
==== Front
Sci Adv
Sci Adv
sciadv
advances
Science Advances
2375-2548
American Association for the Advancement of Science

adp1796
10.1126/sciadv.adp1796
Research Article
Physical and Materials Sciences
SciAdv r-articles
Engineering
Environmental Studies
Engineering
Boosting the photocatalytic decontamination efficiency using a supramolecular photoenzyme ensemble
Pollutant degradation by photoenzyme catalysis
https://orcid.org/0000-0002-0506-2775
Jiang Ruifen Conceptualization Data curation Formal analysis Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing - original draft Writing - review & editing 1 †
https://orcid.org/0009-0006-7073-5223
Luo Gan Conceptualization Data curation Formal analysis Investigation Methodology Resources Software Validation Visualization Writing - original draft Writing - review & editing 1 2 †
https://orcid.org/0000-0002-6540-7675
Chen Guosheng Conceptualization Data curation Formal analysis Funding acquisition Investigation Methodology Project administration Resources Software Supervision Validation Visualization Writing - original draft Writing - review & editing 2 3 *
https://orcid.org/0000-0003-3088-3097
Lin Yuhong Data curation Formal analysis Methodology Visualization 2
Tong Linjing Investigation Resources 2
Huang Anlian Resources 2
https://orcid.org/0009-0005-0635-4744
Zheng Yang Conceptualization Data curation Formal analysis Investigation Methodology Resources Software Validation Visualization Writing - original draft Writing - review & editing 1
https://orcid.org/0000-0002-8605-4848
Shen Yong Formal analysis Methodology Project administration Resources Software 2 *
https://orcid.org/0000-0002-0912-1082
Huang Siming Funding acquisition Investigation Visualization 4
https://orcid.org/0000-0002-0797-6036
Ouyang Gangfeng Conceptualization Funding acquisition Visualization Writing - original draft 2 3 *
1 College of Environment and Climate, Guangdong Provincial Key Laboratory of Environmental Pollution and Health, Jinan University, Guangzhou 511443, China.
2 MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou 510006, China.
3 Guangdong Basic Research Center of Excellence for Functional Molecular Engineering, Sun Yat-sen University, Guangzhou 510006, China.
4 Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences and the Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, China.
* Corresponding author. Email: chengsh39@mail.sysu.edu.cn (G.C.); cessy@mail.sysu.edu.cn (Y.S.); cesoygf@mail.sysu.edu.cn (G.O.)
† These authors contributed equally to this work.

13 9 2024
11 9 2024
10 37 eadp179611 3 2024
06 8 2024
Copyright © 2024 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
2024
The Authors
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Continuous industrialization has raised daunting environmental concerns, and there is an urgent need to develop a sustainable strategy to tackle the contamination issues. Here, we report a supramolecular photoenzyme ensemble enabling the harvest of solar energy to remove contaminations in water. The well-sourced oxidoreductase, laccase, is confined into a photoactive hydrogen-bonded organic framework (PHOF) through an in situ encapsulation method. The direct electron migration between the oxidation center in a PHOF and the reduction center in laccase facilitates synergistic photoenzyme-coupled catalysis, showing two orders of magnitude higher activity than free laccase for pollutant degradation under visible light, without the need for sacrificial agents or costly co-mediators. Such high decontamination efficiency also surpasses the reported catalysts. The structure and decontamination function of this supramolecular photoenzyme ensemble remain highly stable in complex environment matrices, presenting desirable reusability and almost 100% conversion efficiency of pollutants for real sewage samples. Our conceptual photoenzyme hybrid catalyst offers important insights into green and sustainable water decontamination.

A photoenzyme-coupled catalyst is reported by confining laccase in a supramolecular crystal for photocatalytic decontamination.

http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 22174164 http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 22104159 Guangdong Basic and Applied Basic Research Foundation 2024B1515020070 The Fundamental Research Funds for the Central Universities, Sun Yat-sen University 23lgbj005 The Guangdong Basic Research Center of Excellence for Functional Molecular Engineering 31000-42080002
==== Body
pmcINTRODUCTION

The photocatalytic solar energy conversion is regarded as a green approach for the development of sustainable and clean energy, which holds great potential to address global environmental issues (1). This important photochemical reaction has been well harnessed by natural organisms, and one of the most impressive examples can be found in photosynthesis (2, 3). It involves a well-known photoenzyme catalytic system, in which chlorophyll molecules absorb light to generate excited electrons that undergo charge separation and are subsequently stored as reduced form of nicotinamide-adenine dinucleotide (phosphate) [NAD(P)H] and adenosine triphosphate (4). Ultimately, with the assistance of enzymes, these energy-rich molecules drive chemical conversion (5). Inspired by this, scientists have endeavored over the past few decades to pursue the hybrid photoenzyme catalysts (6, 7), which holds considerable promise for water decontamination and environment protection at low cost and energy consumption.

Despite the grand achievements in this field, several ongoing challenges persist. The efficiency of molecule diffusion and electron transfer between the photocatalytic ensembles (PEs) and biocatalytic ensembles (BEs) is of paramount importance for photoenzyme catalytic activity (8). While various traditional organic photosensitizers, semiconductors, quantum dots, and carbon-based nanomaterials have been used as PEs for solar energy harvesting, their limited or nonporous nature hinders efficient molecule diffusion across these materials (9–12). Furthermore, achieving efficient electron transfer from the PE to BE often requires the photochemical regeneration of natural cofactors such as NAD(P)H or artificial mediators like methyl viologen (13). However, both NAD(P)H and artificial mediators are costly and the regeneration process is complex and technically challenging, which notably hampers large-scale industrial application (14, 15). In this context, direct electron transfer between the PE and BE would be highly desirable, but it typically suffers from poor electron transfer efficiency (16). In addition, the incompatibility issue arising from the simultaneous presence of a PE and BE in one system voxel poses another difficulty because the reactive oxygen species (ROS) generated by the PE can potentially damage the fragile enzymes (17–19). Therefore, it still remains a formidable challenge to engineer photoenzyme catalysts with high efficiency and durability (20).

To target this, we herein report a direct electron transfer photoenzyme catalyst by a supramolecular strategy for water decontamination. The oxidoreductase, laccase, is confined into the defective domain of a porous photoactive hydrogen-bonded organic framework (PHOF). This structural confinement results in the formation of a bio-interface that facilitates electron transfer between a PHOF and laccase, eliminating the requirement for natural cofactors or mediators. The redox centers are compartmentalized in the PHOF and laccase domains, and the photoenzyme-coupled catalytic process does not involve any highly devastating ROS like a superoxide anion and hydroxyl radical. This well circumvents the incompatibility issues between photocatalysis and biocatalysis in one system voxel. In addition, compared to the nonporous nanoarchitecture, the long-range ordered mesopores in this supramolecular ensemble facilitate the mass transfer of reactant molecules, favoring the photoenzyme-coupled catalysis. Using bisphenol A (BPA) as a proof-of-concept pollutant, this supramolecular photoenzyme catalyst presents two orders of magnitude improvement on both the catalytic conversion rate and turnover frequency (TOF) compared to free laccase under visible light radiation, without the need of any sacrificial agents. In addition, the decontamination efficiency, in terms of the pollutant conversion amount per gram of catalyst per hour, considerably surpasses the catalysts reported previously. We also demonstrate the structural and functional stability as well as the low cytotoxicity of this supramolecular photoenzyme catalyst, which offers the promise to fulfillment of the water purification and decontamination in an environmentally friendly and sustainable manner.

RESULTS

Rational engineering of a laccase-PHOF hierarchical catalyst

To achieve efficient direct electron transfer in photoenzyme ensemble, careful consideration must be given to the spatial connectivity between a photocatalyst and biocatalyst as well as the accessibility of the catalytic center. The HOFs are a class of porous supramolecular crystals constructed by the hydrogen-bonded linkage of discrete organic modules (21). It well inherits the reticular chemistry property of metal-organic frameworks (MOFs) or covalent organic frameworks (COFs) (22). Compared to MOFs and COFs, which are molecularly linked by relatively strong coordination bonds and covalent bonds, the flexible hydrogen bond linkages enable HOFs to crystallize in milder and more biocompatible environments, rendering the in situ encapsulation of fragile enzymes into a HOF scaffold highly feasible (23). In this study, we used a pyrene-core module with four symmetric benzoic acids as building blocks for constructing the PHOF supramolecular scaffold (Fig. 1A). The choice of pyrene units was motivated by their highly photoactive properties, while the facile hydrogen-bonded dimerization of benzoic acids allowed for the formation of a pyrene-discrete and porous supramolecular scaffold (24). In this PHOF scaffold, the photogenerated charges by the pyrene core can efficiently transfer to the inner surface of adjacent pores with shortened distance, markedly improving exciton utilization (25). Laccase is an important blue multicopper oxidase that consists of a mononuclear type 1 (T1) copper center and a trinuclear type 2 (T2)/type 3 (T3) copper cluster (fig. S1) (26). It can catalyze the degradation of phenolic pollutants using O2 as the electron acceptor, which has been considered as a green biocatalyst for pollutant remediation. Considering this, laccase was in situ encapsulated into this PHOF supramolecular scaffold during its mild crystallization process (27), resulting in a hybrid architecture where laccase was tightly confined to a defective domain of the porous PHOF (Fig. 1B). This spatial organization was reasonable as it not only enhanced the stability of fragile laccase through PHOF confinement but also brought the laccase and PHOF material into close proximity, which facilitated the electron transfer.

Fig. 1. Structural characterization of the designed photoenzyme ensemble.

(A) Molecular structure of the pyrene-core module. (B) Schematic representation of the hierarchical architecture of Lac@PHOF. (C) PXRD and (D) SEM of the as-synthesized Lac@PHOF. a.u., arbitrary units. (E) Cryo-EM image showing the microstructure of Lac@PHOF. (F) CLSM images presenting the spatial distribution of AF350-labbled laccase in the PHOF. Scale bars, 2 μm.

The as-synthesized laccase-PHOF (Lac@PHOF) exhibited high crystallinity, as confirmed by powder x-ray diffraction (PXRD) analysis, which showed excellent agreement with the simulated crystallographic structure (Fig. 1C). Of note, we found that, in the pure PHOF sample, the PXRD peaks around 14° and 18° appeared. This might be attributed to the different crystallization solvents used in the pure PHOF (in methanol) and Lac@PHOF (in water), which could affect the purity of the crystalline phase. Scanning electron microscopy (SEM) revealed that Lac@PHOF adopted a regular rod-like structure, further supporting its crystalline nature (Fig. 1D). To gain insight into the microstructure of Lac@PHOF, low-electron-dose cryo–electron microscopy (cryo-EM) was used (Fig. 1E and fig. S2). The lattice fringes assigned to (011) and (103¯) planes were identified in each crystal. It revealed the presence of long-range ordered one-dimensional channels with an approximate width of 2.0 nm distributed throughout the nanoarchitecture. These penetrable large channels were formed by intermolecular carboxylic dimerization and layer-by-layer π-π stacking interactions, which were molecularly imaged under high-resolution cryo-EM (fig. S3). This made the encapsulated laccase highly accessible and enabling rapid removal of catalytic products.

