Enzyme Immobilization by Inkjet Printing on Reagentless Biosensors for Electrochemical Phosphate Detection.

Enzyme-based biosensors commonly utilize the drop-casting method for their surface modification. However, the drawbacks of this technique, such as low reproducibility, coffee ring effects, and challenges in mass production, hinder its application. To overcome these limitations, we propose a novel surface functionalization strategy of enzyme crosslinking via inkjet printing for reagentless enzyme-based biosensors. This method includes printing three functional layers onto a screen-printed electrode: the enzyme layer, crosslinking layer, and protective layer. Nanomaterials and substrates are preloaded together during our inkjet printing. Inkjet-printed electrodes feature a uniform enzyme deposition, ensuring high reproducibility and superior electrochemical performance compared to traditional drop-casted ones. The resultant biosensors display high sensitivity, as well as a broad linear response in the physiological range of the serum phosphate. This enzyme crosslinking method has the potential to extend into various enzyme-based biosensors through altering functional layer components.

Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-enzymatic Synthesis of Rivastigmine.

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66%. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.

Boosted Enzyme Activity via Encapsulation within Metal-Organic Frameworks with Pores Matching Enzyme Size and Shape.

A novel and versatile approach called "physical imprinting" is introduced to modulate enzyme conformation using mesoporous materials, addressing challenges in achieving improved enzyme activity and stability. Metal-organic frameworks with tailored mesopores, precisely matching enzyme size and shape, are synthesized. Remarkably, enzymes encapsulated within these customized mesopores exhibit over 1670% relative activity compared to free enzymes, maintaining outstanding efficiency even under harsh conditions such as heat, exposure to organic solvents, wide-ranging pH extremes from acidic to alkaline, and exposure to a digestion cocktail. After 18 consecutive cycles of use, the immobilized enzymes retain 80% of their initial activity. Additionally, the encapsulated enzymes exhibit a substantial increase in catalytic efficiency, with a 14.1-fold enhancement in kcat/KM compared to native enzymes. This enhancement is among the highest reported for immobilized enzymes. The improved enzyme activity and stability are corroborated by solid-state UV-vis, electron paramagnetic resonance, Fourier-transform infrared spectroscopy, and solid-state NMR spectroscopy. The findings not only offer valuable insights into the crucial role of size and shape complementarity within confined microenvironments but also establish a new pathway for developing solid carriers capable of enhancing enzyme activity and stability.

HLM chip - A microfluidic approach to study the mechanistic basis of cytochrome P450 inhibition using immobilized human liver microsomes.

Cytochrome P450 (CYP) system is a critical elimination route to most pharmaceuticals in human, but also prone to drug-drug interactions arising from the fact that concomitantly administered pharmaceuticals inhibit one another's CYP metabolism. The most severe form of CYP interactions is irreversible inhibition, which results in permanent inactivation of the critical CYP pathway and is only restored by de novo synthesis of new functional enzymes. In this study, we conceptualize a microfluidic approach to mechanistic CYP inhibition studies using human liver microsomes (HLMs) immobilized onto the walls of a polymer micropillar array. We evaluated the feasibility of these HLM chips for CYP inhibition studies by establishing the stability and the enzyme kinetics for a CYP2C9 model reaction under microfluidic flow and determining the half-maximal inhibitory concentrations (IC50) of three human CYP2C9 inhibitors (sulfaphenazole, tienilic acid, miconazole), including evaluation of their inhibition mechanisms and nonspecific microsomal binding on chip. Overall, the enzyme kinetics of CYP2C9 metabolism on the HLM chip (KM = 127 ± 55 µM) was shown to be similar to that of static HLM incubations (KM = 114 ± 14 µM) and the IC50 values toward CYP2C9 derived from the microfluidic assays (sulfaphenazole 0.38 ± 0.09 µM, tienilic acid 3.4 ± 0.6 µM, miconazole 0.54 ± 0.09 µM) correlated well with those determined using current standard IC50 shift assays. Most importantly, the HLM chip could distinguish between reversible (sulfaphenazole) and irreversible (tienilic acid) enzyme inhibitors in a single, automated experiment, indicating the great potential of the HLM chip to simplify current workflows used in mechanistic CYP inhibition studies. Furthermore, the results suggest that the HLM chip can also identify irreversible enzyme inhibitors, which are not necessarily resulting in a time-dependent inhibition (like suicide inhibitors), but whose inhibition mechanism is based on other kind of covalent or irreversible interaction with the CYP system. With our HLM chip approach, we could identify miconazole as such a compound that nonselectively inhibits the human CYP system with a prolonged, possibly irreversible impact in vitro, even if it is not a time-dependent inhibitor according to the IC50 shift assay.

