.0 mg) is defined as 100% and The relative enzyme activity of un-immobilized enzyme (30 ± 2% mass of total enzyme amount containing both immobilized and un-immobilized enzymes = 0.6 mg enzyme) decreased from 100% to 97 ± 2% in the solution, indicating that the un-immobilized enzymes still retained their 97 ± 2% activity compared to the same amount of free enzyme (0.6 mg). This did not mean that the enzymatic activity was going up with time in solution (i.e., 30 ± 2% mass of the enzyme fraction can produce 97 ± 2% catalytic activity of total enzymes). The 3% loss of relative enzyme activity is due to slight denaturation of the enzyme after storage at room temperature with shaking during the immobilization experiments.\n(2) For the immobilized enzyme in the capsule:\nThe initial activity of the free enzyme before any treatments is defined as 100%. The relative enzyme activity of immobilized enzymes (70 ± 2% mass of total enzyme amount containing both immobilized and un-immobilized enzymes = 1.4 mg enzyme) decreased from 100% to 93 ± 1% after immobilization in the capsule, indicating that the immobilized enzymes still retained their 93 ± 1% activity compared to the same amount of free enzyme (1.4 mg). This did not mean that 70 ± 2% mass of the enzyme fraction (1.4 mg) produced 93 ± 1% catalytic yield of total enzymes (2.0 mg). The 7% loss of relative enzyme activity is because the substrate diffusion rate can decrease due to immobilization by COF capsules, and enzyme exposure at room temperature induces denaturation.\nComment 2: This just does not add up and suggests the authors are not doing the appropriate controls. This also does not agree with the results of Figure 39, where the same activity is observed for the same amount of free and immobilized enzyme.\nResponse: We respectively disagree with the reviewer’s comment. All the control experiments follow the standard protocls widely employed in the literature (e.g., Nat. Protoc. 2023, 18, 2032-2050; Nat. Protoc. 2023, 18, 3080-3125; Nat. Commun. 2022, 13, 4816; J. Am. Chem. Soc. 2019, 141, 2348; J. Am. Chem. Soc. 2019, 141, 14298).\nIn Figure S39, the relative FDH activity of free FDH refers to the relative activity of the enzyme before any treatments. The relative activity of immobilized FDH refers to the relative enzyme activity of the enzymes immobilized in the capsule compared with the free enzymes of the same amount.\nTo avoid misunderstanding, we revised Figure S39.\n(1) .\nFigure S39. The relative activity of free FDH before any treatment and immobilized FDH in FDH&RhCp*&NAD+◎iCOFcap-1. The initial activity of the free FDH before any treatments is defined as 100%. The free FDH (1.4 mg) or the FDH&RhCp*&NAD+ ◎iCOFcap-1 (5 mg material containing 1.4 mg FDH) was added to the 3 mL solution containing PB buffer (50 mM, pH=7.0), 0.16 mM NAD+, and 5 mM sodium formate. The absorbance change of free FDH or FDH&RhCp*&NAD+◎iCOFcap-1 at 340 nm within 5 min was recorded in a UV-Vis. The relative activity of the enzyme refers to the increase in absorbance change of FDH&RhCp*&NAD+◎iCOFcap-1 compared to the increase in absorbance change of free FDH."}, {"round": 5, "reviewer_comments": {"reviewer_5": "I was selected to evaluate the rationale for measuring residual FDH activity in the supernatant after COF encapsulation. The authors have provided compelling evidence and clear justification for their quantification method and the interpretation of the retained formate dehydrogenase (FDH) activity. Their methodological approach is robust, the data are presented clearly, and their conclusions are well-supported.\nThe manuscript meets the high standards of Nature Communications in its novelty, mechanistic insight, and experimental rigor. I recommend acceptance in its present form"}, "author_response": ""}]

Decision letter