Immobilized Enzyme Research Articles

Covalent Immobilization of Cellulase Enzyme on Chitosan and Eudragit S-100 Biopolymers for Recovery and Reusability in Denim Fading Application

Covalent Immobilization of Cellulase Enzyme on Chitosan and Eudragit S-100 Biopolymers for Recovery and Reusability in Denim Fading Application

Advances in Biocatalysis for Sustainable Pharmaceutical Synthesis: Applications, Technological Progress, and Future Directions

Biocatalysis has emerged as a critical tool in pharmaceutical synthesis, offering a range of significant advancements in recent years. This paper provides a comprehensive review of biocatalysis applications in drug synthesis, emphasizing its role in enhancing the efficiency and environmental sustainability of producing complex pharmaceutical molecules. Through detailed case studies, including the biocatalytic synthesis of amoxicillin, paclitaxel, and ribavirin, the paper illustrates the superior reaction efficiency, selectivity, and reduced environmental impact achievable through biocatalysis. The technological advancements discussed include optimizing enzyme activity via genetic engineering, enhancing enzyme stability through covalent modification, and applying enzyme immobilization techniques. Furthermore, the integration of biocatalysis with green chemistry principles is explored, highlighting its potential to reduce harmful byproducts and energy consumption in industrial processes. The paper also delves into the future directions and challenges of scaling biocatalysis for broader industrial application. As an integral part of green chemistry, biocatalysis is poised to play a pivotal role in promoting sustainable development and minimizing environmental impact in the pharmaceutical industry.

Exploring Enzyme Encapsulation Efficiency in MOFs Using Eco-Friendly Approaches.

The encapsulation of protein enzymes in metal-organic frameworks (MOFs) has been recognized as an effective enzyme immobilization approach. In this study, we demonstrated the influence of enzyme amount and the isoelectric points (pI) of different enzymes on the enzyme loading capacity in both mechanochemical (ball-milling) and water-based approaches. We found that increasing enzyme amounts enhances MOF enzyme loading without compromising activity, while the MOF shell protects encapsulated enzymes from proteinase K degradation through its size-sheltering mechanism. However, an excess of enzymes can hinder the formation of ZIF-90. Moreover, enzymes with low pI values (e. g., catalase, pI 5.4) facilitate encapsulation in MOFs, whereas enzymes with high pI values (e. g., lysozyme, pI 11.35) are more challenging to encapsulate. The simulation results revealed that increasing the enzyme amounts and pI values raises the activation energy necessary for MOF formation. This study highlights the crucial role of enzyme properties in the encapsulation process within MOFs, providing valuable insights for fabricating enzyme-MOF biocomposites for diverse applications, such as protein drug delivery.

Self‐Driving Lab for Solid‐Phase Extraction Process Optimization and Application to Nucleic Acid Purification

Sorptive bioprocesses are the basis for numerous biotechnological applications such as enzyme immobilization, biosensors, controlled drug delivery, water treatment, and molecular purification. Yet due to the complexity of these processes, their optimization is still time, labor, and cost‐intensive. This research presents a flexible self‐driving laboratory (SDL) designed for the accelerated development and optimization of solid‐phase extraction processes. As a use case, the SDL was used to optimize a DNA purification process using silica magnetic beads. Through the integration of robotics, machine learning, and data‐driven experimentation, the SDL demonstrates a highly accelerated process optimization with minimal human intervention. In the multistep purification approach, the system is able to optimize buffer compositions for DNA extraction from complex samples, demonstrating effectiveness in both conventional chaotropic salt‐based methods and innovative chaotropic salt‐free buffers. The study highlights the SDL's capability to autonomously refine process parameters, achieving significant enhancements in yield and purity of the product. This blueprint for future self‐driving optimization of bioprocess parameters showcases the potential of autonomous systems to revolutionize biochemical process development, offering insights into scalable, environmentally sustainable, and cost‐effective solutions.