The loading amount of laccase was first examined by inductively coupled plasma mass spectrometry (ICP-MS), where an average of 0.047 wt % Cu was detected in the Lac@PHOF, equal to about 10 wt % laccase on average. In the Fourier transform infrared (FTIR) spectra, the characteristic absorption at 1700 to 1610 cm−1 attributed to the stretching vibration of the protein amide I band was recorded in both Lac@PHOF and laccase (28), which further confirmed the successful incorporation of laccase (fig. S4). Furthermore, the thermogravimetric analysis (TGA) experiments (under N2 atmosphere) supported the integration of laccase, as evidenced by weight loss around 280° to 300°C due to laccase pyrolysis (fig. S5). As the temperature increased above 400°C, both the PHOF and Lac@PHOF masses experienced substantial decreased, attributing to the structural collapse of the PHOF. Of note, the weight loss associated with the laccase pyrolysis was estimated to be ~4 wt % in the TGA test, which was lower than the loading of 10 wt % measured by the ICP-MS method. This was interpretable because laccase was thermally decomposed to carbon under N2 atmosphere.

The insight into the laccase location was then examined by N2 adsorption/desorption isotherms (fig. S6A). The raw PHOF had a typical type I isotherm, with a Brunauer-Emmett-Teller (BET) specific surface area of 588.5 m2/g. However, the N2 adsorption capacity of Lac@PHOF was greatly reduced, with a BET specific surface area of 45.7 m2/g. In addition, the calculated pore volume was also notably decreased in the Lac@PHOF sample compared to the raw PHOF (fig. S6B). This indicated that the laccases were confined inside the PHOF crystal, leading to a reduced porosity. To further verify this point, we carried out a surface adsorption experiment, in which the laccases were incubated in the PHOF-dispersive solution. After 1 hour of stirring, the laccase-adsorbed PHOF solid was collected for the FTIR test (fig. S7). No protein characteristic adsorption band (amide I band) was recorded in the sample, indicating a very limited surface adsorption of laccase onto the PHOF. In addition, this solid sample was decomposed for the ICP-MS test and no Cu signal was detected, excluding the possibility that laccases were simply adsorbed onto the PHOF. These results well evidenced that the laccases were located inside the defect cavity formed in the PHOF crystal.

To further disclose the spatial distribution of laccase within the PHOF nanoarchitecture, a confocal laser scanning microscopy (CLSM) experiment was conducted using a blue dye, AF350, to prelabeled laccase. Figure 1F shows a perfect overlap of the blue fluorescence with the material profile, indicating the uniform distribution of laccase in the PHOF. In addition, laccase retained its catalytic function after confinement in the PHOF, as evidenced by the activity test using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the hydrogen donor under aerobic conditions (fig. S8) (29). These structural examinations confirmed the successful engineering of a porous laccase-PHOF hierarchical architecture as intended.

Interfacial connectivity between the laccase and PHOF

After confirming the hierarchical structure of the laccase-encapsulated PHOF, we proceeded to investigate the interfacial connectivity as it plays a crucial role in electron transfer between the laccase and PHOF. The insight into the 1H solid-state nuclear magnetic resonance (ssNMR) spectrum showed that the carboxyl proton of a pyrene module in the PHOF was blue shifted from δ = 13.7 to 13.9 parts per million (ppm) after laccase encapsulation (Fig. 2A), suggesting the strong hydrogen-bonded interactions between the carboxyl of a pyrene module and enzyme residues (30, 31). Moreover, the carboxyl carbon of a pyrene module, centered at δ = 172 ppm in 13C ssNMR, was shifted toward a low magnetic field (δ = 173 ppm), also suggesting the hydrogen-bonded interaction between the carboxyl of a pyrene module and laccase (Fig. 2B). Such a molecular interaction was also supported by two-dimensional (2D) 1H double quantum magic angle spinning NMR spectroscopy (fig. S9A), in which the characteristic peaks at δ = (13.03 ppm, 13.40 ppm) only appeared in the sample of Lac@PHOF. This characteristic contour plot implied the spatial proximity of the carboxyl proton of pyrene and the carboxyl proton of enzyme residues.

Fig. 2. Interface interactions between the laccase and PHOF.

1H ssNMR (A) and 13C ssNMR spectra (B and C) of the samples of the PHOF, Lac@PHOF, and Lac+PHOF. (D) All-atom explicit solvent MD simulation showing the potential interface formed by hydrogen-bonded and π···H interactions between the laccase and PHOF.

The 13C chemical shifts of the benzene carbons in Lac@PHOF provided evidence of additional interactions between benzene moieties of thePHOF and laccase residues (Fig. 2C). Of specific note, the pyrene core acted as the hydrogen bond receptors, of which the electron density increased such that the tertiary carbon (labeled as “e” in Fig. 2C) chemical shift of the pyrene core decreased from δ = 127.8 to 127.4 ppm (32), which was further supported in the following computational simulation (vide infra). In addition, 2D 1H-13C heteronuclear multiple-quantum coherence NMR was conducted to validate the C-H correlation between the PHOF and enzyme, with the acquisition time of 0.016 s and recycle delay (D1) of 2 s. The contour plots depicted in fig. S9B highlighted several emerging cross-correlation peaks in the Lac@PHOF sample, indicating the interfacial interactions between the benzene carbons of the PHOF and the protons of enzyme residues.

To further get a glimpse of the interfacial interaction, we carried out the all-atom explicit solvent molecular dynamics (MD) simulations (figs. S10 to 12). The simulated results showed that the multicopper center of laccase was retained after being encapsulated by the PHOF (fig. S13). This could well interpret the findings that laccase holds desirable catalytic activity in Lac@PHOF (fig. S8). In addition, several interfacial interactions were identified using MD simulation (Fig. 2D). We found that two glycan chains, highlighted in red and blue boxes in Fig. 2D, formed strong hydrogen-bonded interactions with the carboxyl of pyrene modules arranged in the PHOF. At the same time, the polar residues of Ala497 and Glu496 were also observed to strongly interact with the carboxyl moieties of the PHOF through hydrogen bonds. Such hydrogen-bonded interfaces were well supported by the aforementioned 1H and 13C NMR results (Fig. 2, A and B). The close insights into the interface, highlighted in green box in Fig. 2D, revealed the existence of π···H interaction between the pyrene core of the PHOF and the Pro285 residue of laccase, in line with the 13C NMR analysis (Fig. 2C).

Photoenzyme-coupled catalytic activity of Lac@PHOF

BPA, an industrial chemical used in the production of plastic products, stands as one of the most prevalent endocrine-disrupting chemicals documented by global environmental agencies. BPA exhibits the capacity to infiltrate and accumulate in drinking water and the human body, consequently giving rise to various adverse health effects on sex differentiation, brain development, and immune system (33). Here, it was selected as a model pollutant to investigate the photoenzyme-coupled catalytic behavior of Lac@PHOF. The catalytic conversion time profiles of different catalysts were carried out under both dark and light conditions with same amounts of laccase and/or PHOF materials (Fig. 3, A and B). Considering that both the Lac@PHOF and PHOF exhibited strong light absorption in the visible spectrum of 400 to 500 nm (fig. S14), we first profiled the catalytic kinetics data under 420-nm light-emitting diode (LED) light irradiation (8 mW/cm2) without the addition of any sacrificial agents or costly co-mediators (Fig. 3A). It was found that all the catalytic kinetics data fit well with a first-order kinetic model (fig. S15). The initial conversion rate of BPA was obtained from the slope of the fitting curves within 60 min and was represented by k. Under light conditions, the initial conversion rate of BPA with Lac@PHOF (k = 1.795 hour−1) was ~110 times that of the PHOF (k = 0.016 hour−1) and 250 times that of free laccase under dark conditions (k = 0.007 hour−1). Further comparison of the catalytic rates of Lac@PHOF and Lac+PHOF (k = 0.017 hour−1) suggested that the remarkable activity displayed by Lac@PHOF did not originate from a functional combination of laccase and the PHOF alone. This finding implied the presence of a synergetic mechanism in Lac@PHOF. Notably, the reduced activity of free laccase under irradiation (k = 0.004 hour−1) can be attributed to the irradiation-induced structural damage of enzyme molecules (34). However, we observed that the catalytic function of laccase may not be affected in our Lac@PHOF biohybrid architecture because the catalytic conversion rate of Lac@PHOF was much higher than that of the PHOF under irradiation. This disparity could potentially be attributed to the synergetic effect resulting from the light-harvesting ability of the exterior PHOF and the retained catalytic function of the enzyme. Of note, when the catalytic conversion was implemented under dark conditions, the conversion rates of both the Lac@PHOF and individual PHOF were notably reduced (Fig. 3B). This observation implied the involvement of a light-initiated mechanism, as detailed below. It was worth noting that the free laccase was more active than the Lac@PHOF in the dark because of the inevasible diffusion inhibition by the supramolecular scaffold. In addition, the TOF of Lac@PHOF was calculated to be 1.1 × 104 hour−1, compared to 4.3 × 10 hour−1 of free laccase, indicating a 255-fold enhancement (see the calculation method in Materials and Methods). Furthermore, the conversion efficiency, in terms of BPA degradation amount per gram of catalyst per hour, was compared with the catalysts described in the literature (table S1). Impressively, a conversion efficiency as high as 1125 mg/g per hour was attained by the Lac@PHOF catalyst without the need of any sacrificial agents or costly co-mediators, substantially surpassing the ones by reported catalysts previously (Fig. 3C). At the same time, many molecule fragments were identified by time-of-flight MS, demonstrating the catalytic conversion of BPA by Lac@PHOF (fig. S16A). By virtue of monitoring the dynamitic variations of the intermediates using gas chromatography coupled with MS (GC-MS; details seen in fig. S16), a possible degradation pathway was proposed and described in fig. S17.

Fig. 3. Catalytic efficiency for pollutants degradation.

Conversion kinetics of different catalysts under 420-nm LED light (A) and darkness (B). The data of three independent experiments and calculated error bars (SD) are presented in (A) and (B), SD (n = 3). Data are presented as mean values ± SD. (C) Comparison of the catalytic conversion efficiency of different catalysts reported in the literature and this work for BPA conversion (table S1). (D) Catalytic conversion efficiency of different catalysts for varying organic pollutants under 420-nm LED light except free laccase in the dark. The data of three independent experiments and calculated error bars (SD) are presented in (D), SD (n = 3).