Single-Particle and Single-Molecule Characterization of Immobilized Enzymes: A Multiscale Path toward Optimizing Heterogeneous Biocatalysts.

Heterogeneous biocatalysis is highly relevant in biotechnology as it offers several benefits and practical uses. To leverage the full potential of heterogeneous biocatalysts, the establishment of well-crafted protocols, and a deeper comprehension of enzyme immobilization on solid substrates are essential. These endeavors seek to optimize immobilized biocatalysts, ensuring maximal enzyme performance within confined spaces. For this aim, multidimensional characterization of heterogeneous biocatalysts is required. In this context, spectroscopic and microscopic methodologies conducted at different space and temporal scales can inform about the intraparticle enzyme kinetics, the enzyme spatial distribution, and the mass transport issues. In this Minireview, we identify enzyme immobilization, enzyme catalysis, and enzyme inactivation as the three main processes for which advanced characterization tools unveil fundamental information. Recent advances in operando characterization of immobilized enzymes at the single-particle (SP) and single-molecule (SM) levels inform about their functional properties, unlocking the full potential of heterogeneous biocatalysis toward biotechnological applications.

Bioproduction, purification, partial characterization and phenol removal efficacy of tyrosinase enzyme from Streptomyces sp. strain MR28.

The study focused on the production of the tyrosinase enzyme from Streptomyces sp. MR28 and its potency in removal of phenol content from water using free and immobilized tyrosinase enzyme. The tyrosinase was produced by Streptomyces sp. MR28 in liquid tyrosine broth medium, and it was further purified to near its homogeneity by employing, precipitation, dialysis, and column chromatography. After the purification, 44.49% yield with a 4 fold purification was achieved. The characterization of the purified enzyme showed a single major peak on HPLC and a solitary band on SDS-PAGE. The purified tyrosinase enzyme was active at a pH of 7.0 and a temperature of 30 °C. Further immobilization of purified tyrosinase was performed using the sodium alginate entrapment method. The capacity of the purified tyrosinase to remove phenol in water was evaluated by spectrophotometric method. The free tyrosinase enzyme-treated solutions showed a gradual decrease in the concentration of phenol with increased incubation time at 30 °C and 40 °C, at 90 min of the incubation time, it showed maximum efficacy in removing phenol from the solution. At 50 °C and 60 °C, the free tyrosinase enzyme exhibited very less capacity to remove the phenol. The immobilized enzyme showed good capacity for the removal of phenol from the solutions; the concentration of phenol in the solution decreased with an increase in the incubation time. At temperatures of 40 °C and 50 °C, the immobilized tyrosinase enzyme beads showed significant removal of phenol from the solution, and at temperatures of 30 °C and 60 °C, they also exhibited good capacity for the removal of phenol. At the end of the 90 min incubation period, it exhibited good capability. The current study suggests using immobilized microbial tyrosinase enzyme can be used for the removal of phenol from the contaminated water in a greener manner.

Covalent Organic Frameworks Based Photoenzymatic Nano-reactor for Asymmetric Dynamic Kinetic Resolution of Secondary Amines.

Bio-catalysis represents a highly efficient and stereoselective method for the synthesis of valuable chiral compounds, however, the poor stability and limited reaction types of free enzymes restrict their wide application in industrial production. In this work, to overcome these problems, a multifunctional photoenzymatic nanoreactor CALB@COF-Ir was developed through the encapsulation of Candida antarctica lipase B (CALB) in a photosensitive covalent organic framework COF-Ir. This bio-nanocluster serves as efficient catalysts in asymmetric dynamic kinetic resolution (DKR) of secondary amines to give a series of chiral amines in high yields (up to 99 %) and enantioselectivities (up to 99 % ee). The well-designed COF-Ir not only acts as safety cover to prevent CALB from deactivation but promotes racemization of secondary amines via photo-induced hydrogen atom transfer (HAT) process. Photoelectric characterization and TDDFT calculation revealed that (ppy)2Ir units in COF-Ir play crucial role in this photocatalytic system which enhance its photo-redox properties through facilitating the separation between photoelectrons (e-) and holes (h+). Furthermore, the heterogeneous photoenzymatic nanoreactor could be recycled for five rounds with slight decline of catalytic reactivity.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone.

