Enzyme-Immobilized Porous Crystals for Environmental Applications.

Developing efficient technologies to eliminate or degrade contaminants is paramount for environmental protection. Biocatalytic decontamination offers distinct advantages in terms of selectivity and efficiency; however, it still remains challenging when applied in complex environmental matrices. The main challenge originates from the instability and difficult-to-separate attributes of fragile enzymes, which also results in issues of compromised activity, poor reusability, low cost-effectiveness, etc. One viable solution to harness biocatalysis in complex environments is known as enzyme immobilization, where a flexible enzyme is tightly fixed in a solid carrier. In the case where a reticular crystal is utilized as the support, it is feasible to engineer next-generation biohybrid catalysts functional in complicated environmental media. This can be interpreted by three aspects: (1) the highly crystalline skeleton can shield the immobilized enzyme against external stressors. (2) The porous network ensures the high accessibility of the interior enzyme for catalytic decontamination. And (3) the adjustable and unambiguous structure of the reticular framework favors in-depth understanding of the interfacial interaction between the framework and enzyme, which can in turn guide us in designing highly active biocomposites. This Review aims to introduce this emerging biocatalysis technology for environmental decontamination involving pollutant degradation and greenhouse gas (carbon dioxide) conversion, with emphasis on the enzyme immobilization protocols and diverse catalysis principles including single enzyme catalysis, catalysis involving enzyme cascades, and photoenzyme-coupled catalysis. Additionally, the remaining challenges and forward-looking directions in this field are discussed. We believe that this Review may offer a useful biocatalytic technology to contribute to environmental decontamination in a green and sustainable manner and will inspire more researchers at the intersection of the environment science, biochemistry, and materials science communities to co-solve environmental problems.

A Synergetic Pore Compartmentalization and Hydrophobization Strategy for Synchronously Boosting the Stability and Activity of Enzyme.

Spatial immobilization of fragile enzymes using a nanocarrier is an efficient means to design heterogeneous biocatalysts, presenting superior stability and recyclability to pristine enzymes. An immobilized enzyme, however, usually compromises its catalytic activity because of inevasible mass transfer issues and the unfavorable conformation changes in a confined environment. Here, we describe a synergetic metal-organic framework pore-engineering strategy to trap lipase (an important hydrolase), which confers lipase-boosted stability and activity simultaneously. The hierarchically porous NU-1003, featuring interconnected mesopore and micropore channels, is precisely modified by chain-adjustable fatty acids on its mesopore channel, into which lipase is trapped. The interconnected pore structure ensures efficient communication between trapped lipase and exterior media, while the fatty acid-mediated hydrophobic pore can activate the opening conformation of lipase by interfacial interaction. Such dual pore compartmentalization and hydrophobization activation effects render the catalytic center of trapped lipase highly accessible, resulting in 1.57-fold and 2.46-fold activities as native lipase on ester hydrolysis and enantioselective catalysis. In addition, the feasibility of these heterogeneous biocatalysts for kinetic resolution of enantiomer is also validated, showing much higher efficiency than native lipase.

Developing Covalent Organic Framework Biocatalysts through Enzyme Encapsulation.

Utilizing covalent organic frameworks (COFs) as porous supports to encapsulate enzyme represents an advanced strategy for constructing COFs biocatalysts, which has inspired numerous interests across various applications. As the structural advantages including ultrastable covalent-bonded linkage, tailorable pore structure, and metal-free biocompatibility, the resultant enzyme-COFs biocatalysts showcase functional enhancement in catalytic activity, chemical stability, long-term durability, and recyclability. This Concept describes the recent advances in the methodological strategies for engineering the COFs biocatalysts, with specific emphasis on the pore entrapment and in situ encapsulation strategies. The structural advantages of the COFs hybrid biocatalysts for organic synthesis, environment- and energy-associated applications are also canvassed. Additionally, the remaining challenges and the forward-looking directions in this field are also discussed. We believe that this Concept can offer useful methodological guidance for developing active and robust COFs biocatalysts.