To elucidate the advantage of this Lac@PHOF architecture, we synthesized other biohybrid catalysts where laccase was encapsulated into alternative porous solids, including inorganic nanoparticles of CaCO3 (fig. S18) (35), porous framework materials such as ZIF-8 (fig. S19) (36), and NU-1003 (fig. S20) (37). Both the Lac@CaCO3 and Lac@ZIF-8 showed very limited catalytic ability on BPA conversion (fig. S21). This phenomenon can be attributed to the no light-harvesting ability of both CaCO3 and ZIF-8 (fig. S22). It should be noted that NU-1003 also involved the pyrene unit as the PHOF, but the intermolecular linkage of which was bridged by an extra Zr cluster. It caused an entirely different topology and required the harsh conditions to crystallize (38). Considering this, laccase (4.4 × 4.5 × 6.4 nm) was encapsulated into presynthesized NU-1003 through pore infiltration (~4.7-nm mesopore), forming a laccase-encapsulated NU-1003 (Lac@NU-1003; fig. S20). Although Lac@NU-1003 showed an increased BPA conversion efficiency compared to free laccase under light irradiation, its activity was notably inferior to that of our well-designed Lac@PHOF (fig. S21). This distinct contrast in photoenzyme-coupled efficiency highlights the structural advantages of Lac@PHOF, which facilitated direct electron transfer from the photoactive PHOF to laccase, thereby accelerating the redox reaction (vide infra).

We further used Lac@PHOF for the catalytic conversion of other toxic organic pollutants, including 3-chlorophenol (3-CP), phenol, bisphenol F (BPF), tetrabromobisphenol A (TBBPA), malachite green (MG), and Congo red (CR). Like BPA, Lac@PHOF exhibited a much higher catalytic conversion efficiency toward the aforementioned target pollutants compared to the free laccase, PHOF, and Lac+PHOF, achieving complete conversion of all pollutants within 24 hours (Fig. 3D).

Direct electron transfer photoenzyme catalytic mechanism

To unravel the photoenzyme-coupled catalytic mechanism underlying Lac@PHOF, we first investigated the orbital energy characters. Ultraviolet-visible diffuse reflectance spectroscopy was performed to assess the light absorption behaviors. Both the PHOF and Lac@PHOF had a broad absorption band in the visible region, indicating the efficient harvesting of visible light (fig. S14). On the basis of the Tauc plot of the Kubelka-Munk function, the calculated bandgaps of the PHOF and Lac@PHOF were both 2.40 eV (fig. S23). At the same time, the conduction band (CB) was estimated to be −1.25 and −1.17 V (versus Ag/AgCl electrodes) for the PHOF and Lac@PHOF, respectively, by means of the Mott-Schottky experiment (fig. S24). Therefore, the valence band (VB) of the PHOF and Lac@PHOF can be estimated to be about 1.15 and 1.23 V (versus Ag/AgCl electrodes). On the basis of the orbital character information, we charted out the orbital energy characters (Fig. 4A). To further understand the electronic structures, density functional theory (DFT) calculations was also performed. In the projected density of states (PDOS) profile (Fig. 4B), the dominant feature is C 2p bonding resonances near the Fermi level, which revealed that the VB band was mainly contributed by the 2p orbital of C atoms. This suggested that the excited electron on the PHOF might arise from the well π-delocalization from the 2p orbital of C atoms. The calculated bandgap of the PHOF (1.81 eV) was slightly lower than the experimental values (2.4 eV), which could confirm the semiconductive nature of the PHOF. Clearly, the VB of Lac@PHOF was more positive than the oxidation potential of BPA (0.7 V versus Ag/AgCl electrodes; fig. S25), indicating that the catalytic oxidative degradation of BPA was thermodynamically favorable.

Fig. 4. Investigation of the catalytic mechanism.

(A) Orbital energy characters of the PHOF and Lac@PHOF. (B) Electronic structures of the PHOF based on DFT calculations and corresponding DOS. (C) Photocurrents of the PHOF and Lac@PHOF under 420-nm irradiation in a 0.1 M Na2SO4 solution. (D) EIS of the PHOF and Lac@PHOF. GCE: glassy carbon electrode. (E) Time-resolved fluorescence spectra of the PHOF and Lac@PHOF excited at 450 nm and probed at 540 nm. (F) ESR spectra at 5 K of laccase and the Lac@PHOF catalysts under different gas atmospheres. (G) Dissolved oxygen consumption curves in the presence of different inhibitors and (H) the BPA conversion curves of Lac@PHOF in the presence of different inhibitors. The data of three independent experiments and calculated error bars (SD) are presented in (G) and (H), SD (n = 3). (I) Proposed direct electron transfer photoenzyme-catalytic mechanism.

Subsequently, the electron transfer behavior was carefully evaluated by optical and photoelectric characterizations. The photocurrent test of Lac@PHOF presented three times as high as that of the PHOF, indicating the more efficient electron transfer stemming from the encapsulated laccase (Fig. 4C). To gain further insight into the electron transfer, we carried out photoluminescence (PL) spectroscopy, electrochemical impedance spectroscopy (EIS), and time-resolved PL (tr-PL). Compared to the PHOF, Lac@PHOF exhibited a lower fluorescence intensity (fig. S26) and a smaller electrochemical impedance curve radius (Fig. 4D), indicating that Lac@PHOF had a lower charge transfer resistance and more efficient charge carrier separation, consistent with the result from the photocurrent test. The insight into the tr-PL spectra further evidence this conclusion, in which the average fluorescence lifetime of Lac@PHOF (10.27 ns) was substantially higher than that of the PHOF (4.36 ns) (Fig. 4E). These results demonstrated that the confined laccase suppressed the recombination of electron-hole pairs in the PHOF, facilitating the efficient transfer of photogenerated electrons that participates in photoenzyme-coupled reactions.

Attachment of conductive nanoparticles or polymers to enzymes has showed potential to facilitate the direct electron transfer in enzymes through electron tunneling events (39, 40). Figure 2 clarified the interfacial connectivity between a large π-conjugated PHOF and laccase by means of hydrogen-bonded and π···H interactions. We inferred that the promoted electron-hole separation efficiency of Lac@PHOF was attributed to the facile electron mobility from the PHOF to laccase. This deduction was further validated by the electron spin resonance (ESR) experiment. Laccase is a multicopper oxidase enzyme that uses molecular oxygen as the electron acceptor through its trinuclear T2/T3 Cu cluster (41). Illumination of Lac@PHOF at 420 nm under N2 atmosphere resulted in a notable disappearance of both the Cu(II) T1 and T2 ESR signatures (Fig. 4F). It indicated that the photogenerated electron transferred to the Cu center of laccase, leading to the reduction in Cu(II) to Cu(I) (41). Conversely, when Lac@PHOF was illuminated at 420 nm under oxygen atmosphere, the Cu(II) T1 and T2 ESR signatures were maintained, suggesting the electron transfer process from the PHOF to laccase and subsequently to the laccase trinuclear Cu cluster–captured O2.

To further attest that the trinuclear Cu cluster–captured O2 served as the electron acceptor in photoenzyme-coupled catalysis, we monitored the time-dependent O2 concentration in an enclosed container using a luminescent dissolved oxygen probe (fig. S27). A substantial decrease in O2 concentration (about 54% after 120 min) was observed when BPA was added to the Lac@PHOF solution under light irradiation at 420 nm (fig. S28). The rate of O2 consumption in this system was much higher compared to the system involving only Lac@PHOF and the one comprising PHOF and BPA (about 12% after 120 min). Such a great difference on O2 consumption rate indicated the crucial role of a well-designed Lac@PHOF architecture for photogenerated electron transfer, which facilitated the catalytic oxidation of BPA using O2 as the electron acceptor.

In addition, the electron paramagnetic resonance (EPR) experiment confirmed that no highly oxidative ROS such as hydroxyl radicals (•OH) and superoxide radical (•O2−) were generated during the catalytic process (fig. S29, A and B). Only singlet oxygen (1O2) was detected (fig. S29C). This observation aligned with the O2 reduction mediated by the trinuclear T2/T3 Cu cluster of laccase. Laccase reduces O2 to H2O through a two-step, two-electron reduction pathway, without generating the superoxide radical (42). Notably, the absence of a superoxide anion and hydroxyl radical is crucial for efficient photoenzyme-coupled catalysis as these highly active superoxide anion and hydroxyl radical will damage the enzyme (18).

AgNO3 has been reported as an electron scavenger capable of quenching the photogenerated electrons (43), while NaN3 acted as a laccase inhibitor by binding to the T1 copper site and blocking the electrons transfer to the T2/T3 center for O2 reduction (44). We then added AgNO3 (20 g/liter) and NaN3 (0.5 g/liter) to the reaction systems containing Lac@PHOF and BPA under the light exposure and monitored the consumption of dioxygen. The results showed that both AgNO3 and NaN3 substantially decreased the dioxygen consumption rate (Fig. 4G). At the same time, the conversion rates of BPA by Lac@PHOF in the presence of NaN3 and AgNO3 inhibitors were decreased to 0.03776 and 0.07889 hour−1, respectively, representing almost complete inhibition compared to the one without an inhibitor (1.795 hour−1) (Fig. 4H). Furthermore, we further test the photocatalytic activity in the presence of the scavenger of holes [ammonium oxalate (AO)]. When the holes were quenched, a notable inhibition of BPA catalytic conversion was observed (fig. S30), demonstrating that the oxidation center originated from the photogenerated holes. This observation was further supported by the orbital energy characters revealed in Fig. 4A, in which the more positive CB thermodynamically satisfied the catalytic oxidation of BPA.

On the basis of these experimental results, we inferred that the photogenerated holes in the PHOF serve as the catalytic sites for BPA oxidation while the trinuclear T2/T3 copper center of laccase functioned as the reduction center for O2 (Fig. 4I). Such a compartmentalized catalytic mechanism effectively overcame the electron-hole recombination and the incompatibility between photogenerated holes and fragile enzymes, thereby greatly enhancing the efficiency of photoenzyme catalytic efficiency without the requirement of sacrificial agents or costly co-mediators.