Properties and kinetic behavior of free and immobilized laccase from Oudemansiella canarii: Emphasis on the effects of NaCl and Na2SO4 on catalytic activities

Studies have highlighted the great potential of Oudemansiella canarii laccase in degrading synthetic dyes for reducing their toxicity. Immobilization of enzymes improves usability in degradation processes and the present work succeeded in immobilizing this laccase onto MANAE-agarose. Immobilization improved pH, thermal, and storage stabilities. Both, free and immobilized enzymes presented Michaelis-Menten kinetics with the substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) with Km values of 0.056 ± 0.003 and 0.195 ± 0.022 mM, respectively. Immobilization increased Vmax 1.27-fold. NaCl caused incomplete (hyperbolic) inhibition, which was satisfactorily described by the one-substrate one-modifier mechanism. Immobilization reduced the maximal inhibition by NaCl from 80.2 to 55.7 %. The effect of Na2SO4 was predominantly stimulation, but inhibition of the free enzyme occurred at high substrate concentrations. Stimulation of the immobilized enzyme by Na2SO4 was much more pronounced. It strongly depended on the substrate concentration and was much stronger (up to 300 %) at low substrate concentrations. The combined effects of substrate and sulfate on the immobilized laccase could be satisfactorily described by the one-substrate one-modifier mechanism. The modified response of the immobilized O. canarii laccase to NaCl and Na2SO4 considerably favors its use as a tool in bioremediation processes because environmental contamination by salts frequently represents a strong operational challenge.

Improving enzyme immobilization: A new carrier-based magnetic polymer for enhanced covalent binding of laccase enzyme

In the quest for effective enzyme immobilization methods, this study focuses on synthesizing carrier-based magnetic polymer to enhance the covalent binding of T. versicolor laccase. Utilizing chitosan (CS) and alginate (ALG) composites, modified with Fe3O4 magnetic nanoparticles (MNPs), we aimed to improve the enzyme's stability, reusability, and performance under varying conditions. The ionic gelation method was employed to prepare CS-ALG and CS-ALG-Fe3O4 MNPs composites, resulting in an 84 % and 91 % immobilization efficiency. The immobilized enzyme demonstrated superior thermal stability, retaining 48 % activity for CS-ALG-laccase and 67 % for CS-ALG-Fe3O4 MNPs-laccase at 70 °C, compared to 29 % for the free enzyme. Scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy were used to characterize the composites, revealing significant morphological changes and successful enzyme integration. Kinetic studies indicated that immobilization increased the Vmax to 141 μmol/min for CS-ALG-laccase and 111 μmol/min for CS-ALG-Fe3O4 MNPs-laccase, while slightly reducing substrate affinity (Km) to 1.42 mM−1 and 1.32 mM−1, respectively. The immobilized laccase retained higher activity after ten reaction cycles (81 % activity for CS-ALG-Fe3O4 MNPs-laccase) and during prolonged storage (75 % activity retention), showcasing its potential for industrial applications. Additionally, the enzyme exhibited increased resistance to various organic solvents, enhancing its practical utility.

Expanding the high-pH range of the sucrose synthase reaction by enzyme immobilization

The glycosylation of an alcohol group from a sugar nucleotide substrate involves proton release, so the reaction is favored thermodynamically at high pH. Here, we explored expansion of the alkaline pH range of sucrose synthase (SuSy; EC 2.4.1.13) to facilitate enzymatic glycosylation from uridine 5’-diphosphate (UDP)-glucose. The apparent equilibrium constant of the SuSy reaction (UDP-glucose + fructose ↔ sucrose + UDP) at 30 °C increases by ∼4 orders of magnitude as the pH is raised from 5.5 to 9.0. However, the SuSy in solution loses ≥80% of its maximum productivity at pH ∼7 when alkaline reaction conditions (pH 9.0) are used. We therefore immobilized the SuSy on nanocellulose-based biocomposite carriers (∼48 U/g carrier; ≥ 50% effectiveness) and reveal in the carrier-bound enzyme a substantial broadening of the pH-productivity profile to high pH, with up to 80% of maximum capacity retained at pH 9.5. Using reaction by the immobilized SuSy with automated pH control at pH ∼9.0, we demonstrate near-complete conversion (≥ 96%) of UDP-glucose and fructose (each 100mM) into sucrose, as expected from the equilibrium constant (Keq = ∼7 × 102) under these conditions. Collectively, our results support the idea of glycosyltransferase-catalyzed synthetic glycosylation from sugar nucleotide donor driven by high pH; and they showcase a marked adaptation to high pH of the operational activity of the soybean SuSy by immobilization.