Ionic Liquid-Mediated Dynamic Polymerization for Facile Aqueous-Phase Synthesis of Enzyme-Covalent Organic Framework Biocatalysts.

Utilizing covalent organic framework (COF) as a hypotoxic and porous scaffold to encapsulate enzyme (enzyme@COF) has inspired numerous interests at the intersection of chemistry, materials, and biological science. In this study, we report a convenient scheme for one-step, aqueous-phase synthesis of highly crystalline enzyme@COF biocatalysts. This facile approach relies on an ionic liquid (2 µL of imidazolium ionic liquid)-mediated dynamic polymerization mechanism, which can facilitate the in situ assembly of enzyme@COF under mild conditions. This green strategy is adaptive to synthesize different biocatalysts with highly crystalline COF "exoskeleton", as well evidenced by the low-dose cryo-EM and other characterizations. Attributing to the rigorous sieving effect of crystalline COF pore, the hosted lipase shows non-native selectivity for aliphatic acid hydrolysis. In addition, the highly crystalline linkage affords COF "exoskeleton" with higher photocatalytic activity for in situ production of H2 O2 , enabling us to construct a self-cascading photo-enzyme coupled reactor for pollutants degradation, with a 2.63-fold degradation rate as the poorly crystalline photo-enzyme reactor. This work showcases the great potentials of employing green and trace amounts of ionic liquid for one-step synthesis of crystalline enzyme@COF biocatalysts, and emphasizes the feasibility of diversifying enzyme functions by integrating the reticular chemistry of a COF.

Structural and Functional Insights into the Biomineralized Zeolite Imidazole Frameworks.

Biomineralization is a natural process of mineral formation mediated by biomacromolecules, allowing access to hierarchical structures integrating biological, chemical, and material properties. In this contribution, we comprehensively investigate the biomineralization of zeolite imidazole frameworks (ZIFs) for one-step synthesis of an enzyme-MOF biocomposite, in terms of differential crystallization behaviors, fine microstructure of resultant ZIF biominerals, the enzyme's conformation evolution, and protective effect of ZIF mineral. We discover that the biomineralization ability is ZIF organic linker dependent and the biocatalytic function is highly related to the ZIF mineral species and their distinguishable topologies and defect structures. Importantly, a side-by-side analysis suggests that the protective effect of ZIF mineral toward the hosted enzyme is highly associated with the synergistic effect of size dimension and chemical microenvironment of the ZIF pores. This work provides important insight into the ZIF-dependent biomineralization behaviors and highlights the important role of the ZIF microstructure in its biocatalytic activity and durability, which has been underestimated previously.

Protocol for mechanochemistry-guided assembly strategy for enzyme encapsulation using covalent organic frameworks.

Enzyme immobilization into porous frameworks is an emerging strategy for enhancing the stability of dynamic conformation and prolonging the lifespan of enzymes. Here, we present a protocol for a de novo mechanochemistry-guided assembly strategy for enzyme encapsulation using covalent organic frameworks. We describe steps for mechanochemical synthesis, enzyme loading measurements, and material characterizations. We then detail evaluations of biocatalytic activity and recyclability. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).1.

Pore-Environment-Dependent Photoresponsive Oxidase-Like Activity in Hydrogen-Bonded Organic Frameworks.

Mimicking the bioactivity of native enzymes through synthetic chemistry is an efficient means to advance the biocatalysts in a cell-free environment, however, remains long-standing challenges. Herein, we utilize structurally explicit hydrogen-bonded organic frameworks (HOFs) to mimic photo-responsive oxidase, and uncover the important role of pore environments on mediating oxidase-like activity by means of constructing isostructural HOFs. We discover that the HOF pore with suitable geometry can stabilize and spatially organize the catalytic substrate into a favorable catalytic route, as with the function of the native enzyme pocket. Based on the desirable photo-responsive oxidase-like activity, a visual and sensitive HOFs biosensor is established for the detection of phosphatase, an important biomarker of skeletal and hepatobiliary diseases. This work demonstrates that the pore environments significantly influence the nanozymes' activity in addition to the active center.