Structural and functional stability

The structural and functional stability are crucial for industrial applications in complicated water environments. The supramolecular scaffolds can stabilize fused enzyme by means of a nanoconfinement effect (45). Various environmental factors, including temperature, pH, ionic strength, and natural organic matter (NOM), are widely recognized to induce enzyme deactivation in the field of biocatalysis. Here, we comprehensively assessed the impact of these environmental factors on the catalytic activity of Lac@PHOF and free laccase under same amounts of laccase (catalytic activity was estimated based on the change of initial catalytic rate). From the initial conversion rates of BPA, it was evident that Lac@PHOF presented improved tolerance toward pH, incubation temperature, salt, and NOM compared to free laccase. Lac@PHOF well preserved its activity in solution with a pH range of 4 to 8.5. When the pH was further decreased to 2.5 or increased to 11, more than 87% of its initial catalytic rate was still retained (Fig. 5A and fig. S31A). At the same time, PXRD and SEM imaging experiments validated that the Lac@PHOF could maintain its crystallographic structure under a wide range of pH solution (figs. S32 and S33). The slightly reduced activity at pH = 2.5 and pH = 11 solutions was due to the highly structural susceptibility of enzymes under strong acid and alkaline solutions. In contrast, free laccase lost more activity at the same pH solutions and was completely inactive under extreme pH conditions of 2.5 and 11 (Fig. 5A and fig. S34A). In addition, Lac@PHOF could maintain more than 78 and 86% catalytic activity even with high salt concentrations up to 20% and NOM amounts up to 100 mg/liter, respectively (Fig. 5, B and C, and fig. S31, B and C). In contrast, free laccase was markedly affected by these experimental conditions. It became completely deactivated with salt concentrations above 10% and NOM amounts exceeding 100 mg/liter (Fig. 5, B and C, and fig. S34, B and C). Furthermore, the thermal stability was investigated by incubating the catalyst suspensions at different temperatures for 24 hours. Lac@PHOF retained almost 100% catalytic activity even when exposed to a temperature as high as 80°C, while free laccase experienced completely deactivation at a temperature of 60°C (Fig. 5D and figs. S31D and S34D).

Fig. 5. Structural and functional stability.

The effect of pH (A), NaCl (B), NOM (C), and incubation temperature (D) on the initial conversion rates of BPA with Lac@PHOF and free laccase catalysts. (E) Recyclability of the Lac@PHOF catalyst for BPA degradation. (F) Effect of storage time on the initial conversion rate of BPA using Lac@PHOF and free laccase catalysts. The catalytic degradation experiments with Lac@PHOF were carried out under 420-nm irradiation (8 mW/cm2), while those with laccase were conducted in the dark. The data of three independent experiments and calculated error bars (SD) are presented in (A) to (F), SD (n = 3).

The reusability of Lac@PHOF for pollutant degradation was investigated by periodically adding a BPA stock solution to the system containing Lac@PHOF catalysts (8 mg/liter) every 2 hours under 420-nm lighting conditions. The catalytic activity of Lac@PHOF remained stable even after 10 cycles, indicating its high reusability (Fig. 5E and fig. S35). In addition, analysis of crystallinity and morphology confirmed that the supramolecular ensemble was structurally intact after being reused 10 times (fig. S36). Of specific note, it is difficult to estimate the reusability of free laccase because (i) the degradation rate of free laccase is much slower such that the BPA cannot be completely eliminated in each cycle; (ii) laccase is highly soluble in water, and it is infeasible to recover the laccase after catalysis. Next, we assessed the storage stability of Lac@PHOF hybrid catalysts by storing them at room temperature for varying durations (7, 14, 21, and 30 days) before performing the catalytic activity test. The results demonstrated that Lac@PHOF exhibited remarkable stability even after being stored for 30 days (Fig. 5F and fig. S37A). However, the activity of free laccase decreased with increasing storage time and became almost completely deactivated after 30 days (fig. S37B).

These results verified that the photoenzyme-coupled catalysis organized by this supramolecular ensemble was functionally stable in a variety of biologically incompatible scenarios, highlighting the great potential of this photoenzyme catalyst in real-world applications.

Water decontamination in real samples

The depletion of fossil resources has led to a growing need for abundant, economical, and sustainable solar energy as an ideal alternative to traditional energy sources. Considering the strong absorption of visible light by Lac@PHOF, we also investigated its photocatalytic decontamination ability under sunlight (17 May 2022, Panyu District, Guangzhou). Unexpectedly, using Lac@PHOF as the catalyst, the BPA conversion rate (1.098 hour−1) was comparable to that under 420-nm LED light irradiation and was also more than 40-fold higher than those of the PHOF and Lac+PHOF under this condition (Fig. 6A). After exposing to sunlight for 4 hours, BPA achieved completely conversion by the catalysis of Lac@PHOF. Notably, even on a cloudy or raining day, significant BPA conversion efficiencies were more than 82 or 54.3% after 8 hours using Lac@PHOF as the catalyst (fig. S38).

Fig. 6. Photocatalytic decontamination in real water samples.

(A) Conversion curves of different catalysts under sunlight except free laccase in the dark. (B) BPA conversion curves in different real water samples under sunlight using Lac@PHOF as the catalyst. (C) Schematic representation of the synthesis of Lac@PHOF@AH. (D) Real picture of the BPA degradation using easy-to-separate Lac@PHOF@AH under sunlight. (E) BPA conversion curves of Lac@PHOF@AH in pure water and sewage under 420-nm light and sunlight. (F) Recyclability of Lac@PHOF@AH in pure water and the sewage in terms of BPA initial conversion rate under 420-nm light. The data of three independent experiments and calculated error bars (SD) are presented in (A), (B), (E), and (F), SD (n = 3).

We next used the Lac@PHOF ensemble to degrade BPA-involved real water samples collected from various sources, including the campus lake, Pearl River, and a sewage treatment plant in Guangzhou, China (fig. S39). Despite the complex matrix present in these real water samples, nearly 100% conversion of BPA was achieved within a 6-hour exposure to natural sunlight (Fig. 6B). At the same time, the Lac@PHOF ensemble retained their intact structure after sewage treatment (fig. S40), highlighting the exceptional decontamination capability and functional stability of our synergic photoenzyme catalysts.

The phase separation problem after the use of nanocatalysts has been a key factor restricting their practical application. We also presented easy-to-separate catalytic microspheres by means of loading Lac@PHOF nanoparticles into alginate hydrogels (named Lac@PHOF@AH) (Fig. 6C and fig. S41, A to C). The prepared Lac@PHOF@AH was structurally stable in water solution for 7 days (fig. S41, D to F) and was facile to be separated by a simple decantation method. The Lac@PHOF@AH enabled the complete conversion of BPA in the sewage after 4 hours of sunlight exposure (Fig. 6, D and E). To further investigate the reusability of the Lac@PHOF@AH, we conducted 10 cycles of conversion tests in both pure water and the sewage and examined the first-order kinetic catalytic rates of Lac@PHOF@AH in each cycle (figs. S42 and S43). The results indicated that, even after 10 repeated uses in pure water, the Lac@PHOF@AH retained almost 90% catalytic activity (Fig. 6F). It was noteworthy that, when being used in the sewage, the catalytic capability started to decrease after seven repeated uses, with the conversion rate decreasing to 44% of the initial conversion rate after 10 times of recycling. The decreased recyclability in the sewage was caused by the attachment of a high concentration of dissolved organic matter (DOM) and suspended particles onto the catalytic microspheres, which weakened the light absorption capacity of the PHOF. This phenomenon was also observed in the stability assay of DOM, in which a high concentration of DOM led to an activity inhibition of Lac@PHOF (Fig. 5C). Nevertheless, such recyclability in complex sewage samples was still encouraging as the Lac@PHOF@AH was structurally stable and easily recoverable, which holds huge potential in large-scale sewage treatment (fig. S44).

Apart from the decontamination efficiency, the biosafety of catalyst materials is a crucial consideration for sustainable applications. Here, we evaluated the toxicity of Lac@PHOF using HeLa cells. Lac@PHOF exhibited limited cytotoxicity, with cell mortality remaining below 20% even at high concentrations of up to 400 mg/liter (fig. S45). This suggested that the metal-free scaffold of the PHOF and the inherent biocompatibility of laccase rendered this hybrid catalyst highly safe. These findings underscored the immense potential of the Lac@PHOF as an environmentally friendly and sustainable catalyst for real-world water treatment applications.

DISCUSSION

In this contribution, we showcase the feasibility of using a supramolecular photoenzyme ensemble for photocatalytic water decontamination under visible light, without the need of any sacrificial agents or costly co-mediators. The efficient electron transfer between photoharvesting units and enzyme activity centers promotes the synergetic photoenzyme-coupled catalysis, achieving two orders of magnitude higher activity than free laccase for pollutant conversion under visible light. This supramolecular photoenzyme ensemble is very stable even in complex environment matrices, rendering the decontamination ability well preserved after repeated cycling. The practicability of this hybrid catalyst is evidenced by its successful application in achieving almost 100% pollutant conversion in real water samples, including river and sewage treatment plant effluent. Considering the well-sourced and low-cost attributes of laccase as well as the easy preparation of this supramolecular ensemble, our findings may offer the promise to fulfillment of the water purification and decontamination in an environmentally friendly and sustainable manner.

MATERIALS AND METHODS

Reagent and materials

All chemicals and reagents were purchased from commercial sources and used without further purification. Laccase from Trametes versicolor (≥0.5 U/mg) was purchased from Yuanye Bio-Technology Co. Ltd. (Shanghai, China). 1,3,6,8-Tetrakis(p-benzoic acid)pyrene (H4TBAPy; 98%), zinc acetate dihydrate (ZnAc2·2H2O; 99%), 2-methylimidazole (HmIm; 99%), ABTS (99%), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 2,2,6,6-tetramethylpiperidine (TEMP) were purchased from J&K Scientific (Beijing, China). TBBPA (98%) was purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). BPA [chromatographic pure (GC), 99%], BPF [analytical reagent (AR), 98%], MG (AR, 97%), phenol (GC, 99.5%), 3-CP (AR, 98%), sodium azide, AO, and CR (AR, 98%) were purchased from Macklin Inc. (Shanghai, China). Silver nitrate (SN) was purchased from Xinhong Trading Co. Ltd. (Guangzhou, China). Other organic solvents including N,N-dimethylformamide (DMF) and ethanol were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China).

Synthesis of the PHOF and Lac@PHOF

PHOF were synthesized based on recrystallization according to a reported method (46). A solution of 40 mg of H4TBAPy in 2 ml of DMF was prepared using ultrasonication. Subsequently, 24 ml of methanol was rapidly added to the H4TBAPy solution, followed by stirring for 12 hours. The resulting yellow precipitate was collected through centrifugation at 8500 rpm for 5 min and subjected to three washes with methanol. Last, the precipitate was dried at room temperature under vacuum.

Lac@PHOF was synthesized with slight modifications based on an in situ encapsulation method (27, 47). A suitable amount of hydrochloric acid was added to ultrapure water to obtain a pH 3 to 4 aqueous solution. Then, 10 mg of laccase was dissolved in 18 ml of this aqueous solution. Subsequently, the freshly prepared laccase solution was added to 2 ml of DMF containing 20 mg of H4TBAPy (H4TBAPy was completely dissolved in DMF), followed by stirring at room temperature. The mixed solution was stirred for 5 min and then allowed to stand for 25 min. The resulting yellow precipitate was collected by centrifugation at 12,000 rpm for 5 min, washed twice with water and once with methanol, and then dried under vacuum at room temperature.