Biochar as an Enzyme Immobilization Support and Its Application for Dye Degradation

Wastewaters generated by the textile industry often contain significant amounts of harmful (carcinogenic and mutagenic) cationic dyes, whose efficient removal is of crucial importance. This study investigates the laccase immobilization on biochar obtained from sour cherry stones (SCS-B), as a cost effective adsorbent, and evaluates its application for brilliant green (BG) degradation. The successful immobilization of laccase on biochar was achieved via adsorption and confirmed using scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and Fourier transform infrared spectroscopy (FTIR). An immobilization efficiency of 66% was achieved using 0.274 U/mL of laccase at pH 5 and a temperature of 40 °C. The adsorption kinetics of laccase followed a pseudo-second-order model, indicating that chemical adsorption plays a significant role in the immobilization process. The BG degradation by immobilized system was further optimized by evaluating effects of pH, temperature, dye concentration, and contact time. More than 92% of BG (50 mg/L) was removed within 4 h at pH 5 and temperature of 30 °C. These findings suggest that SCS-B can effectively be used as an enzyme carrier and be further utilized for the removal of emerging pollutants, positioning it as a sustainable solution for wastewater treatment.

ENHANCEMENT OF SENSITIVITY, DETECTION RANGE, AND DETECTION LIMIT USING GA METHOD POTENTIOMETRIC BIOSENSOR WITH UREASE ENZYME IMMOBILIZATION TECHNIQUE ON PVA

This study aims to increase the sensitivity range of the width of the UV-Vis absorbance peak with Glutaraldehyde (GA) crosslinking and useful to determine the level of urea in the urine. The potentiometer cell consists of an indicator electrode and a reference electrode. The indicator electrode is denoted PVA-Enzyme/GA/PVC-KTpCIPB, meaning PVA-Enzim as the first layer, GA as the second layer and PVC-KTpCIPB as the third layer. Biosensor with urease enzyme immobilization technique analyzed variations of urea 10-7-10-1 M. The potentiometric method of the biosensor detects signal and voltage (potentiometer cell). The signals that were analyzed for signal variables were symmetrical up and down signals of 2000 signals/second. Voltage was carried out by linear curve analysis, the results of linear curve analysis were the range of detection from a concentration of 10-4-10-2 M with cross-linked GA which increased the absorbance, the detection limit was 10-4 M, the sensitivity was 46.67 mV/ war reversibly and R squared (R2) which is 0.9839 is close to linear.

Immobilization of Coupled Enzymes onto Metalated Hierarchical Organic Microspheres.

Hierarchical organic microspheres (HOMs) have emerged as an ideal carrier for immobilizing biomacromolecules. In this research, an in-depth investigation into the structural characteristics of a striated HOM, known as HOM-15, has revealed the assembly mechanism of microspheres through weakly stacked two-dimensional structural units that are composed of V-shaped small organic molecules. With the leverage of this understanding, HOM-15 was adopted as a stable and reusable platform for co-immobilizing of ene-reductases and glucose dehydrogenases via metal ion bridging onto the surface of HOMs. The research demonstrates that metal ion bridging can finely tune the surface properties of HOM-15, thereby facilitating the immobilization of enzymes that would otherwise be impeded by electrostatic repulsion. Comparing HOM-15 to other microspherical variants revealed its superior biocatalytic performance, attributed to the reduction of the mass transfer barrier facilitated by its lamellar-stacking morphology. This novel biocatalytic system underscores the potential applications of HOMs in broader biocatalytic processes.