Enzymes-Encapsulated Defective Metal-Organic Framework Hydrogel Coupling with a Smartphone for a Portable Glucose Biosensor.

Enzymes featuring high catalytic efficiency and selectivity have been widely used as the sensing element in analytical chemistry. However, the structural fragility and poor machinability of an enzyme significantly limit its practicability in biosensors. Herein, we develop a robust and sensitive hybrid biosensor by means of co-encapsulating enzymes into a defective metal-organic framework (MOF), followed by a double-crosslinked alginate gelatinization. The defective MOF encapsulation can enhance the stability of enzymes, yet well preserve their biocatalytic function, while the alginate gelatinization allows the MOF biohybrid high stretchability and mechanical strength, which facilitates the integration of a bead-, fiber-, and sheet-like portable biosensor. In this work, the enzymes consisting of glucose oxidase and peroxidase are co-encapsulated into this MOF hydrogel, and it can efficiently convert glucose into a blue-violet product through the biocatalytic cascade of encapsulated enzymes, enabling the colorimetric biosensing of glucose on a miniaturized MOF hydrogel when coupling with a smartphone. Interestingly, this MOF biohybrid hydrogel outputs a stronger sensing signal than the free biohybrid powders, attributed to the catalytic product-accumulated effect of the highly hydrophilic microenvironment of the hydrogel. As a result, this portable biosensor can sensitively and selectively sense glucose with a linear range from 0.05 to 4 mM. Importantly, both the hydrophilic hydrogel and MOF "armor" endow enzymes with high durability, and its sensing activity was well-maintained even after placing the biosensor at room temperature for 30 d. We believe that this MOF biohybrid hydrogel has huge potential for the engineering of next-generation portable biosensors.

Hierarchically mesoporous Ce-based MOFs with enhanced alkaline phosphatase-like activity for phosphorylated biomarker sensing.

Herein, we develop a hierarchically mesoporous cerium metal-organic framework (Ce-HMMOF) nanozyme with enhanced ALP-mimicking activity for the naked-eye detection of phosphorylated biomarkers. The long-range ordered mesochannels (9.18 nm) throughout the Ce-HMMOF promote both the mass transfer and the accessibility of interior active sites, permitting the rapid and sensitive sensing of phosphorylated biomarkers through ALP-like biocatalysis. This work provides a new insight into the engineering of highly active nanozymes for disease-associated biomarker screening and diagnosis.

Photodynamic Hydrogen-Bonded Biohybrid Framework: A Photobiocatalytic Cascade Nanoreactor for Accelerating Diabetic Wound Therapy.

A diabetic wound causes thousands of infections or deaths around the world each year, and its healing remains a critical challenge because of the ease of multidrug-resistant (MDR) bacterial infection, as well as the intrinsic hyperglycemic and hypoxia microenvironment that inhibits the therapeutic efficiency. Herein, we pioneer the design of a photobiocatalytic cascade nanoreactor via spatially organizing the biocatalysts and photocatalysts utilizing a hydrogen-bonded organic framework (HOF) scaffold for diabetic wound therapy. The HOF scaffold enables it to disperse and stabilize the host cargos, and the formed long-range-ordered mesochannels also facilitate the mass transfer that enhances the cascade activity. This integrated HOF nanoreactor allows the continuous conversion of overexpressed glucose and H2O2 into toxic reactive oxygen species by the photobiocatalytic cascade. As a result, it readily reverses the microenvironment of the diabetes wound and exhibits an extraordinary capacity for wound healing through synergistic photodynamic therapy. This work describes the first example of constructing an all-in-one HOF bioreactor for antimicrobial diabetes wound treatment and showcases the promise of combined biocatalysis and photocatalysis achieved by using an HOF scaffold in biomedicine applications.

Application of the NU-1000 coated SPME fiber on analysis of trace organochlorine pesticides in water.

