A magnetic beads-based ligand fishing method for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from Hunteria zeylanica.

In this study, a novel magnetic bead-based ligand fishing method was developed for rapid discovery of monoterpene indoles as monoamine oxidase A inhibitors from natural products. In order to improve the screening efficiency, two different magnetic beads, i.e. amine and carboxyl terminated magnetic beads, were comprehensively compared in terms of their ability to immobilize monoamine oxidase A (MAOA), biocatalytic activity and specific adsorption rates for affinity ligands. Carboxyl terminated magnetic beads performed better for MAOA immobilization and demonstrated superior performance in ligand fishing. The MAOA immobilized magnetic beads were applied to screen novel monoamine oxidase inhibitors in an alkaloid-rich plant, Hunteria zeylanica. Twelve MAOA affinity ligands were screened out, and ten of them were identified as monoterpene indole alkaloids by HPLC-Obitrap-MS/MS. Among them, six ligands, namely geissoschizol, vobasinol, yohimbol, dihydrocorynanthenol, eburnamine and (+)-isoeburnamine which exhibited inhibitory activity against MAOA with low IC50 values. To further explore their inhibitory mechanism, enzyme kinetic analysis and molecular docking studies were conducted.

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.

L-Lactate Electrochemical Biosensor Based on an Integrated Supramolecular Architecture of Multiwalled Carbon Nanotubes Functionalized with Avidin and a Recombinant Biotinylated Lactate Oxidase.

L-Lactate is an important bioanalyte in the food industry, biotechnology, and human healthcare. In this work, we report the development of a new L-lactate electrochemical biosensor based on the use of multiwalled carbon nanotubes non-covalently functionalized with avidin (MWCNT-Av) deposited at glassy carbon electrodes (GCEs) as anchoring sites for the bioaffinity-based immobilization of a new recombinant biotinylated lactate oxidase (bLOx) produced in Escherichia coli through in vivo biotinylation. The specific binding of MWCNT-Av to bLOx was characterized by amperometry, surface plasmon resonance (SPR), and electrochemical impedance spectroscopy (EIS). The amperometric detection of L-lactate was performed at -0.100 V, with a linear range between 100 and 700 µM, a detection limit of 33 µM, and a quantification limit of 100 µM. The proposed biosensor (GCE/MWCNT-Av/bLOx) showed a reproducibility of 6.0% and it was successfully used for determining L-lactate in food and enriched serum samples.

Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

DNA Framework-Programmed Nanoscale Enzyme Assemblies.

Multienzyme assemblies mediated by multivalent interaction play a crucial role in cellular processes. However, the three-dimensional (3D) programming of an enzyme complex with defined enzyme activity in vitro remains unexplored, primarily owing to limitations in precisely controlling the spatial topological configuration. Herein, we introduce a nanoscale 3D enzyme assembly using a tetrahedral DNA framework (TDF), enabling the replication of spatial topological configuration and maintenance of an identical edge-to-edge distance akin to natural enzymes. Our results demonstrate that 3D nanoscale enzyme assemblies in both two-enzyme systems (glucose oxidase (GOx)/horseradish peroxidase (HRP)) and three-enzyme systems (amylglucosidase (AGO)/GOx/HRP) lead to enhanced cascade catalytic activity compared to the low-dimensional structure, resulting in ∼5.9- and ∼7.7-fold enhancements over homogeneous diffusional mixtures of free enzymes, respectively. Furthermore, we demonstrate the enzyme assemblies for the detection of the metabolism biomarkers creatinine and creatine, achieving a low limit of detection, high sensitivity, and broad detection range.

Encapsulation of an enzyme-immobilized smart polymer membrane in a metal-organic framework for enhancement of catalytic performance.