Synthesis of Lac@CaCO3

Laccase was incorporated into CaCO3 through de novo encapsulation (48). Two milligrams of laccase was dissolved in 4 ml of deionized water solution of CaCl2 (330 mM), followed by adding 4 ml of deionized water solution of Na2CO3 (330 mM). The mixture system was vigorously stirred for 30 s at room temperature and aged for 15 min without stirring. The obtained Lac@CaCO3 precipitate was collected by centrifugation at 8000 rpm, washed twice with water and once with methanol, and then dried under vacuum at room temperature.

Synthesis of Lac@ZIF-8

Lac@ZIF-8 was synthesized through a coprecipitation method (49). Two milligrams of laccase was dispersed into 1 ml of deionized water and then stirred for 10 s. Two milliliters of zinc acetate solution (0.1 M) and 2 ml of HmIm solution (1.2 M) was orderly added into the laccase solution. The mixture was aged for 4 hours, and the formed Lac@ZIF-8 were collected by centrifugation at 8000 rpm, washed twice with water and once with methanol, and then dried under vacuum at room temperature.

Synthesis of Lac@NU-1003

Lac@NU-1003 was synthesized through the infiltration of laccase into the mesopore of presynthesized NU-1003.

Preparation and activation of NU-1003

A stock solution A was prepared by adding ZrOCl2·8H2O (200 mg), benzoic acid (2.5 g), and 50 ml of DMF into a 250-ml bottle. The mixture solution was heated at 80°C for 1 hour and then allowed to cool to room temperature. Then, 80 mg of organic linker 1,3,6,8-tetra(6-carboxynaphthalen-2-yl)pyrene and 50 ml of DMF were added to the 250-ml bottle to form a clear solution.

The stock solution A is evenly distributed into five 8-dram vials. For each vial, 500 μl of trifluoroacetic acid (14.72 mmol) was added and the solution was sonicated for 10 min. The reaction mixture was then placed in an oven at 120°C for 18 hours. The light yellow NU-1003 products were separated by a centrifuge and further washed by DMF (3 × 20 ml) and acetone (3 × 20 ml) and soaked in fresh acetone (20 ml) for 6 hours.

For activation of the NU-1003 samples, the obtained NU-1003 solids were soaked in 100 ml of DMF and 10 ml of 8 M HCl solution is added and placed in 100°C for 10 hours.

The MOF solid is further washed by DMF (3 × 20 ml) and ethanol (3 × 20 ml) and soaked in fresh ethanol (20 ml), and fresh ethanol was replaced six times every 2 hours.

Laccase infiltration

Laccase has a molecular dimension of ~4.4 × 4.5 × 6.4 nm. It allowed the infiltration of laccase into the mesopores (~4.7 nm) of NU-1000 in the specific orientation. A 1.5-mg laccase and 7.5 mg of NU-1003 were dispersed in 7.5 ml of 0.90 M Tris buffer (pH = 7.5). The mixed system was left in an incubator shaker at 350 rpm for 24 hours at room temperature. The precipitate was collected by centrifugation at 8000 rpm, washed twice with water and once with methanol, and then dried under vacuum at room temperature.

Estimation of laccase loading using ICP-MS

Laccase is a copper protein; thereby, the loading contents of laccase within PHOFs can be estimated by ICP-MS. A certain amount of Lac@PHOF, Lac@ZIF-8, or Lac@NU-1003 samples were digested with concentrated HNO3 and then dissolved with 1 ml of 2% HNO3. The contents of the dissolved Cu in these samples were detected and quantified using the standard curve method. Because one laccase molecule (molecular weight = 56 kDa) contains four Cu atoms, the ratio of Cu in one laccase molecule is ~0.46%. Given this ratio value, the laccase content in PHOFs could be calculated.

Structural characterization of the synthesized biohybrid catalysts

PXRD patterns were acquired at room temperature using a Bruker D8 Advance diffractometer equipped with a copper Kα radiation source. The data were collected with a step size of 0.02° and a collection time of 0.06 s per step.

The N2 adsorption/desorption isotherms were obtained using a JW-DX surface area analyzer at a temperature of −196°C. Before the measurements, all samples were subjected to activation at 100°C for a duration of 12 hours.

FTIR spectroscopy was performed using a Bruker EQUINOX 55 spectrometer using the potassium bromide (KBr) method. The spectra were acquired within the spectral range of 400 to 4000 cm−1, with a spectral resolution of 4 cm−1, and 32 repeated scans were conducted.

Cryo-EM experiments were conducted using an FEI Titan Krios G3i (D3845) transmission electron microscope operating at 300 kV. The images were acquired at a nominal magnification of 350,000, resulting in a pixel size of 0.34 Å by 0.34 Å. The total dose rate applied during data collection was ~30 e−/Å2 for each micrograph. For a more detailed description of the experimental procedure, please refer to our previous report (47).

Fluorescence labeling and CLSM experiment

For visualizing the spatial distribution of encapsulated laccase, the raw laccase was prelabeled by a fluorescence dye.

Rhodamine B labeling

The rhodamine B labeling was based on the chemical conjugation between the amino of the lysine residue of laccase and the thiocarbmide of rhodamine B isothiocyanate (RhBTC; a red fluorescence dye). In brief, 20 mg of laccase was dispersed into 10 ml of carbonate buffer solution (pH = 9.0, 0.5 M), followed by adding 1 mg of RhBTC. The mixed solution was then stirred for 12 hours in the dark. Last, the RhB-labeled laccase was obtained three times through ultrafiltration by a centrifugal filter device [molecular weight cutoff (MWCO) = 8 kDa] to remove excess reaction reagents and salts.

AF350 labeling

AF350 labeling was based on the amidation between the amino of the lysine residue of laccase and N-hydroxysuccinimide ester of AF350. Twenty milligrams of laccase was dispersed into 10 ml of phosphate buffer (pH = 8.0, 0.5 M), followed by adding 1 mg of AF350. The mixed solution was then stirred for 4 hours in the dark. Last, the AF350-labeled laccase was obtained three times through ultrafiltration by a centrifugal filter device (MWCO = 8 kDa) to remove excess reaction reagents and salts.

The raw laccase was replaced by the dye-labeled laccase to prepare the Lac@PHOF. The distribution of dye-labeled laccase in the PHOF was surveyed by a confocal laser scanning microscope (LSM 880 NLO, Carl Zeiss, Göttingen, Germany).

Laccase activity test

ABTS has been widely used as a standardized substrate for measuring the enzyme activity based on the formation of a stable ABTS radical cation, which serves as a chemically stable chromophore and exhibits pronounced absorption at a wavelength of 420 or 734 nm. Because the PHOF also showed a strong adsorption peak at about 420 nm, 734 nm was chosen to indicate the generation of the ABTS radical cation.

A solution containing 2 mM ABTS solution was prepared by dissolving 2.2 mg of ABTS in 2 ml of deionized water. Subsequently, an aliquot of 200 μl of the ABTS solution was transferred to a 96-well plate, followed by the addition of 20 μl of the free laccase solution or Lac@PHOF suspension (the laccase contents in both the laccase solution and Lac@PHOF suspension were 100 μg/ml). All the samples were kept in the dark before analysis. The absorbance at a wavelength of 734 nm was then monitored using a microplate reader. This absorbance value was directly correlated to the concentration of ABTS radical cations formed as well as the catalytic activity of laccase.

The DFT calculation of PDOS

The PDOS profile was calculated using the Vienna Ab initio Simulation Package (VASP) (50, 51) with the frozen-core projector-augmented wave method (52, 53). The exchange-correlation energy was estimated based on the generalized gradient approximation in the Perdew-Burke-Ernzerhof function (54). A cutoff energy was set to 400 eV for the plane-wave expansion. The convergence criteria for the force and electronic self-consistent iteration were set to 0.03 eV/Å and 10−4 eV, respectively. The gamma k-point was used to sample the Brillouin zone.

All-atom explicit solvent MD simulations

Laccase (Protein Data Bank ID: 1gyc) exhibits an ellipsoidal structure with a molecular size of ~4.4 × 4.5 × 6.4 nm. Given the substantial size the laccase molecule, we constructed a 25-layer 5 × 5 HOF hollow model featuring a 3 × 3 hollow cavity (~6.37 nm in width and ~9.05 nm in depth) to accommodate laccase. Of note, both the top and bottom layers were set as an intact HOF monolayer. A docking process was performed by AutoDock v4.2.6, yielding 50 conformations of Lac@PHOF, which were further categorized into 11 clusters based on a similar docking energy (fig. S10). The conformation with the lowest energy was selected for subsequent MD simulations.

MD simulations were performed with Amber FF14SB force field and the TIP3P water model, while the hollowed HOF model was described with GAFF2 force field by the Amber20 package. In the simulation process, water and ions are equilibrated first, followed by successive release of restraints on the side chains of the protein and then the entire protein. The equilibrated process included an energy minimization, heating in the NVT ensemble, and equilibration in the NPT ensemble. After equilibration steps, a 100-ns simulation in the NPT ensemble was performed, generating a total of 1000 snapshots. The equilibrated state between 60 and 100 ns was used to investigate the interfacial interactions between the laccase guest and HOF host.

Catalytic degradation of BPA in the water sample

BPA samples were prepared by adding 100 μl of a stock solution with a concentration of 1000 mg/liter in ethanol to 5 ml of water in a 10-ml clear vial. Subsequently, 20 μl of the catalyst suspension involving the Lac@PHOF, PHOF, or free laccase (all the laccase contents were kept at 0.2 mg/ml) was added to each sample. The samples were then placed in a light irradiation chamber equipped with an LED light with a wavelength of 420 nm.

BPA monitoring by DI-SPME coupled with GC-MS

The concentration change of BPA during the catalytic degradation process was monitored using direct immersion solid-phase microextraction (DI-SPME) coupled with GC-MS (55). The sample solution was initially diluted into a 10-ml sample vial. Subsequently, a polyacrylate (PA) SPME coating was used for sampling the BPA for 20 min with direct immersion mode. After extraction, the PA fiber was desorbed in an Agilent 7890 GC-5977 MS equipped with a DB-5MS column to quantify the extracted amount of BPA. The SPME extraction and desorption procedures described were performed by a GERTEL MPS autosampler. The BPA concentration was quantified by comparing the peak area with the water sample spiked with a known concentration of BPA.

Evaluation of TOF

TOF refers to the number of substrate molecules converted into product per unit time by a single active site of the enzyme. It represents the catalytic efficiency or activity of the enzyme and can be calculated as followsTOF=degradation rate of BPA in mol/houramounts of enzyme molecules

For Lac@PHOF, the degradation rate of BPA in mol/hour = 8.8 × 10−5 M × 5 × 10−3 liters × 1.795 hour−1 = 7.90 × 10−7 mol/hour, where 1.795 hour−1 is the initial conversion rate of BPA obtained from the degradation curves under the 420-nm LED light.