Green and Fast Synthesis of NiCo-MOF for Simultaneous Purification-Immobilization of Bienzyme to Catalyze the Synthesis of Ginsenoside Rh2.

Traditional metal-organic frameworks (MOFs) preparation is generally time-consuming, polluting, and lacking specificity for enzyme immobilization. This paper introduced a facile, rapid, and green method to produce three MOFs subsequently employed to purify and coimmobilize recombinant glycosyltransferase (UGT) and recombinant sucrose synthetase (SUSy) using histidine tag (His-tag) for the specific adsorption of Ni2+ and Co2+ from MOFs. This method simplified enzyme purification from crude extracts and enabled enzymes to be reused. The results demonstrated that NiCo-MOF exhibited a higher enzyme load (115.9 mg/g) than monometallic MOFs. Additionally, the NiCo-MOF@UGT&SUSy demonstrated excellent stability and efficiently produced the rare ginsenoside Rh2 by catalyzing a coupling reaction (95.6 μg/mL), solving the problem of the substrate cost of uridine diphosphate glucose (UDPG). The NiCo-MOF@UGT&SUSy retained 68.97% of the initial activity after 10 cycles. Finally, molecular docking studies elucidated the conversion mechanism of the target product Rh2. This technique is important in the industrialization of ginsenoside production and enzyme purification.

Recent Advances in Enzyme‐based Biofuel Cells Using Glucose Fuel: Achieving High Power Output and Enhanced Operational Stability

AbstractAn enzyme‐based biofuel cell (EBFC) is widely regarded as one of the most efficient power sources for bio‐friendly and implantable medical devices, capable of converting electrochemical reactions into electrical currents under physiological conditions. However, despite its potential, the practical and commercial use of EBFCs is limited by their low power output and operational instability. Therefore, significant research efforts have focused on increasing power output and stability by improving electron transfer between enzymes and host electrodes and developing efficient enzyme immobilization techniques. However, most EBFCs produced by current methods still deliver unsatisfactory performance. A promising approach to address these challenges is the use of conductive linkers that promote favorable interfacial interactions between adjacent enzymes and between enzymes and host electrodes. These linkers can facilitate electron transfer and ensure robust enzyme immobilization. In addition, designing the host electrode with a 3D structure and a large surface area can further improve the areal energy performance. This perspective reviews the working principles, types, and electron transfer mechanisms of EBFC electrodes and explores how conductive linkers and 3D host electrodes can enhance the performance of EBFC electrodes. Finally, recent advances in integrating EBFCs into biomedical devices are described.

Rapid Assembly of Ultrafine Palladium Nanoparticle-Decorated HOF-101 Triggered by Guest Enzyme Encapsulation.

Rapid enzyme immobilization is essential for enzyme catalysis and sensing applications, yet constructing effective immobilization systems is challenging due to the need to balance enzyme activity with the properties of the surrounding framework. Herein, taking glucose oxidase (GOx) as a model, a rapid and straightforward approach was presented for synthesizing palladium nanoparticles (PdNPs)-decorated GOx encapsulated in HOF-101 nanocomposite materials (designated as PdNPs/GOx@HOF-101) through an in situ photoreduction and enzyme-triggering HOF-101 encapsulation. The enzyme's surface residues trigger the nucleation of HOF-101 around it through the hydrogen-bonded bio interface, completing the self-assembly of HOF-101 in 0.5 h. Furthermore, the biocomposites loaded with ultrafine PdNPs show satisfactory photoelectrochemical (PEC) properties. As a proof-of-concept, a PEC biosensor was constructed by utilizing PdNPs/GOx@HOF-101 as a photoactive probe, which can quickly and sensitively detect glucose and simultaneously remain stable within the circumstance of 30-60 °C and pH 4-8. These attributes pave the way for diverse applications, including improved enzyme immobilization techniques, advanced biosensors, and more efficient biocatalytic processes.