Encapsulation of enzymes within porous materials has shown great promise for protecting enzymes from denaturation, increasing their tolerance to harsh environments and promoting their industrialization. However, controlling the conformational freedom of the encapsulated enzymes to enhance their catalytic performance remains a great challenge. To address this issue, herein, following immobilization of GOx and HRP on a thermo-responsive porous poly(styrene-maleic-anhydride-N-isopropylacrylamide) (PSMN) membrane, a GOx-HRP@PSMN@HZIF-8 composite was fabricated by encapsulating GOx-HRP@PSMN in hollow ZIF-8 (HZIF-8) with liposome (L) as the sacrificial template. The improved conformational freedom for enzymes arising from the hollow cavity formed in ZIF-8 through the removal of L enhanced the mass transfer and dramatically promoted the catalytic activity of the composite. Interestingly, at high temperature, the coiled PN moiety in PSMN provided the confinement effect for GOx-HRP, which also significantly boosted the catalytic performance of the composites. Compared to the maximum catalytic reaction rates (Vmax) of GOx-HRP@PSMN@LZIF-8, the free enzyme and GOx-HRP@ZIF-8, the Vmax of the GOx-HRP@PSMN@HZIF-8 composite exhibited an impressive 17.8-fold, 10.8-fold and 6.0-fold enhancement at 37 °C, respectively. The proposed composites successfully demonstrated their potential as catalytic platforms for the colorimetric detection of glucose in a cascade reaction. This study paves a new way for overcoming the current limitations of immobilizing enzymes in porous materials and the use of smart polymers for the potential fabrication of enzyme@polymer@MOF composites with tunable conformational freedom and confinement effect.

Immobilized lipase enzyme on green synthesized magnetic nanoparticles using Psidium guava leaves for dye degradation and antimicrobial activities.

Zinc ferrite nanoparticles (ZnF NPs) were synthesized by a green method using Psidium guava Leaves extract and characterized via structural and optical properties. The surface of ZnF NPs was stabilized with citric acid (CA) by a direct addition method to obtain (ZnF-CA NPs), and then lipase (LP) enzyme was immobilized on ZnF-CA NPs to obtain a modified ZnF-CA-LP nanocomposite (NCs). The prepared sample's photocatalytic activity against Methylene blue dye (MB) was determined. The antioxidant activity of ZnF-CA-LP NCs was measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) as a source of free radicals. In addition, the antibacterial and antibiofilm capabilities of these substances were investigated by testing them against gram-positive Staphylococcus aureus (S. aureus ATCC 25923) and gram-negative Escherichia coli (E. coli ATCC 25922) bacterial strains. The synthesized ZnF NPs were discovered to be situated at the core of the material, as determined by XRD, HRTEM, and SEM investigations, while the CA and lipase enzymes were coated in this core. The ZnF-CA-LP NCs crystallite size was around 35.0 nm at the (311) plane. Results obtained suggested that 0.01 g of ZnF-CA-LP NCs achieved 96.0% removal of 5.0 ppm of MB at pH 9.0. In-vitro zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) results verified that ZnF-CA-LP NCs exhibited its encouraged antimicrobial activity against S. aureus and E. coli (20.0 ± 0.512, and 27.0 ± 0.651 mm ZOI, respectively) & (1.25, and 0.625 µg/ml MIC, respectively). ZnF-CA-LP NPs showed antibiofilm percentage against S. aureus (88.4%) and E. coli (96.6%). Hence, ZnF-CA-LP NCs are promising for potential applications in environmental and biomedical uses.

An enzymatic continuous-flow reactor based on a pore-size matching nano- and isoporous block copolymer membrane.

Continuous-flow biocatalysis utilizing immobilized enzymes emerged as a sustainable route for chemical synthesis. However, inadequate biocatalytic efficiency from current flow reactors, caused by non-productive enzyme immobilization or enzyme-carrier mismatches in size, hampers its widespread application. Here, we demonstrate a general-applicable and robust approach for the fabrication of a high-performance enzymatic continuous-flow reactor via integrating well-designed scalable isoporous block copolymer (BCP) membranes as carriers with an oriented and productive immobilization employing material binding peptides (MBP). Densely packed uniform enzyme-matched nanochannels of well-designed BCP membranes endow the desired nanoconfined environments towards a productive immobilized phytase. Tuning nanochannel properties can further regulate the complex reaction process and fortify the catalytic performance. The synergistic design of enzyme-matched carriers and efficient enzyme immobilization empowers an excellent catalytic performance with >1 month operational stability, superior productivity, and a high space-time yield (1.05 × 105 g L-1 d-1) via a single-pass continuous-flow process. The obtained performance makes the designed nano- and isoporous block copolymer membrane reactor highly attractive for industrial applications.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.

Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Octadecyl and sulfonyl modification of diatomite synergistically improved the immobilization efficiency of lipase and its application in the synthesis of pine sterol esters.