The amounts of enzyme molecules (m/M) = 40 × 10% × 10−6 g ÷ 56,000 g/mol = 7.14 × 10−11 mol, where m (g) is the mass of enzymes added to the reaction solution; M (56,000 g/mol) is the relative molecular mass of the laccase enzymes.

For laccase, the degradation rate of BPA in mol/hour = 8.8 × 10−5 M × 5 × 10−3 liters × 0.007 hour−1 = 3.08 × 10−9 mol/hour, where 0.007 hour−1 is the conversion rate obtained from the degradation curves in the dark.

The amounts of enzyme molecules (m/M) = 4 × 10−6 g ÷ 56,000 g/mol = 7.14 × 10−11 mol, where m (g) is the mass of enzymes added to the reaction solution; M (56,000 g/mol) is the relative molecular mass of the laccase enzymes.

TOF = 1.1 × 104 hour−1 for Lac@PHOF

TOF = 4.3 × 10 hour−1 for laccase

Determination of the oxidation potential of BPA

The oxidation potential of BPA was obtained from linear sweep voltammograms, which was done by loading Lac@PHOF onto a glassy carbon electrode, using Ag/AgCl as the reference electrode and Pt electrode as the counter electrode. The Lac@PHOF was loaded on to the electrode by applying an appropriate amount of the Lac@PHOF suspension onto the electrode and then vaporizing the solvent by an infrared heat lamp. The control group consisted of a 0.5 M Na2SO4 solution at pH 7, while the test group involved the addition of BPA to the control group, resulting in a concentration of 20 mg/liter. The scanning voltage was set from −0.2 to 0.8 V, with a scanning rate of 0.1 V/s.

Monitoring the dissolved oxygen

The dissolved oxygen detection was carried out in 40-ml transparent glass vials containing 20 ml of a solution of BPA (a concentration of 20 mg/liter). Both the PHOF and Lac@PHOF were added at a concentration of 8 mg/liter. The luminescent dissolved oxygen probe (LDO101101 Electrode for Portable Water Quality Analyzer HQ-30d, HACH Company) was positioned at the center of the liquid level and fixed to the cap of the vial. To ensure a relatively enclosed condition, Parafilm was used to seal the cap. Subsequently, the samples were subjected to reaction under 420-nm LED light and dark conditions, and the dissolved oxygen in the samples was recorded at different time intervals. For samples containing AgNO3 and NaN3, all conditions were identical to those mentioned above, except for the addition of AgNO3 (20 g/liter) and NaN3 (0.5 g/liter) in the exposure samples. The schematic diagram of experimental operation was shown in fig. S27.

Ultralow-temperature ESR experiment

The Cu valence change of laccase in Lac@PHOF solids under light irradiation at 420 nm in N2 and O2 atmosphere was explored by a Bruker EMX plus 10/12 equipped with an Oxford ESR910 Liquid Helium cryostat. The test temperature was set at 5 K.

EPR test

EPR was used to probe possible reactive species in the reaction systems. The measurement was performed using a Bruker Elexsys E500 EPR equipped with both a standard resonator and a CoolEdge cryo system (Billerica, United States). The instrument settings were as follows: 20.0-mW microwave power, 9.8-GHz microwave frequency, 100-kHz modulation frequency, 1.00-G modulation amplitude, 3515-G center field, 150-G sweep width, and 40.0-s sweep time. A 50.0 mM DMPO solution was used as the spin trapping agent for the hydroxyl radical (•OH) and super oxygen (•O2−), while 50.0 mM TEMP was used as the spin trapping agent for singlet oxygen (1O2). The EPR measurement for •OH was conducted using a 1:1 mixture of ultrapure water and acetonitrile as the solvent. To avoid the interference of •OH signal, the EPR measurement for •O2− was performed in pure methanol. On the other hand, the measurement for 1O2 was conducted in pure water. Twenty microliters of each catalytic material including the Lac@PHOF, PHOF, laccase, and Lac+PHOF was added into the solutions. After 10 min of irradiation under the LED lamp at 420 nm, the reaction solution was extracted by a 1-mm quartz tube and then placed into a 4-mm quartz EPR tube, which was immediately loaded into the EPR.

Mott-Schottky measurement and on-off cycle measurement

In the Mott-Schottky measurements, a quartz cell filled with a 0.5 M Na2SO4 electrolyte solution was used. Three electrodes were inserted into the cell, and the measurements were performed at three different frequencies: 1000, 2000, and 3000 Hz.

For the on-off cycle measurement, a quartz cell filled with a 0.1 M Na2SO4 electrolyte solution was used. A standard three-electrode setup was used, with a Pt grid electrode as the counter electrode and an Ag/AgCl electrode (saturated with KCl) serving as the reference electrode. The measurements were conducted on a CHI 750E instrument. To measure the photocurrent response curve under dark and light conditions, a xenon lamp (600 W/m2) was used. The light was switched on and off every 20 s, and the measurements were performed using the aforementioned three-electrode setup.

Quenching experiment of active species

In this experiment, a 5-ml solution containing AO (20 g/liter) and SN (20 g/liter) was first prepared. AO and SN were used as holes and photogenerated electron scavengers, respectively. Then, 20 μl of the BPA stock solution with a concentration of 5000 mg/liter and 20 μl of the Lac@PHOF stock solution with a concentration of 2 mg/ml were added to the above solutions. Subsequently, all the samples were exposed to 420-nm LED light for different durations. Last, the amount of BPA left in the samples was quantified by DI-SPME coupled with GC-MS.

Effects of environmental factors on the catalytic efficiency of Lac@PHOF and laccase

To evaluate the impact of pH on the degradation performance, spiking solutions with pH values of 2.5, 4.0, 5.5, 7.5, 8.5, and 11 were prepared using orthophosphoric acid and phosphate buffer solutions. Subsequently, Lac@PHOF and laccase were separately added to 5 ml of each stock solution and incubated in a shaker for 24 hours. To assess the influence of temperature on the degradation performance, Lac@PHOF and laccase were added to 5 ml of deionized water. The solutions were then placed in a water bath set to temperatures of 30°, 40°, 60°, and 80°C and incubated for 24 hours. To investigate the effect of salt concentration, appropriate amounts of sodium chloride were dissolved in deionized water to prepare solutions with mass fractions of 3.5, 10, and 20% sodium chloride. Subsequently, Lac@PHOF and laccase were added to 5 ml of each sodium chloride solution with different mass fractions and incubated for 24 hours. To examine the impact of NOM, suitable amounts of NOM were dissolved in deionized water to create solutions with NOM concentrations of 10, 50, and 100 mg/liter. Lac@PHOF and laccase were then added to 5 ml of each NOM solution with different concentrations and incubated for 24 hours.

All the aforementioned experiments were conducted in 10-ml transparent glass bottles, with Lac@PHOF and laccase added at a laccase amount of 0.8 mg/liter and the BPA concentration of 20 mg/liter. Samples containing Lac@PHOF were exposed to 420-nm LED light for 5, 10, 20, 40, 60, 120, and 240 min, while samples containing laccase underwent dark reaction for 12, 24, 36, 48, and 72 hours. Subsequently, DI-SPME coupled to GC-MS analysis was performed for quantification of the BPA in the exposed solutions. The conversion curves at each group were presented in figs. S31 and S34.

Evaluation of the reusability and storage stability of the Lac@PHOF and laccase

Reusability test

Ten milliliters of deionized water was added into a 20-ml clear vial, and 40 μl of the BPA stock solution (5000 mg/liter) and 40 μl of the Lac@PHOF suspension (2 mg/ml) were subsequently added. The vial was placed in the chamber equipped by a 420-nm LED light and exposed for 2 hours. During this period, 0.2 ml of the exposed solution was taken at intervals of 5, 10, 20, 40, 60, and 120 min for BPA residue quantification. After 2 hours of each exposure, an amount of the BPA stock solution was reintroduced so that the initial BPA concentration remained unchanged, and the irradiation process was repeated for another 2 hours. This procedure was repeated 10 times, and the BPA concentration in the solution was quantified after each 2-hour exposure. The conversion curves at each cycle were presented in fig. S35.

Storability test

The Lac@PHOF and laccase were prepared in deionized water at a concentration of 2 and 0.4 mg/ml, respectively. Subsequently, they were stored at room temperature (23° ± 2°C) for durations of 7, 14, 21, and 30 days. After specified storage periods, the catalytic activity of these stored catalysts was examined by degradation of BPA with a concentration of 20 mg/liter. The conversion curves were presented in fig. S37.

Degradation of BPA by Lac@PHOF under sunlight

Exposure solutions (5 ml) with a BPA concentration of 20 mg/liter and a Lac@PHOF concentration of 8 mg/liter were prepared using ultrapure water. The solutions were exposed under the sunlight from 9:00 a.m. and collected after 2, 4, 6, and 8 hours. Cloudy day was selected on 8 May 2023, rainy day on 12 May 2023, and sunny day on 27 May 2023. Experiments were conducted at the Panyu Campus, Jinan University, Guangzhou, China with a longitude of 113.414221° and a latitude of 23.018181°.

Degradation of BPA spiked in the real water samples under sunlight

Real water samples were collected from the campus lake, Pearl River, and a sewage treatment plant. Detailed location information can be found in fig. S39A.

A 0.01-g EDTA solution was added into 5 ml of the untreated real water samples. Then, BPA with a concentration of 20 mg/liter and Lac@PHOF with a concentration of 8 mg/liter were added into the samples. The spiked samples were exposed to sunlight from 10:00 a.m. and collected after 1, 2, 4, and 6 hours. The experiments were carried out on 2 August 2023 at the Panyu Campus, Jinan University, Guangzhou, China with a longitude of 113.414221° and a latitude of 23.018181°.

Synthesis of hydrogel microspheres loaded with Lac@PHOF

The synthesis method of hydrogels was adapted from the previous literature with slight modification (56). Initially, 40 mg of Lac@PHOF biocomposite powder was uniformly dispersed in 1 ml of ultrapure water through ultrasonication. Subsequently, 20 mg of sodium alginate was added to the suspension under magnetic stirring, forming a mixed solution A. Following this, a 0.05 M polyacrylic acid [molecular weight (MW): 2000 Da] solution dissolved with CaCl2 (60 mM) was prepared as solution B. Next, solution A was loaded into a 1-ml disposable syringe and cautiously dripped into solution B to create the hydrogel microspheres. These microspheres were then filtered, washed three times with pure water, and stored for subsequent use.

Acknowledgments

We thank X. Ma from the Southern University of Science and Technology for the help of cryo-EM imaging.

Funding: This work was supported by the National Natural Science Foundation of China 22174164 (G.C.); the National Natural Science Foundation of China 22104159 (S.H.); the Guangdong Basic and Applied Basic Research Foundation 2024B1515020070 (G.C.); the Fundamental Research Funds for the Central Universities, Sun Yat-sen University 23lgbj005 (G.C.); and the Guangdong Basic Research Center of Excellence for Functional Molecular Engineering 31000-42080002 (G.O.).