Herein, a novel solid-phase microextraction (SPME) fiber based on the NU-1000 sorbent was developed for direct immersion extraction of organochlorine pesticides (OCPs) in water samples. As a kind of metal-organic framework, the NU-1000 possessed the mesoporous channels which were beneficial for the mass transfer of target analytes. Extraction equilibrium was achieved rapidly with the optimal extraction time of 30 min. The NU-1000 coated fiber with a high specific surface area showed better extraction efficiencies than commercial fibers (65 µm PDMS/DVB or 85 µm PA) towards OCPs, with the enrichment factors of the NU-1000 coated fiber 2-20 times higher than the latter. NU-1000 coated fiber showed higher extraction efficiencies toward polycyclic aromatic hydrocarbons (PAHs) than OCPs and nitrobenzenes. This indicated that π-π interaction and CH-π interaction between pollutants and aromatic groups of the NU-1000 contributed to the high extraction efficiencies. Under the optimal conditions (extraction at 40 °C for 30 min and desorption at 260 °C for 6 min), the NU-1000 coated fiber coupled with gas chromatography-mass spectrometry (GC-MS) exhibited satisfied analytical performance on analysis of OCPs, with a wide linear range (0.1-2000 ng L-1), low limits of detections (LODs, 0.011-0.058 ng L-1), and good reproducibility and repeatability. The established method has been successfully applied to the determination of OCPs in surface water with good sensitivity and recoveries, which proved the great promise of the NU-1000 on the extraction of organic pollutants with conjugated groups.

Machine learning directed discrimination of virgin and recycled poly(ethylene terephthalate) based on non-targeted analysis of volatile organic compounds.

The use of non-decontaminated recycled poly(ethylene terephthalate) (PET) in food packages arouses consumer safety concerns, and thus is a major obstacle hindering PET bottle-to-bottle recycling in many developing regions. Herein, machine learning (ML) algorithms were employed for the discrimination of 127 batches of virgin PET and recycled PET (rPET) samples based on 1247 volatile organic compounds (VOCs) tentatively identified by headspace solid-phase microextraction comprehensive two-dimensional gas chromatography quadrupole-time-of-flight mass spectrometry. 100% prediction accuracy was achieved for PET discrimination using random forest (RF) and support vector machine (SVM) algorithms. The features of VOCs bearing high variable contributions to the RF prediction performance characterized by mean decrease Gini and variable importance were summarized as high occurrence rate, dominant appearance and distinct instrument response. Further, RF and SVM were employed for PET discrimination using the simplified input datasets composed of 62 VOCs with the highest contributions to the RF prediction performance derived by the AUCRF algorithm, by which over 99% prediction accuracy was achieved. Our results demonstrated ML algorithms were reliable and powerful to address PET adulteration and were beneficial to boost food-contact applications of rPET bottles.

Silencing of Pyruvate Kinase M2 via a Metal-Organic Framework Based Theranostic Gene Nanomedicine for Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is typically associated with poor prognosis due to its only partial response to chemotherapy and lack of clinically established targeted therapies coupled with an aggressive disease course. Aerobic glycolysis is a hallmark of reprogrammed metabolic activity in cancer cells, which can be repressed by small-interfering RNA (siRNA). However, the lack of effective carriers to deliver vulnerable siRNA restricts the clinical potentials of glycolysis-based gene therapy for TNBC. Herein, we develop a tumor-targeted, biomimetic manganese dioxide (MnO2)-shrouded metal-organic framework (MOF) based nanomedicine to deliver siRNA against pyruvate kinase muscle isozyme M2 (siPKM2), wherein PKM2 is a rate-limiting enzyme in glycolysis, to inhibit the reprogrammed glycolysis of TNBC. This MOF-based genetic nanomedicine shows excellent monodispersity and stability and protects siPKM2 against degradation by nucleases. The nanomedicine not only substantially blocks the glycolytic pathway but also improves intracellular hypoxia in TNBC cells, with a resultant O2-enhanced anticancer effect. In the mice orthotopic TNBC model, the nanomedicine shows a remarkable therapeutic effect. Meanwhile, the Mn2+ ions released from acid microenvironment-responsive MnO2 enable in vivo monitoring of the therapeutic process with magnetic resonance imaging (MRI). Our study shows great promise with this MRI-visible MOF-based nanomedicine for treating TNBC by inhibition of glycolysis via the RNA interference.