Phytosterols usually have to be esterified to various phytosterol esters to avoid their disadvantages of unsatisfactory solubility and low bioavailability. The enzymatic synthesis of phytosterol esters in a solvent-free system has advantages in terms of environmental friendliness, sustainability, and selectivity. However, the limitation of the low stability and recyclability of the lipase in the solvent-free system, which often requires a relatively high temperature to induce the viscosity, also increased the industrial production cost. In this context, a low-cost material, namely diatomite, was employed as the support in the immobilization of Candida rugosa lipase (CRL) due to its multiple modification sites. The Fe3 O4 was also then introduced to this system for quick and simple separation via the magnetic field. Moreover, to further enhance the immobilization efficiency of diatomite, a modification strategy which involved the octadecyl and sulfonyl group for regulating the hydrophobicity and interaction between the support and lipase was successfully developed. The optimization of the ratio of the modifiers suggested that the -SO3 H/C18 (1:1.5) performed best with an enzyme loading and enzyme activity of 84.8 mg·g-1 and 54 U·g-1 , respectively. Compared with free CRL, the thermal and storage stability of CRL@OSMD was significantly improved, which lays the foundation for the catalytic synthesis of phytosterol esters in solvent-free systems. Fortunately, a yield of 95.0% was achieved after optimizing the reaction conditions, and a yield of 70.0% can still be maintained after six cycles.

Extending the computational and experimental analysis of lipase active site selectivity.

Molecular docking is an important computational analysis widely used to predict the interaction of enzymes with several starting materials for developing new valuable products from several starting materials, including oils and fats. In the present study, molecular docking was used as an efficient in silico screening tool to select biocatalysts with the highest catalytic performance in butyl esters production in a solvent-free system, an eco-friendly approach, via direct esterification of free fatty acids from Licuri oil with butanol. For such purpose, three commercial lipase preparations were used to perform molecular docking studies such as Burkholderia cepacia (BCL), Porcine pancreatic (PPL), and Candida rugosa (CRL). Concurrently, the results obtained in BCL and CRL are the most efficient in the esterification process due to their higher preference for catalyzing the esterification of lauric acid, the main fatty acid found in the licuri oil composition. Meanwhile, PPL was the least efficient because it preferentially interacts with minor fatty acids. Molecular docking with the experimental results indicated the better performance in the synthesis of esters was BCL. In conclusion, experimental results analysis shows higher enzymatic productivity in esterification reactions of 1294.83 µmol/h.mg, while the CRL and PPL demonstrated the lowest performance (189.87 µmol / h.mg and 23.96 µmol / h.mg, respectively). Thus, molecular docking and experimental results indicate that BCL is a more efficient lipase to produce fatty acids and esters from licuri oil with a high content of lauric acid. In addition, this study also demonstrates the application of molecular docking as an important tool for lipase screening to achieve more sustainable production of butyl esters with a view synthesis of biolubricants.

Enzymes in "Green" Synthetic Chemistry: Laccase and Lipase.

Enzymes play an important role in numerous natural processes and are increasingly being utilized as environmentally friendly substitutes and alternatives to many common catalysts. Their essential advantages are high catalytic efficiency, substrate specificity, minimal formation of byproducts, and low energy demand. All of these benefits make enzymes highly desirable targets of academic research and industrial development. This review has the modest aim of briefly overviewing the classification, mechanism of action, basic kinetics and reaction condition effects that are common across all six enzyme classes. Special attention is devoted to immobilization strategies as the main tools to improve the resistance to environmental stress factors (temperature, pH and solvents) and prolong the catalytic lifecycle of these biocatalysts. The advantages and drawbacks of methods such as macromolecular crosslinking, solid scaffold carriers, entrapment, and surface modification (covalent and physical) are discussed and illustrated using numerous examples. Among the hundreds and possibly thousands of known and recently discovered enzymes, hydrolases and oxidoreductases are distinguished by their relative availability, stability, and wide use in synthetic applications, which include pharmaceutics, food and beverage treatments, environmental clean-up, and polymerizations. Two representatives of those groups-laccase (an oxidoreductase) and lipase (a hydrolase)-are discussed at length, including their structure, catalytic mechanism, and diverse usage. Objective representation of the current status and emerging trends are provided in the main conclusions.

UCST-Type Soluble Immobilized Cellulase: A New Strategy for the Efficient Degradation and Improved Recycling Performance of Wastepaper Cellulose.