Coral-like Ti3C2Tx/PANI Binary Nanocomposite Wearable Enzyme Electrochemical Biosensor for Continuous Monitoring of Human Sweat Glucose

With the continuous advancement of contemporary medical technology, an increasing number of individuals are inclined towards self-monitoring their physiological health information, specifically focusing on monitoring blood glucose levels. However, as an emerging flexible sensing technique, continuous and non-invasive monitoring of glucose in sweat offers a promising alternative to conventional invasive blood tests for measuring blood glucose levels, reducing the risk of infection associated with blood testing. In this study, we fabricated a flexible and wearable electrochemical enzyme sensor based on a two-dimensional Ti3C2Tx MXene nanosheets and coral-like polyaniline (PANI) binary nanocomposite (denoted as Ti3C2Tx/PANI) for continuous, non-invasive, real-time monitoring of sweat glucose. The exceptional conductivity of Ti3C2Tx MXene nanosheets, in conjunction with the mutual doping effect facilitated by coral-like PANI, significantly enhances electrical conductivity and specific surface areas of Ti3C2Tx/PANI. Consequently, the fabricated sensor exhibits remarkable sensitivity (25.16 μA·mM−1·cm−2), a low detection limit of glucose (26 μM), and an extensive detection range (0.05 mM ~ 1.0 mM) in sweat. Due to the dense coral-like structure of Ti3C2Tx/PANI binary nanocomposite, a larger effective area is obtained to offer more active sites for enzyme immobilization and enhancing enzymatic catalytic activity. Moreover, the sensor demonstrates exceptional mechanical performance, enabling a 60° bend in practical applications, thus satisfying the rigorous demands of human sweat detection applications. The results obtained from continuous 60 min in vitro monitoring of sweat glucose levels demonstrate a robust correlation with the data of blood glucose levels collected by a commercial glucose meter. Furthermore, the fabricated Ti3C2Tx/PANI/GOx sensor demonstrated agreement with HPLC findings regarding the actual concentration of added glucose. This study presents an efficient and practical approach for the development of a highly reliable MXene glucose biosensor, enabling stable and long-term monitoring of glucose levels in human sweat.

Extraction and immobilization of the urease enzyme in polyvinylpyrrolidone films: applications in biosensors

Enzymes play a fundamental role in the manufacture of biosensors, acting as biological recognition elements due to their sensitivity and selectivity in the reactions they catalyze. Among them, urease stands out as an efficient enzyme found in nature, whose function involves the hydrolysis of urea. This enzyme has been used when extracted from legume seeds, as well as various bacteria and fungi. The objective of this study was the extraction and immobilization of the urease enzyme in polyvinylpyrrolidone (PVP) films. Crude jack bean extracts were prepared at concentrations of 15, 25 and 35% in PBS/PVP, centrifuged and refrigerated. Subsequently, aliquots of each solution were applied to plates to form films, being characterized by enzymatic activity, pH, FTIR and UV-vis. The affinity with urea was evidenced by enzymatic activity, pH and UV-vis, compatible with the Michaelis-Menten model. In FTIR, the polypeptide bands characteristic of the amino acids present in the structure of the enzymes, such as those of amides I, II and III, were confirmed.

Thermothelomyces thermophilus cultivated with residues from the fruit pulp industry: enzyme immobilization on ionic supports of a crude cocktail with enhanced production of lichenase.