Iron-Mineralization-Induced Mesoporous Metal-Organic Frameworks Enable High-Efficiency Synergistic Catalysis of Natural/Nanomimic Enzymes.

Metal-organic frameworks (MOFs) have become a promising accommodation for enzyme immobilization and protection. However, the integration of multienzymes into MOFs may result in compromise of individual enzymatic activity. In this work, we report an iron mineralization strategy to facilely construct a mesoporous MOF, possessing excellent peroxidase-mimic bioactivity. Furthermore, the feasibility of in situ encapsulating natural enzymes within the developed mesoporous MOF nanozymes endows these natural/nanomimic enzyme hybrids with remarkably enhanced synergistic catalysis ability. Such activity enhancement is mainly due to (1) the fast flux rate of substances through the interconnected mesoporous channels and (2) the simultaneously increased loading amount of enzymes and iron within the MOFs caused by the iron mineralization process.

Embedding Functional Biomacromolecules within Peptide-Directed Metal-Organic Framework (MOF) Nanoarchitectures Enables Activity Enhancement.

Rationally tailoring a robust artificial coating can enhance the life-time of fragile biomacromolecules. However, the coating also can restrain the activity of the guest because of the decreased substrate accessibility. Herein, we report a peptide-directed strategy that enables in situ tailoring of the MOF-shrouded biohybrids into controllable nanoarchitectures. The MOF biohybrid can be shaped from different 3D microporous architectures into a 2D mesoporous layer by a peptide modulator. Using this mild strategy, we show that the nanoarchitectures of the MOF coatings significantly affect the biological functions of the contained biomacromolecules. The biomacromolecules entrapped within the novel 2D mesoporous spindle-shaped MOFs (2D MSMOFs) have significantly increased bioactivity compared to when encased within the hitherto explored 3D microporous MOFs. The improvement results from the shortened diffusion path and enlarged pore channel in 2D MSMOFs. Meanwhile, the thin 2D MSMOF layer also can provide excellent protection of the hosted biomacromolecules or protein-scaffolded biominerals through structural confinement.

Metal-Organic Frameworks: A New Platform for Enzyme Immobilization.

Metal-organic frameworks (MOFs) with attractive properties such as high surface area, tunable porosity, designable functionality and excellent stability, have aroused great interest from researchers as the matrices for enzyme immobilization. Recently, several efficient strategies including surface immobilization, post-synthetic infiltration and in situ encapsulation have been explored. MOF-immobilized enzymes, named enzymes@MOFs, show remarkably enhanced stability and recyclability, accelerating cell-free biocatalysis in diverse applications. This concept will impart the typical strategies for enzyme immobilization with MOFs, and their potential applications.

Smartphone-assisted robust enzymes@MOFs-based paper biosensor for point-of-care detection.

Portable devices featured with fast analysis and affordable methodologies for clinical diagnostics have stimulated the rapid development of point-of-care (POC) technologies, potentially lowering the mortality rate. Herein, we demonstrated a portable, robust, and user-friendly intelligent metal-organic frameworks (MOFs) paper device, called smartphone-assisted biomimetic MOFs nanoreactor colorimetric paper (SBMCP), for on-demand POC detection of endogenous biomolecules. The concept of this paper platform was analogous to the intracellular cascades signal transduction, wherein the single/multiple enzymes components trapped within a ZIF-8 exoskeleton allowed the sensitive and selective recognition of target analyte via the accessible micropores network of ZIF-8, and then transferred the recognition event to a visual color signal based on the cascade reaction. Meanwhile, the ZIF-8 exoskeleton also endowed the enzymes with significantly elevated stability. As a result, this robust and portable SBMCP sensor enabled the on-site analysis of different important disease-related biomolecules through modulating the enzyme cascades, combining with a custom-designed smartphone application for signal readout. In the SBMCP assay, no sophisticated instruments or professional skill of the user was required, only 5 µL sample volume was needed, and the whole analysis process could be achieved within a portable MOFs paper and pervasive smartphone, endowing this new assay with the merits of low-cost, time-saving and easy-to-use. We demonstrated this SBMCP sensor was capable of real-time colorimetric detection of glucose and uric acid in diabetes and gout events. It is believed that this portable biosensor platform proposed herein potentially represents promising alternatives for POC diagnosis, especially applicable in developing world and resource-limited settings.