This paper reports an innovative study that aims to address key issues in the efficient recycling of wastepaper cellulose. The research team utilized the temperature-responsive upper critical solution temperature (UCST) polymer P(NAGA-b-DMA) in combination with the LytA label's affinity for choline analogs. This innovative approach enabled them to successfully develop a novel soluble immobilized enzyme, P(NAGA-b-DMA)-cellulase. This new enzyme has proven highly effective, significantly enhancing the degradation of wastepaper cellulose while demonstrating exceptional stability. Compared with the traditional insoluble immobilized cellulase, the enzyme showed a significant improvement in the pH, temperature stability, recycling ability, and storage stability. A kinetic parameter calculation showed that the enzymatic effectiveness of the soluble immobilized enzyme was much better than that of the traditional insoluble immobilized cellulase. After the immobilization reaction, the Michaelis constant of the immobilized enzyme was only increased by 11.5%. In the actual wastepaper degradation experiment, the immobilized enzyme was effectively used, and it was found that the degradation efficiency of wastepaper cellulose reached 80% of that observed in laboratory conditions. This novel, thermosensitive soluble immobilized cellulase can efficiently catalyze the conversion of wastepaper cellulose into glucose under suitable conditions, so as to further ferment into environmentally friendly biofuel ethanol, which provides a solution to solve the shortage of raw materials and environmental protection problems in the paper products industry.

Pyrite-assisted degradation of methoxychlor by laccase immobilized on Fe3S4/earthworm-like mesoporous SiO2.

Laccase immobilized and cross-linked on Fe3S4/earthworm-like mesoporous SiO2 (Fe3S4/EW-mSiO2) was used to degrade methoxychlor (MXC) in aqueous environments. The effects of various parameters on the degradation of MXC were determined using free and immobilized laccase. Immobilization improved the thermal stability and reuse of laccase significantly. Under the conditions of pH 4.5, temperature 40 °C, and reaction time 8 h, the degradation rate of MXC by immobilized laccase reached a maximum value of 40.99% and remained at 1/3 of the original after six cycles. The excellent degradation performance of Fe3S4/EW-mSiO2 was attributable to the pyrite (FeS2) impurity in Fe3S4, which could act as an electron donor in reductive dehalogenation. Sulfide groups and Fe2+ reduced the activation energy of the system resulting in pyrite-assisted degradation of MXC. The degradation mechanism of MXC in aqueous environments by laccase immobilized on Fe3S4/EW-mSiO2 was determined via mass spectroscopy of the degradation products. This study is a new attempt to use pyrite to support immobilized laccase degradation.

Enhancing enzyme immobilization: Fabrication of biosilica-based organic-inorganic composite carriers for efficient covalent binding of D-allulose 3-epimerase.

D-allulose, an ideal low-calorie sweetener, is primarily produced through the isomerization of d-fructose using D-allulose 3-epimerase (DAE; EC 5.1.3.30). Addressing the gap in available immobilized DAE enzymes for scalable commercial D-allulose production, three core-shell structured organic-inorganic composite silica-based carriers were designed for efficient covalent immobilization of DAE. Natural inorganic diatomite was used as the core, while 3-aminopropyltriethoxysilane (APTES), polyethyleneimine (PEI), and chitosan organic layers were coated as the shells, respectively. These tailored carriers successfully formed robust covalent bonds with DAE enzyme conjugates, cross-linked via glutaraldehyde, and demonstrated enzyme activities of 372 U/g, 1198 U/g, and 381 U/g, respectively. These immobilized enzymes exhibited an expanded pH tolerance and improved thermal stability compared to free DAE. Particularly, the modified diatomite with PEI exhibited a higher density of binding sites than the other carriers and the PEI-coated immobilized DAE enzyme retained 70.4 % of its relative enzyme activity after ten cycles of reuse. This study provides a promising method for DAE immobilization, underscoring the potential of using biosilica-based organic-inorganic composite carriers for the development of robust enzyme systems, thereby advancing the production of value-added food ingredients like D-allulose.

Enhancing biocatalyst performance through immobilization of lipase (Eversa® Transform 2.0) on hybrid amine-epoxy core-shell magnetic nanoparticles.