In this study, we prepared a functional organic-inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme's terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 âˆ¼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3-11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

Cross-linked enzyme aggregates of polyethylene terephthalate hydrolyse (PETase) from Ideonella sakaiensis for the improvement of plastic degradation.

Polyethylene terephthalate (PET) is one of the most produced plastics globally and its accumulation in the environment causes harm to the ecosystem. Polyethylene terephthalate hydrolyse (PETase) is an enzyme that can degrade PET into its monomers. However, free PETase lacks operational stabilities and is not reusable. In this study, development of cross-linked enzyme aggregate (CLEA) of PETase using amylopectin (Amy) as cross-linker was introduced to solve the limitations of free PETase. PETase-Amy-CLEA exhibited activity recovery of 81.9 % at its best immobilization condition. Furthermore, PETase-Amy-CLEA exhibited 1.37-, 2.75-, 2.28- and 1.36-fold higher half-lives than free PETase at 50 °C, 45 °C, 40 °C and 35 °C respectively. Moreover, PETase-Amy-CLEA showed broader pH stability from pH 5 to 10 and could be reused up to 5 cycles. PETase-Amy-CLEA retained >70 % of initial activity after 40 days of storage at 4 °C. In addition, lower Km of PETase-Amy-CLEA indicated better substrate affinity than free enzyme. PETase-Amy-CLEA corroded PET better and products yielded was 66.7 % higher than free PETase after 32 h of treatment. Hence, the enhanced operational stabilities, storage stability, reusability and plastic degradation ability are believed to make PETase-Amy-CLEA a promising biocatalyst in plastic degradation.

Direct binding and characterization of laccase onto iron oxide nanoparticles.

Iron oxide nanoparticles (IONPs) exhibit unique magnetic properties and possess a high surface-to-volume ratio, making them ideal candidates for the conjugation of substances, including enzymes. Laccase (EC 1.10.3.2), an oxidative enzyme with diverse applications, presents an opportunity for enhancing stability and reusability through innovative immobilization techniques, thus reducing overall process costs. In this study, we employed a direct binding procedure via carbodiimide activation to conjugate laccase onto IONPs synthesized using thermal chemical coprecipitation. Stabilization of the nanoparticles was achieved using thioglycerol and polyvinyl alcohol (PVA) as capping agents. Characterization of the synthesized nanoparticles was conducted using UV-spectroscopy, Fourier transform infrared spectroscopy (FTIR), x-ray diffraction, scanning electron microscopy, and energy dispersive x-ray spectroscopy. FTIR spectroscopy analysis confirmed successful laccase binding to magnetic nanoparticles, with binding efficiencies of 90.65% and 73.02% observed for thioglycerol and PVA capped IONPs, respectively. Furthermore, the conjugated enzyme exhibited remarkable stability, retaining nearly 50% of its initial activity after 20 reuse cycles. This research demonstrates that immobilizing laccase onto IONPs enhances its activity, stability, and reusability, with the potential for significant cost savings and expanded applications in various fields.

Immobilization of Enological Pectinase on Magnetic Sensitive Polyamide Microparticles for Wine Clarification.