Author contributions: Conceptualization: G.C. Methodology: G.C., R.J., Y.S., and G.O. Investigation: R.J., G.L., G.C., Y.S., Y.L., L.T., A.H., Y.Z., and S.H. Visualization: R.J., G.L., G.C., S.H., and G.O. Supervision: G.C., Y.S., and G.O. Writing—original draft: R.J. and G.C. Writing—review and editing: G.C., R.J., G.L., Y.S., and G.O.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S45

Table S1

References
==== Refs
REFERENCES AND NOTES

1 S. Ghosh, N. A. Kouame, L. Ramos, S. Remita, A. Dazzi, A. Deniset-Besseau, P. Beaunier, F. Goubard, P. H. Aubert, H. Remita, Conducting polymer nanostructures for photocatalysis under visible light. Nat. Mater. 14 , 505–511 (2015).25774954
2 K. Kalyanasundaram, M. Graetzel, Artificial photosynthesis: Biomimetic approaches to solar energy conversion and storage. Curr. Opin. Biotechnol. 21 , 298–310 (2010).20439158
3 J. Lv, J. Xie, A. G. A. Mohamed, X. Zhang, Y. Feng, L. Jiao, E. Zhou, D. Yuan, Y. Wang, Solar utilization beyond photosynthesis. Nat. Rev. Chem. 7 , 91–105 (2023).37117911
4 E. Romero, V. I. Novoderezhkin, R. van Grondelle, Quantum design of photosynthesis for bio-inspired solar-energy conversion. Nature 543 , 355–365 (2017).28300093
5 Y. Wu, J. Shi, D. Li, S. Zhang, B. Gu, Q. Qiu, Y. Sun, Y. Zhang, Z. Cai, Z. Jiang, Synergy of electron transfer and electron utilization via metal-organic frameworks as an electron buffer tank for nicotinamide regeneration. ACS Catal. 10 , 2894–2905 (2020).
6 L. Schmermund, V. Jurkaš, F. F. Özgen, G. D. Barone, H. C. Büchsenschütz, C. K. Winkler, S. Schmidt, R. Kourist, W. Kroutil, Photo-biocatalysis: Biotransformations in the presence of light. ACS Catal. 9 , 4115–4144 (2019).
7 Z. Wang, Y. Hu, S. Zhang, Y. Sun, Artificial photosynthesis systems for solar energy conversion and storage: Platforms and their realities. Chem. Soc. Rev. 51 , 6704–6737 (2022).35815740
8 S. Zhang, S. Liu, Y. Sun, S. Li, J. Shi, Z. Jiang, Enzyme-photo-coupled catalytic systems. Chem. Soc. Rev. 50 , 13449–13466 (2021).34734949
9 S.-H. Lee, J.-K. Ryu, D.-H. Nam, C.-B. Park, Photoenzymatic synthesis through sustainable NADH regeneration by SiO2-supported quantum dots. Chem. Commun. 47 , 4643, 4645 (2011).
10 J. Liu, M. Antonietti, Bio-inspired NADH regeneration by carbon nitride photocatalysis using diatom templates. Energy Environ. Sci. 6 , 1486 (2013).
11 R. K. Yadav, J. O. Baeg, G. H. Oh, N. J. Park, K. J. Kong, J. Kim, D. W. Hwang, S. K. Biswas, A photocatalyst-enzyme coupled artificial photosynthesis system for solar energy in production of formic acid from CO2. J. Am. Chem. Soc. 134 , 11455–11461 (2012).22769600
12 K. A. Brown, D. F. Harris, M. B. Wilker, A. Rasmussen, N. Khadka, H. Hamby, S. Keable, G. Dukovic, J. W. Peters, L. C. Seefeldt, P. W. King, Light-driven dinitrogen reduction catalyzed by a CdS: Nitrogenase MoFe protein biohybrid. Science 352 , 448–450 (2016).27102481
13 S. H. Lee, D. S. Choi, S. K. Kuk, C. B. Park, Photobiocatalysis: Activating redox enzymes by direct or indirect transfer of photoinduced electrons. Angew. Chem. Int. Ed. Engl. 57 , 7958–7985 (2018).29194901
14 M. A. Emmanuel, S. G. Bender, C. Bilodeau, J. M. Carceller, J. S. DeHovitz, H. Fu, Y. Liu, B. T. Nicholls, Y. Ouyang, C. G. Page, T. Qiao, F. C. Raps, D. R. Sorigue, S. Z. Sun, J. Turek-Herman, Y. Ye, A. Rivas-Souchet, J. Cao, T. K. Hyster, Photobiocatalytic strategies for organic synthesis. Chem. Rev. 123 , 5459–5520 (2023).37115521
15 X. Wang, T. Saba, H. H. P. Yiu, R. F. Howe, J. A. Anderson, J. Shi, Cofactor NAD(P)H regeneration inspired by heterogeneous pathways. Chem 2 , 621–654 (2017).
16 X. Ji, Z. Su, P. Wang, G. Ma, S. Zhang, Integration of artificial photosynthesis system for enhanced electronic energy-transfer efficacy: A case study for solar-energy driven bioconversion of carbon dioxide to methanol. Small 12 , 4753–4762 (2016).27273818
17 S. Zhang, Y. Zhang, Y. Chen, D. Yang, S. Li, Y. Wu, Y. Sun, Y. Cheng, J. Shi, Z. Jiang, Metal hydride-embedded titania coating to coordinate electron transfer and enzyme protection in photo-enzymatic catalysis. ACS Catal. 11 , 476–483 (2020).
18 W. Zhang, E. Fernandez-Fueyo, Y. Ni, M. van Schie, J. Gacs, R. Renirie, R. Wever, F. G. Mutti, D. Rother, M. Alcalde, F. Hollmann, Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalisations. Nat. Catal. 1 , 55–62 (2018).29430568
19 Z. C. Litman, Y. Wang, H. Zhao, J. F. Hartwig, Cooperative asymmetric reactions combining photocatalysis and enzymatic catalysis. Nature 560 , 355–359 (2018).30111790
20 N. Yang, Y. Tian, M. Zhang, X. Peng, F. Li, J. Li, Y. Li, B. Fan, F. Wang, H. Song, Photocatalyst-enzyme hybrid systems for light-driven biotransformation. Biotechnol. Adv. 54 , 107808 (2022).34324993
21 R. B. Lin, B. Chen, Hydrogen-bonded organic frameworks: Chemistry and functions. Chem 8 , 2114–2135 (2022).
22 B. Wang, R.-B. Lin, Z. Zhang, S. Xiang, B. Chen, Hydrogen-bonded organic frameworks as a tunable platform for functional materials. J. Am. Chem. Soc. 142 , 14399–14416 (2020).32786796
23 W. Liang, F. Carraro, M. B. Solomon, S. G. Bell, H. Amenitsch, C. J. Sumby, N. G. White, P. Falcaro, C. J. Doonan, Enzyme encapsulation in a porous hydrogen-bonded organic framework. J. Am. Chem. Soc. 141 , 14298–14305 (2019).31426638
24 Q. Yin, P. Zhao, R. J. Sa, G. C. Chen, J. Lu, T. F. Liu, R. Cao, An ultra-robust and crystalline redeemable hydrogen-bonded organic framework for synergistic chemo-photodynamic therapy. Angew. Chem. Int. Ed. Engl. 57 , 7691–7696 (2018).29696754
25 Q. Zhou, Y. Guo, Y. Zhu, Photocatalytic sacrificial H2 evolution dominated by micropore-confined exciton transfer in hydrogen-bonded organic frameworks. Nat. Catal. 6 , 574–584 (2023).
26 S. Riva, Laccases: Blue enzymes for green chemistry. Trends Biotechnol. 24 , 219–226 (2006).16574262
27 G. Chen, S. Huang, Y. Shen, X. Kou, X. Ma, S. Huang, Q. Tong, K. Ma, W. Chen, P. Wang, J. Shen, F. Zhu, G. Ouyang, Protein-directed, hydrogen-bonded biohybrid framework. Chem 7 , 2722–2742 (2021).
28 A. Barth, Infrared spectroscopy of proteins. Biochim. Biophys. Acta Bioenerg. 1767 , 1073–1101 (2007).
29 C. Madzak, M. C. Mimmi, E. Caminade, A. Brault, S. Baumberger, P. Briozzo, C. Mougin, C. Jolivalt, Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis. Protein Eng. Des. Sel. 19 , 77–84 (2006).16368720
30 W. Huang, H. Yuan, H. Yang, X. Ma, S. Huang, H. Zhang, S. Huang, G. Chen, G. Ouyang, Green synthesis of stable hybrid biocatalyst using a hydrogen-bonded, π-π-stacking supramolecular assembly for electrochemical immunosensor. Nat. Commun. 14 , 3644 (2023).37339954
31 G. Chen, L. Tong, S. Huang, S. Huang, F. Zhu, G. Ouyang, Hydrogen-bonded organic framework biomimetic entrapment allowing non-native biocatalytic activity in enzyme. Nat. Commun. 13 , 4816 (2022).35974100
32 S. Grimme, Do special noncovalent π-π stacking interactions really exist? Angew. Chem. Int. Ed. Engl. 47 , 3430–3434 (2008).18350534
33 S. Li, Y. Sheng, S. Xiao, Q. Liu, K. Sun, Exolaccase propels humification to decontaminate bisphenol A and create humic-like biostimulants. J. Agric. Food Chem. 71 , 11386–11395 (2023).37470251
34 C. Wang, Q. Zhang, X. Wang, H. Chang, S. Zhang, Y. Tang, J. Xu, R. Qi, Y. Cheng, Dynamic modulation of enzyme activity by near-infrared light. Angew. Chem. Int. Ed. Engl. 56 , 6767–6772 (2017).28481023
35 D. V. Volodkin, N. I. Larionova, G. B. Sukhorukov, Protein encapsulation via porous CaCO3 microparticles templating. Biomacromolecules 5 , 1962–1972 (2004).15360312
36 L. Tong, S. Huang, Y. Shen, S. Liu, X. Ma, F. Zhu, G. Chen, G. Ouyang, Atomically unveiling the structure-activity relationship of biomacromolecule-metal-organic frameworks symbiotic crystal. Nat. Commun. 13 , 951 (2022).35177632
37 P. Li, S. Y. Moon, M. A. Guelta, L. Lin, D. A. Gomez-Gualdron, R. Q. Snurr, S. P. Harvey, J. T. Hupp, O. K. Farha, Nanosizing a metal-organic framework enzyme carrier for accelerating nerve agent hydrolysis. ACS Nano 10 , 9174–9182 (2016).27701869
38 P. Li, Q. Chen, T. C. Wang, N. A. Vermeulen, B. L. Mehdi, A. Dohnalkova, N. D. Browning, D. Shen, R. Anderson, D. A. Gómez-Gualdrón, F. M. Cetin, J. Jagiello, A. M. Asiri, J. F. Stoddart, O. K. Farha, Hierarchically engineered mesoporous metal-organic frameworks toward cell-free immobilized enzyme systems. Chem 4 , 1022–1034 (2018).