β-Glucans comprise a group of β-D-glucose polysaccharides (glucans) that occur naturally in the cell walls of bacteria, fungi, and cereals. Its degradation is catalyzed by β-glucanases, enzymes that catalyze the breakdown of β-glucan into cello-oligosaccharides and glucose. These enzymes are classified as endo-glucanases, exo-glucanases, and glucosidases according to their mechanism of action, being the lichenases (β-1,3;1,4-glucanases, EC 3.2.1.73) one of them. Hence, we aimed to enhance lichenase production by Thermothelomyces thermophilus through the application of response surface methodology, using tamarind (Tamarindus indica) and jatoba (Hymenaea courbaril) seeds as carbon sources. The crude extract was immobilized, with a focus on improving lichenase activity, using various ionic supports, including MANAE (monoamine-N-aminoethyl), DEAE (diethylaminoethyl)-cellulose, CM (carboxymethyl)-cellulose, and PEI (polyethyleneimine)-agarose. Regarding lichenase, the optimal conditions yielding the highest activity were determined as 1.5% tamarind seeds, cultivation at 50°C under static conditions for 72h. Moreover, transitioning from Erlenmeyer flasks to a bioreactor proved pivotal, resulting in a 2.21-fold increase in activity. Biochemical characterization revealed an optimum temperature of 50°C and pH of 6.5. However, sustained stability at varying pH and temperature levels was challenging, underscoring the necessity of immobilizing lichenase on ionic supports. Notably, CM-cellulose emerged as the most effective immobilization medium, exhibiting an activity of 1.01 U/g of the derivative (enzyme plus support), marking a substantial enhancement. This study marks the first lichenase immobilization on these chemical supports in existing literature.

Magnetic protein aggregates generated by supramolecular assembly of ferritin cages - a modular strategy for the immobilization of enzymes.

Efficient and cost-effective immobilization methods are crucial for advancing the utilization of enzymes in industrial biocatalysis. To this end, in vivo immobilization methods relying on the completely biological production of immobilizates represent an interesting alternative to conventional carrier-based immobilization methods. This study aimed to introduce a novel immobilization strategy using in vivo-produced magnetic protein aggregates (MPAs). MPA production was achieved by expressing gene fusions of the yellow fluorescent protein variant citrine and ferritin variants, including a magnetically enhanced Escherichia coli ferritin mutant. Cellular production of the gene fusions allows supramolecular assembly of the fusion proteins in vivo, driven by citrine-dependent dimerization of ferritin cages. Magnetic properties were confirmed using neodymium magnets. A bait/prey strategy was used to attach alcohol dehydrogenase (ADH) to the MPAs, creating catalytically active MPAs (CatMPAs). These CatMPAs were purified via magnetic columns or centrifugation. The fusion of the mutant E. coli ferritin to citrine yielded fluorescent, insoluble protein aggregates, which are released upon cell lysis and coalesce into MPAs. MPAs display magnetic properties, as verified by their attraction to neodymium magnets. We further show that these fully in vivo-produced protein aggregates can be magnetically purified without ex vivo iron loading. Using a bait/prey strategy, MPAs were functionalized by attaching alcohol dehydrogenase post-translationally, creating catalytically active magnetic protein aggregates (CatMPAs). These CatMPAs were easily purified from crude extracts via centrifugation or magnetic columns and showed enhanced stability. This study presents a modular strategy for the in vivo production of MPAs as scaffold for enzyme immobilization. The approach eliminates the need for traditional, expensive carriers and simplifies the purification process by leveraging the insoluble nature and the magnetic properties of the aggregates, opening up the potential for novel, streamlined applications in biocatalysis.

Unlocking of Hidden Mesopores for Enzyme Encapsulation by Dynamic Linkers in Stable Metal-Organic Frameworks.