The use of free pectinases as clarification biocatalysts constitutes a well-established practice in the large-scale production of various types of wines. However, when in the form of free enzymes, the recovery and reusability of pectinases is difficult if not impossible. To address these limitations, the present study focuses on the noncovalent adsorption immobilization of a commercial pectinolytic preparation onto highly porous polyamide 6 (PA6) microparticles, both with and without magnetic properties, prepared via activated anionic polymerization. The two pectinase complexes resulting after immobilization underwent comparative activity and kinetic studies, contrasting them with the free enzyme preparation. In comparison with the free enzyme, the PA6-immobilized pectinase complexes exhibited more than double the specific activity toward the pectin substrate. They displayed a slightly higher affinity to the substrate while acting as faster catalysts that were more resistant to inhibition. Furthermore, the immobilized complexes were applied in the clarification process of industrial rosé must, whereby they demonstrated accelerated performance as compared with the free enzyme. Moreover, the PA6-immobilized pectinase biocatalysts offered the potential for three consecutive cycles of reuse, achieving complete rosé must clarification within relevant timeframes in the range of 3-36 h. All these results suggest the potential industrial application of the pectinases noncovalently immobilized upon PA6 microparticles.

"High-throughput screening of catalytically active inclusion bodies using laboratory automation and Bayesian optimization".

BACKGROUND: In recent years, the production of inclusion bodies that retain substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) are formed by genetic fusion of an aggregation-inducing tag to a gene of interest via short linker polypeptides. The resulting CatIBs are known for their easy and cost-efficient production, recyclability as well as their improved stability. Recent studies have outlined the cooperative effects of linker and aggregation-inducing tag on CatIB activities. However, no a priori prediction is possible so far to indicate the best combination thereof. Consequently, extensive screening is required to find the best performing CatIB variant. RESULTS: In this work, a semi-automated cloning workflow was implemented and used for fast generation of 63 CatIB variants with glucose dehydrogenase of Bacillus subtilis (BsGDH). Furthermore, the variant BsGDH-PT-CBDCell was used to develop, optimize and validate an automated CatIB screening workflow, enhancing the analysis of many CatIB candidates in parallel. Compared to previous studies with CatIBs, important optimization steps include the exclusion of plate position effects in the BioLector by changing the cultivation temperature. For the overall workflow including strain construction, the manual workload could be reduced from 59 to 7 h for 48 variants (88%). After demonstration of high reproducibility with 1.9% relative standard deviation across 42 biological replicates, the workflow was performed in combination with a Bayesian process model and Thompson sampling. While the process model is crucial to derive key performance indicators of CatIBs, Thompson sampling serves as a strategy to balance exploitation and exploration in screening procedures. Our methodology allowed analysis of 63 BsGDH-CatIB variants within only three batch experiments. Because of the high likelihood of TDoT-PT-BsGDH being the best CatIB performer, it was selected in 50 biological replicates during the three screening rounds, much more than other, low-performing variants. CONCLUSIONS: At the current state of knowledge, every new enzyme requires screening for different linker/aggregation-inducing tag combinations. For this purpose, the presented CatIB toolbox facilitates fast and simplified construction and screening procedures. The methodology thus assists in finding the best CatIB producer from large libraries in short time, rendering possible automated Design-Build-Test-Learn cycles to generate structure/function learnings.

Immobilization of fumarase from thermophilic eukaryotic red alga Cyanidioschyzon merolae on ceramic carrier.

Fumarase is an enzyme catalyzing reversible reaction between fumarate and L-malate in the citric acid cycle. Fumarase is used in the industrial production of L-malate, and its immobilization is required for reuse of the fumarases to reduce the cost. Accordingly, understanding the properties of immobilized fumarase is crucial, and several groups report on the storage stability and kinetic parameters of immobilized fumarase. Here we have immobilized fumarase from the thermophilic red alga Cyanidioschyzon merolae (CmFUM) on ceramic beads and investigated its biochemical and physical properties. CmFUM demonstrated sufficient stability and reusability for industry use after immobilization. Notably, the thermostability was dramatically enhanced through immobilization. The Km value and kcat of immobilized CmFUM for fumarate were 1.7 mM and 22.7 s-1 respectively. The Km value for fumarate was lower than that of other reported immobilized fumarases, indicating a high substrate affinity of immobilized CmFUM. Furthermore, the enhanced stability resulting from immobilization partially compensated for the decrease in activity. The high affinity towards fumarate and good thermostability of immobilized CmFUM revealed in this study are advantageous traits for improving enzyme-mediated isomer-specific L-malate production.

Expression and Immobilization of Tannase for Tannery Effluent Treatment from Lactobacillus plantarum and Staphylococcus lugdunensis: A Comparative Study.