39 J. T. Holland, C. Lau, S. Brozik, P. Atanassov, S. Banta, Engineering of glucose oxidase for direct electron transfer via site-specific gold nanoparticle conjugation. J. Am. Chem. Soc. 133 , 19262–19265 (2011).22050076
40 F. Mao, N. Mano, A. Heller, Long tethers binding redox centers to polymer backbones enhance electron transport in enzyme “wiring” hydrogels. J. Am. Chem. Soc. 125 , 4951–4957 (2003).12696915
41 T. Lazarides, I. V. Sazanovich, A. J. Simaan, M. C. Kafentzi, M. Delor, Y. Mekmouche, B. Faure, M. Reglier, J. A. Weinstein, A. G. Coutsolelos, T. Tron, Visible light-driven O2 reduction by a porphyrin-laccase system. J. Am. Chem. Soc. 135 , 3095–3103 (2013).23330664
42 A. J. Augustine, C. Kjaergaard, M. Qayyum, L. Ziegler, D. J. Kosman, K. O. Hodgson, B. Hedman, E. I. Solomon, Systematic perturbation of the trinuclear copper cluster in the multicopper oxidases: The role of active site asymmetry in its reduction of O2 to H2O. J. Am. Chem. Soc. 132 , 6057–6067 (2010).20377263
43 Y. Wen, A. Renteria-Gomez, G. S. Day, M. F. Smith, T. H. Yan, R. O. K. Ozdemir, O. Gutierrez, V. K. Sharma, X. Ma, H. C. Zhou, Integrated photocatalytic reduction and oxidation of perfluorooctanoic acid by metal-organic frameworks: Key insights into the degradation mechanisms. J. Am. Chem. Soc. 144 , 11840–11850 (2022).35732040
44 W. Du, C. Sun, J. Liang, Y. Han, J. Yu, Z. Liang, Improvement of laccase production and its characterization by mutagenesis. J. Food Biochem. 39 , 101–108 (2015).
45 M. Vázquez-González, C. Wang, I. Willner, Biocatalytic cascades operating on macromolecular scaffolds and in confined environments. Nat. Catal. 3 , 256–273 (2020).
46 L. Tong, Y. Lin, X. Kou, Y. Shen, Y. Shen, S. Huang, F. Zhu, G. Chen, G. Ouyang, Pore-environment-dependent photoresponsive oxidase-like activity in hydrogen-bonded organic frameworks. Angew. Chem. Int. Ed. Engl. 62 , e202218661 (2023).36719177
47 G. Chen, S. Huang, X. Ma, R. He, G. Ouyang, Encapsulating and stabilizing enzymes using hydrogen-bonded organic frameworks. Nat. Protoc. 18 , 2032–2050 (2023).37198321
48 A. I. Petrov, D. V. Volodkin, G. B. Sukhorukov, Protein-calcium carbonate coprecipitation: A tool for protein encapsulation. Biotechnol. Prog. 21 , 918–925 (2005).15932274
49 G. Chen, S. Huang, X. Kou, F. Zhu, G. Ouyang, Embedding functional biomacromolecules within peptide-directed metal-organic framework (MOF) nanoarchitectures enables activity enhancement. Angew. Chem. Int. Ed. Engl. 59 , 13947–13954 (2020).32400001
50 G. Kresse, J. Furthmüller, Efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set. Phys. Rev. B 54 , 11169–11186 (1996).
51 G. Kresse, J. Furthmüller, Efficiency of ab-initio total energy calculations for metals and semiconductors using a plane-wave basis set. Comput. Mater. Sci. 6 , 15–50 (1996).
52 G. Kresse, D. Joubert, From ultrasoft pseudopotentials to the projector augmented-wave method. Phys. Rev. B 59 , 1758–1775 (1999).
53 P. E. Blöchl, Projector augmented-wave method. Phys. Rev. B 50 , 17953–17979 (1994).
54 J. P. Perdew, K. Burke, M. Ernzerhof, Generalized gradient approximation made simple. Phys. Rev. Lett. 77 , 3865–3868 (1996).10062328
55 Q.-Z. Su, P. Vera, C. Nerín, Direct immersion-solid-phase microextraction coupled to gas chromatography-mass spectrometry and response surface methodology for nontarget screening of (semi-) volatile migrants from food contact materials. Anal. Chem. 92 , 5577–5584 (2020).32186366
56 N. Zhong, R. Gao, Y. Shen, X. Kou, J. Wu, S. Huang, G. Chen, G. Ouyang, Enzymes-encapsulated defective metal–organic framework hydrogel coupling with a smartphone for a portable glucose biosensor. Anal. Chem. 94 , 14385–14393 (2022).36205458
57 M. Rani, Rachna, U. Shanker, Efficient photocatalytic degradation of Bisphenol A by metal ferrites nanoparticles under sunlight. Environ. Technol. Innov. 19 , 100792 (2020).
58 G. F. Schirinzi, I. Pérez-Pomeda, J. Sanchís, C. Rossini, M. Farré, D. Barceló, Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells. Environ. Res. 159 , 579–587 (2017).28898803
59 S. T. Hossain, S. K. Mukherjee, Toxicity of cadmium sulfide (CdS) nanoparticles against Escherichia coli and HeLa cells. J. Hazard. Mater. 260 , 1073–1082 (2013).23892173
60 K. M. Ramkumar, C. Manjula, G. GnanaKumar, M. A. Kanjwal, T. V. Sekar, R. Paulmurugan, P. Rajaguru, Oxidative stress-mediated cytotoxicity and apoptosis induction by TiO2 nanofibers in HeLa cells. Eur. J. Pharm. Biopharm. 81 , 324–333 (2012).22446064
61 G. Wang, H. Li, N. Li, D. Chen, J. He, Q. Xu, J. Lu, Construction of perylene-based amphiphilic micelle and its efficient adsorption and in situ photodegradation of bisphenol A in aqueous solution. Angew. Chem. Int. Ed. Engl. 61 , e202210619 (2022).35972462
62 L. Yang, J. Yuan, G. Wang, Q. Cao, C. Zhang, M. Li, J. Shao, Y. Xu, H. Li, J. Lu, Construction of tri-functional HOFs material for efficient selective adsorption and photodegradation of bisphenol A and hydrogen production. Adv. Funct. Mater. 33 , 2300954 (2023).
63 L. Dekanovsky, H. Huang, S. Akir, Y. Ying, Z. Sofer, B. Khezri, Light-driven MXene-based microrobots: Mineralization of bisphenol A to CO2 and H2O. Small Methods 7 , e2201547 (2023).37075736
64 S. Zhang, Y. Liu, R. Ma, D. Jia, T. Wen, Y. Ai, G. Zhao, F. Fang, B. Hu, X. Wang, Molybdenum (VI)-oxo clusters incorporation activates g-C3N4 with simultaneously regulating charge transfer and reaction centers for boosting photocatalytic performance. Adv. Funct. Mater. 32 , 2204175 (2022).
65 J. Liang, F. Liu, M. Li, W. Liu, M. Tong, Facile synthesis of magnetic Fe3O4@BiOI@AgI for water decontamination with visible light irradiation: Different mechanisms for different organic pollutants degradation and bacterial disinfection. Water Res. 137 , 120–129 (2018).29547775
66 E. Garcia-Diaz, D. Zhang, Y. Li, R. Verduzco, P. J. J. Alvarez, TiO2 microspheres with cross-linked cyclodextrin coating exhibit improved stability and sustained photocatalytic degradation of bisphenol A in secondary effluent. Water Res. 183 , 116095 (2020).32645579
67 J. Guo, H. Sun, X. Yuan, L. Jiang, Z. Wu, H. Yu, N. Tang, M. Yu, M. Yan, J. Liang, Photocatalytic degradation of persistent organic pollutants by Co-Cl bond reinforced CoAl-LDH/Bi12O17Cl2 photocatalyst: Mechanism and application prospect evaluation. Water Res. 219 , 118558 (2022).35569278
68 S. Zhang, H. Lan, Y. Cui, X. An, H. Liu, J. Qu, Insight into the key role of Cr intermediates in the efficient and simultaneous degradation of organic contaminants and Cr(VI) reduction via g-C3N4-assisted photocatalysis. Environ. Sci. Technol. 56 , 3552–3563 (2022).35212521
69 H. Huang, X. Li, J. Wang, F. Dong, P. K. Chu, T. Zhang, Y. Zhang, Anionic group self-doping as a promising strategy: Band-gap engineering and multi-functional applications of high-performance CO32−-doped Bi2O2CO3. ACS Catal. 5 , 4094–4103 (2015).
70 P. Xu, P. Wang, Q. Wang, R. Wei, Y. Li, Y. Xin, T. Zheng, L. Hu, X. Wang, G. Zhang, Facile synthesis of Ag2O/ZnO/rGO heterojunction with enhanced photocatalytic activity under simulated solar light: Kinetics and mechanism. J. Hazard. Mater. 403 , 124011 (2021).33265040
71 J. Han, Z. Zhu, N. Li, D. Chen, Q. Xu, H. Li, J. He, J. Lu, Metalloporphyrin-based D-A type conjugated organic polymer nanotube for efficient photocatalytic degradation. Appl. Catal. B Environ. 291 , 120108 (2021).
72 T. Zeng, S. Xia, S. Li, X. Hong, Y. Wang, L. Wang, X. Huang, Kinetic and mechanistic investigations of the oxidation of organics by near-infrared light driven thermocatalytic activation of peroxydisulfate with Fe3O4. Chem. Eng. J. 455 , 140629 (2023).
73 Z. Huang, H. Yu, L. Wang, X. Liu, S. Ren, J. Liu, Ferrocene-modified UiO-66-NH2 hybrids with g-C3N4 as enhanced photocatalysts for degradation of bisphenol A under visible light. J. Hazard. Mater. 436 , 129052 (2022).35580498
74 J. Han, N. Li, D. Chen, Q. Xu, J. Lu, Boosting photocatalytic activity for porphyrin-based D-A conjugated polymers via dual metallic sites regulation. Appl. Catal. B Environ. 317 , 121724 (2022).
75 S. K. S. Kumar, R. D. Kaushik, L. P. Purohit, ZnO-CdO nanocomposites incorporated with graphene oxide nanosheets for efficient photocatalytic degradation of bisphenol A, thymol blue and ciprofloxacin. J. Hazard. Mater. 424 , 127332 (2022).34607025