Facile construction of superhydrophobic hybrids of metal-organic framework grown on nanosheet for high-performance extraction of benzene homologues.

Encapsulating functional nanomaterials within the bulk of metal-organic frameworks (MOFs) offers the opportunity to construct high-performance hybrid coating materials for solid phase microextraction (SPME). In this work, we proposed the facile synthesis of a superhydrophobic MOF composite material (NSZIF-8Si) by growing ZIF-8 on MnxOy nanosheet (NS) and subsequently depositing short-chain polysiloxane on the surface of the composite. A novel SPME fiber was successfully prepared based on the NSZIF-8Si composite. The NSZIF-8Si fiber possessed outstanding thermal stability (up to 450 °C). In headspace SPME of BTEX, the home-made fiber exhibited extraction efficiencies much higher than the commercially available PDMS fiber. This phenomenon was due to the synergetic cooperation of the π-π stacking and the hydrophobic interactions between the NSZIF-8Si coating and the analyte molecules, as well as the increased aspect ratio of the MOF grown on the nanosheet. The established method achieved wide linearity (5-2000 ng L-1) and low LODs (0.02 ng L-1 to 0.21 ng L-1). Satisfactory recoveries were obtained in the analysis of real water samples collected from the Pearl River, indicative of the good reliability of the established method for real-scenario applications. This work might provide critical insights in constructing novel NS/MOF composite materials for the development of high-performance SPME fiber coatings.

"Armor-Plating" Enzymes with Metal-Organic Frameworks (MOFs).

Cell-free enzymatic catalysis (CFEC) is an emerging biotechnology that enable the biological transformations in complex natural networks to be imitated. This biomimetic approach allows industrial products such as biofuels and biochemical to be manufactured in a green manner. Nevertheless, the main challenge in CFEC is the poor stability, which restricts the effectiveness and lifetime of enzymes in sophisticated applications. Immobilization of the enzymes within solid carriers is considered an efficient strategy for addressing these obstacles. Specifically, putting an "armor-like" porous metal-organic framework (MOF) exoskeleton tightly around the enzymes not only shields the enzymes against external stimulus, but also allows the selective transport of guests through the accessible porous network. Herein we present the concept of this biotechnology of MOF-entrapped enzymes and its cutting-edge applications.

Modulating the Biofunctionality of Metal-Organic-Framework-Encapsulated Enzymes through Controllable Embedding Patterns.

Embedding an enzyme within a MOF as exoskeleton (enzyme@MOF) offers new opportunities to improve the inherent fragile nature of the enzyme, but also to impart novel biofunctionality to the MOF. Despite the remarkable stability achieved for MOF-embedded enzymes, embedding patterns and conversion of the enzymatic biofunctionality after entrapment by a MOF have only received limited attention. Herein, we reveal how embedding patterns affect the bioactivity of an enzyme encapsulated in ZIF-8. The enzyme@MOF can maintain high activity when the encapsulation process is driven by rapid enzyme-triggered nucleation of ZIF-8. When the encapsulation is driven by slow coprecipitation and the enzymes are not involved in the nucleation of ZIF-8, enzyme@MOF tends to be inactive owing to unfolding and competing coordination caused by the ligand, 2-methyl imidazole. These two embedding patterns can easily be controlled by chemical modification of the amino acids of the enzymes, modulating their biofunctionality.