Agro-industrial discharges have higher concentrations of tannins and have been a significant cause of pollution to water bodies and soil surrounding the agro-industries. So in this study, toxic tannic acid is into commercially valuable gallic acid from the tannery effluent using immobilized microbial tannase. Tannase genes were isolated from Lactobacillus plantarum JCM 1149 (tanLpl) and Staphylococcus lugdunensis MTCC 3614 (tanA). Further, these isolated tannese genes were cloned and expressed in BL 21 host using pET 28a as an expression vector,  and immobilized in sodium alginate beads. Vegetable tannery effluent was treated by tannase-immobilized beads at 25 °C and 37 °C, where liberated gallic acid was analyzed using TLC and NMR to confirm the tannin reduction. Further, both immobilized tannases exhibited excellent reusability up to 15 cycles of regeneration without significant reduction in their activity. Moreover, we also showed that immobilized tannases tanLpl and tanA activity remained unaffected compared to the free enzyme in the presence of metal ions. Further, tanA activity remained unaffected over a wide range of pH, and tanLpl showed high thermal stability. Thus, immobilized tannase tanLpl and tanA provide a possible solution for tannery effluent treatment depending upon industry requirements and reaction composition/effluent composition, one can choose a better-immobilized tannase among the two as per the need-based requirement.

Enhanced High-Fructose Corn Syrup Production: Immobilizing Serratia marcescens Glucose Isomerase on MOF (Co)-525 Reduces Co2+ Dependency in Glucose Isomerization to Fructose.

The escalating demand for processed foods has led to the widespread industrial use of glucose isomerase (GI) for high-fructose corn syrup (HFCS) production. This reliance on GIs necessitates continual Co2+ supplementation to sustain high catalytic activity across multiple reaction cycles. In this study, Serratia marcescens GI (SmGI) was immobilized onto surfaces of the metal-organic framework (MOF) material MOF (Co)-525 to generate MOF (Co)-525-GI for use in catalyzing glucose isomerization to generate fructose. Examination of MOF (Co)-525-GI structural features using scanning electron microscopy-energy dispersive spectroscopy, Fourier-transform infrared spectroscopy, and ultraviolet spectroscopy revealed no structural changes after SmGI immobilization and the addition of Co2+. Notably, MOF (Co)-525-GI exhibited optimal catalytic activity at pH 7.5 and 70 °C, with a maximum reaction rate (Vmax) of 37.24 ± 1.91 µM/min and Km value of 46.25 ± 3.03 mM observed. Remarkably, immobilized SmGI exhibited sustained high catalytic activity over multiple cycles without continuous Co2+ infusion, retaining its molecular structure and 96.38% of its initial activity after six reaction cycles. These results underscore the potential of MOF (Co)-525-GI to serve as a safer and more efficient immobilized enzyme technology compared to traditional GI-based food-processing technologies.

Biocatalytic Performance of ß-Glucosidase Immobilized on 3D-Printed Single- and Multi-Channel Polylactic Acid Microreactors.

Microfluidic devices have attracted much attention in the current day owing to the unique advantages they provide. However, their application for industrial use is limited due to manufacturing limitations and high cost. Moreover, the scaling-up process of the microreactor has proven to be difficult. Three-dimensional (3D) printing technology is a promising solution for the above obstacles due to its ability to fabricate complex structures quickly and at a relatively low cost. Hence, combining the advantages of the microscale with 3D printing technology could enhance the applicability of microfluidic devices in the industrial sector. In the present work, a 3D-printed single-channel immobilized enzyme microreactor with a volume capacity of 30 µL was designed and created in one step via the fused deposition modeling (FDM) printing technique, using polylactic acid (PLA) as the printing material. The microreactor underwent surface modification with chitosan, and ß-glucosidase from Thermotoga maritima was covalently immobilized. The immobilized biocatalyst retained almost 100% of its initial activity after incubation at different temperatures, while it could be effectively reused for up to 10 successful reaction cycles. Moreover, a multi-channel parallel microreactor incorporating 36 channels was developed, resulting in a significant increase in enzymatic productivity.

Enzymatic interfacial conversion of acylglycerols in Pickering emulsions stabilized by hydrogel microparticles.