Magnetic nanoparticles were functionalized with polyethylenimine (PEI) and activated with epoxy. This support was used to immobilize Lipase (Eversa® Transform 2.0) (EVS), optimization using the Taguchi method. XRF, SEM, TEM, XRD, FTIR, TGA, and VSM performed the characterizations. The optimal conditions were immobilization yield (I.Y.) of 95.04 ± 0.79 %, time of 15 h, ionic load of 95 mM, protein load of 5 mg/g, and temperature of 25 °C. The maximum loading capacity was 25 mg/g, and its stability in 60 days of storage showed a negligible loss of only 9.53 % of its activity. The biocatalyst demonstrated better stability at varying temperatures than free EVS, maintaining 28 % of its activity at 70 °C. It was feasible to esterify free fatty acids (FFA) from babassu oil with the best reaction of 97.91 % and ten cycles having an efficiency above 50 %. The esterification of produced biolubricant was confirmed by NMR, and it displayed kinematic viscosity and density of 6.052 mm2/s and 0.832 g/cm3, respectively, at 40 °C. The in-silico study showed a binding affinity of -5.8 kcal/mol between EVS and oleic acid, suggesting a stable substrate-lipase combination suitable for esterification.

Magnetic nanoparticles immobilized thrombin ligand fishing to screen thrombin inhibitors in natural products.

In this study, thrombin was immobilized with magnetic particles modified by glutaraldehyde. The changes in secondary structures of immobilized enzyme revealed an increment in conformational rigidity and stability, which can be reflected in temperature and pH stability as well as the tolerance of organic reagents. The optimal reutilization times of magnetic particle immobilized thrombin were 7 times, and the half-life of enzyme activity preserved at room temperature was 5 days, which was 2.5 times higher than that of free enzyme. Ligusticum chuanxiong and Anemarrhenae Rhizoma with high enzyme inhibitory activity were selected for primary screening, and six potential inhibitors of thrombin were identified by HPLC/MS. The results showed that three compounds in Anemarrhenae Rhizoma had better predictive thrombin inhibitory activity. Through the in vitro thrombin activity inhibition experiment, it was also verified that mangiferin and neo-mangiferin had an ideal thrombin activity inhibition effect, which was consistent with the results of molecular docking.

Combinatorial High-Throughput Screening of Complex Polymeric Enzyme Immobilization Supports.

Recent advances have demonstrated the promise of complex multicomponent polymeric supports to enable supra-biological enzyme performance. However, the discovery of such supports has been limited by time-consuming, low-throughput synthesis and screening. Here, we describe a novel combinatorial and high-throughput platform that enables rapid screening of complex and heterogeneous copolymer brushes as enzyme immobilization supports, named combinatorial high-throughput enzyme support screening (CHESS). Using a 384-well plate format, we synthesized arrays of three-component polymer brushes in the microwells using photoactivated surface-initiated polymerization and immobilized enzymes in situ. The utility of CHESS to identify optimal immobilization supports under thermally and chemically denaturing conditions was demonstrated usingBacillus subtilisLipase A (LipA). The identification of supports with optimal compositions was validated by immobilizing LipA on polymer-brush-modified biocatalyst particles. We further demonstrated that CHESS could be used to predict the optimal composition of polymer brushes a priori for the previously unexplored enzyme, alkaline phosphatase (AlkP). Our findings demonstrate that CHESS represents a predictable and reliable platform for dramatically accelerating the search of chemical compositions for immobilization supports and further facilitates the discovery of biocompatible and stabilizing materials.

Calcium-based MOFs as scaffolds for shielding immobilized lipase and enhancing its stability.

The enzyme immobilization technology has become a key tool in the field of enzyme applications; however, improving the activity recovery and stability of the immobilized enzymes is still challenging. Herein, we employed a magnetic carboxymethyl cellulose (MCMC) nanocomposite modified with ionic liquids (ILs) for covalent immobilization of lipase, and used Ca-based metal-organic frameworks (MOFs) as the support skeleton and protective layer for immobilized enzymes. The ILs contained long side chains (eight CH2 units), which not only enhanced the hydrophobicity of the carrier and its hydrophobic interaction with the enzymes, but also provided a certain buffering effect when the enzyme molecules were subjected to compression. Compared to free lipase, the obtained CaBPDC@PPL-IL-MCMC exhibited higher specific activity and enhanced stability. In addition, the biocatalyst could be easily separated using a magnetic field, which is beneficial for its reusability. After 10 cycles, the residual activity of CaBPDC@PPL-IL-MCMC could reach up to 86.9%. These features highlight the good application prospects of the present immobilization method.