Mesoporous metal-organic frameworks (MOFs) are promising supports for the immobilization of enzymes, yet their applications are often limited by small pore apertures that constrain the size of encapsulated enzymes to below 5 nm. In this study, we introduced labile linkers (4,4',4''-(2,4,6-boroxintriyl)-tribenzoate, TBTB) with dynamic boroxine bonds into mesoporous PCN-333, resulting in PCN-333-TBTB with enhanced enzyme loading and protection capabilities. The selective breaking of B-O bonds creates defects in PCN-333, which effectively expands both window and cavity sizes, thereby unlocking hidden mesopores for enzyme encapsulation. Consequently, this strategy not only increases the adsorption kinetics of small enzymes (<5 nm) such as cytochrome c (Cyt C) and horseradish peroxidase (HRP), but also enables the immobilization of various large-sized enzymes (>5 nm), such as glycoenzymes. The glycoenzymes@PCN-333-TBTB platform was successfully applied to synthesize thirteen complex oligosaccharides and polysaccharides, demonstrating high activity and enhanced enzyme stability. The dynamic linker-mediated enzyme encapsulation strategy enables the immobilization of enzymes exceeding the inherent pore size of MOFs, thus broadening the scope of enzymatic catalytic reactions achievable with MOF materials.

Unlocking of Hidden Mesopores for Enzyme Encapsulation by Dynamic Linkers in Stable Metal‐Organic Frameworks

AbstractMesoporous metal‐organic frameworks (MOFs) are promising supports for the immobilization of enzymes, yet their applications are often limited by small pore apertures that constrain the size of encapsulated enzymes to below 5 nm. In this study, we introduced labile linkers (4,4′,4′′‐(2,4,6‐boroxintriyl)‐tribenzoate, TBTB) with dynamic boroxine bonds into mesoporous PCN‐333, resulting in PCN‐333‐TBTB with enhanced enzyme loading and protection capabilities. The selective breaking of B−O bonds creates defects in PCN‐333, which effectively expands both window and cavity sizes, thereby unlocking hidden mesopores for enzyme encapsulation. Consequently, this strategy not only increases the adsorption kinetics of small enzymes (&lt;5 nm) such as cytochrome c (Cyt C) and horseradish peroxidase (HRP), but also enables the immobilization of various large‐sized enzymes (&gt;5 nm), such as glycoenzymes. The glycoenzymes@PCN‐333‐TBTB platform was successfully applied to synthesize thirteen complex oligosaccharides and polysaccharides, demonstrating high activity and enhanced enzyme stability. The dynamic linker‐mediated enzyme encapsulation strategy enables the immobilization of enzymes exceeding the inherent pore size of MOFs, thus broadening the scope of enzymatic catalytic reactions achievable with MOF materials.

Simultaneous Degradation of AFB1 and ZEN by CotA Laccase from Bacillus subtilis ZJ-2019-1 in the Mediator-Assisted or Immobilization System.

The global prevalence of aflatoxin B1 (AFB1) and zearalenone (ZEN) contamination in food and feed poses a serious health risk to humans and animals. Recently, enzymatic detoxification has received increasing attention, yet most enzymes are limited to degrading only one type of mycotoxin, and free enzymes often exhibit reduced stability and activity, limiting their practicality in real-world applications. In this study, the laccase CotA gene from ZEN/AFB1-degrading Bacillus subtilis ZJ-2019-1 was cloned and successfully expressed in Escherichia coli BL21, achieving a protein yield of 7.0 mg/g. The recombinant CotA (rCotA) completely degraded AFB1 and ZEN, with optimal activity at 70 °C and pH 7.0. After rCotA treatment, neither AFB1 nor ZEN showed significantly cytotoxicity to mouse macrophage cell lines. Additionally, the AFB1/ZEN degradation efficiency of rCotA was significantly enhanced by five natural redox mediators: acetosyringone, syringaldehyde, vanillin, matrine, and sophoridin. Among them, the acetosyringone-rCotA was the most effective mediator system, which could completely degrade 10 μg of AFB1 and ZEN within 1 h. Furthermore, the chitosan-immobilized rCotA system exhibited higher degradation activity than free rCotA. The immobilized rCotA degraded 27.95% of ZEN and 41.37% of AFB1 in contaminated maize meal within 12 h, and it still maintained more than 40% activity after 12 reuse cycles. These results suggest that media-assisted or immobilized enzyme systems not only boost degradation efficiency but also demonstrate remarkable reusability, offering promising strategies to enhance the degradation efficiency of rCotA for mycotoxin detoxification.