HYPOTHESIS: A critical challenge in the enzymatic conversion of acylglycerols is the limited exposure of the enzyme dissolved in the aqueous solution to the hydrophobic substrate in the oil phase. Positioning the enzyme in a microenvironment with balanced hydrophobicity and hydrophilicity in Pickering emulsion will facilitate the acylglycerol-catalyzing reactions at the interface between the oil and liquid phases. EXPERIMENTS: In this work, to overcome the challenge of biphasic catalysis, we report a method to immobilize enzymes in polyethylene glycol (PEG)-based hydrogel microparticles (HMPs) at the interface between the oil and water phases in Pickering emulsion to promote the enzymatic conversion of acylglycerols. FINDINGS: 3 wt% of HMPs can stabilize the oil-in-water Pickering emulsion for at least 14 days and increase the viscosity of emulsions. Lipase-HMP conjugates showed significantly higher hydrolytic activity in Pickering emulsion; HMP-immobilized lipase SMG1 showed an activity about three times that of free lipase SMG1. Co-immobilization of a lipase and a fatty acid photodecarboxylase from Chlorella variabilis (CvFAP) in Pickering emulsion enables light-driven cascade conversion of triacylglycerols to hydrocarbons, transforming waste oil to renewable biofuels in a green and sustainable approach. HMPs stabilize the Pickering emulsion and promote interfacial biocatalysis in converting acylglycerols to renewable biofuels.

A Protocol for Custom Biomineralization of Enzymes in Metal-Organic Frameworks (MOFs).

Enzyme immobilization offers a number of advantages that improve biocatalysis; however, finding a proper way to immobilize enzymes is often a challenging task. Implanting enzymes in metal-organic frameworks (MOFs) via co-crystallization, also known as biomineralization, provides enhanced reusability and stability with minimal perturbation and substrate selectivity to the enzyme. Currently, there are limited metal-ligand combinations with a proper protocol guiding the experimental procedures. We have recently explored 10 combinations that allow custom immobilization of enzymes according to enzyme stability and activity in different metals/ligands. Here, as a follow-up of that work, we present a protocol for how to carry out custom immobilization of enzymes using the available combinations of metal ions and ligands. Detailed procedures to prepare metal ions, ligands, and enzymes for their co-crystallization, together with characterization and assessment, are discussed. Precautions for each experimental step and result analysis are highlighted as well. This protocol is important for enzyme immobilization in various research and industrial fields. Key features • A wide selection of metal ions and ligands allows for the immobilization of enzymes in metal-organic frameworks (MOFs) via co-crystallization. • Step-by-step enzyme immobilization procedure via co-crystallization of metal ions, organic linkers, and enzymes. • Practical considerations and experimental conditions to synthesize the enzyme@MOF biocomposites are discussed. • The demonstrated method can be generalized to immobilize other enzymes and find other metal ion/ligand combinations to form MOFs in water and host enzymes.

Enhancement of enzyme activity by laser-induced energy propulsion of upconverting nanoparticles under near-infrared light: A comprehensive methodology for in vitro and in vivo applications.

If the appropriate immobilization method and carrier support are not selected, partial decreases in the activity of enzymes may occur after immobilization. Herein, to overcome this challenge, an excitation mechanism that enables energy transfer was proposed. Modified upconverting nanoparticles (UCNPs) were constructed and the important role of near-infrared (NIR) excitation in enhancing the catalytic activity of the enzyme was demonstrated. For this purpose, UCNPs were first synthesized via the hydrothermal method, functionalized with isocyanate groups, and then, PEG-L-ASNase was immobilized via covalent binding. UCNPs with and without PEG-L-ASNase were extensively characterized by different methods. These supports had immobilization yield and activity efficiency of >96 % and 78 %, respectively. Moreover, immobilized enzymes exhibited improved pH, thermal, and storage stability. In addition, they retained >65 % of their initial activity even after 20 catalytic cycles. Biochemical and histological findings did not indicate a trend of toxicity in rats due to UCNPs. Most importantly, PEG-L-ASNase activity was triggered approximately 5- and 2-fold under in vitro and in vivo conditions, respectively. Overall, it is anticipated that this pioneering work will shed new light on the realistic and promising usage of NIR-excited UCNPs for the immobilization of enzymes in expensive and extensive applications.

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.