Infiltration of porcine pancreatic lipase into magnetic hierarchical mesoporous UiO-66-NH2 metal-organic frameworks for efficient detoxification of patulin from apple juice.

Patulin (PAT) is a mycotoxin known to globally contaminate fruits. The economic losses and health hazards caused by PAT desires a safe and efficient strategy for detoxifying PAT. Here, a magnetic core-shell hierarchical mesoporous metal-organic framework (Fe3O4@HMUiO-66-NH2) was synthesized via a salt-assisted nanoemulsion guided assembly method. This mesoporous structure (centered at 4.25 nm) allowed porcine pancreatic lipase (PPL) to infiltrate into the MOF shell at an immobilized amount of 255 mg/g, providing protection for PPL and enabling rapid separation and recovery. Compared with free PPL, PPL/Fe3O4@HMUiO-66-NH2 at 70 °C possessed 4.7 folds improved thermal stability in terms of half-life. The detoxification rates of immobilized enzyme for PAT in neutral water, acidic water, and apple juice were 99.6%, 60.9%, and 52.6%, respectively. Moreover, the so designed PPL/Fe3O4@HMUiO-66-NH2 showed extraordinary storage stability, reusability, and biocompatibility. Crucially, the quality of apple juice did not change significantly after PPL/Fe3O4@HMUiO-66-NH2 treatment, which facilitated its application in apple juice. The magnetic core-shell mesoporous structure along with the revealed mechanism of immobilized enzyme detoxification of PAT provide tremendous opportunity for designing a safe and efficient PAT detoxification method.

Mixed-mode column allows simple direct coupling with immobilized enzymatic reactor for on-line protein digestion.

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, ß-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

Cellulase immobilization to enhance enzymatic hydrolysis of lignocellulosic biomass: An all-inclusive review.

Cellulase-mediated lignocellulosic biorefinery plays a crucial role in the production of high-value biofuels and chemicals, with enzymatic hydrolysis being an essential component. The advent of cellulase immobilization has revolutionized this process, significantly enhancing the efficiency, stability, and reusability of cellulase enzymes. This review offers a thorough analysis of the fundamental principles underlying immobilization, encompassing various immobilization approaches such as physical adsorption, covalent binding, entrapment, and cross-linking. Furthermore, it explores a diverse range of carrier materials, including inorganic, organic, and hybrid/composite materials. The review also focuses on emerging approaches like multi-enzyme co-immobilization, oriented immobilization, immobilized enzyme microreactors, and enzyme engineering for immobilization. Additionally, it delves into novel carrier technologies like 3D printing carriers, stimuli-responsive carriers, artificial cellulosomes, and biomimetic carriers. Moreover, the review addresses recent obstacles in cellulase immobilization, including molecular-level immobilization mechanism, diffusion limitations, loss of cellulase activity, cellulase leaching, and considerations of cost-effectiveness and scalability. The knowledge derived from this review is anticipated to catalyze the evolution of more efficient and sustainable biocatalytic systems for lignocellulosic biomass conversion, representing the current state-of-the-art in cellulase immobilization techniques.

Improving the Bioactivity and Stability of Embedded Enzymes by Covalent Organic Frameworks.

De novo embedding enzymes within reticular chemistry materials have shown the enhancement of physical and chemical stability for versatile catalytic reactions. Compared to metal-organic frameworks (MOFs), covalent organic frameworks (COFs) are usually considered to be the more superior host of enzymes because of their large channels with low diffusion barriers, outstanding chemical/thermal stability, and metal-free nature. However, detailed investigations on the comparison of COFs and MOFs in enhancing biocatalytic performance have not been explored. Here, we de novo encapsulated enzymes within two COFs via a mechanochemical strategy, which avoided the extreme synthetic conditions of COFs and highly maintained the biological activities of the embedded enzymes. The enzymes@COFs biocomposites exhibited a much higher activity (3.4-14.7 times higher) and enhanced stability than those in MOFs (ZIF-8, ZIF-67, HKUST-1, MIL-53, and CaBDC), and the rate parameter (kcat/Km) of enzyme@COFs was 41.3 times higher than that of enzyme@ZIF-8. Further explorations showed that the conformation of enzymes inside MOFs was disrupted, owing to the harmful interfacial interactions between enzymes and metal ions as confirmed by ATR-FTIR, fluorescence spectroscopy, and XPS data. In contrast, enzymes that were embedded in metal-free COFs highly preserved the natural conformation of free enzymes. This study provides a better understanding of the interfacial interactions between reticular supports and enzymes, which paves a new road for optimizing the bioactivities of immobilized enzymes.

Immobilized Enzymes on Magnetic Beads for Separate Mass Spectrometric Investigation of Human Phase II Metabolite Classes.

The human body has evolved to remove xenobiotics through a multistep clearance process. Non-endogenous metabolites are converted through a series of phase I and different phase II enzymes into compounds with higher hydrophilicity. These compounds are important for diverse research fields such as toxicology, nutrition, biomarker discovery, doping control, and microbiome metabolism. One of the challenges in these research fields has been the investigation of the two major phase II modifications, sulfation and glucuronidation, and the corresponding unconjugated aglycon independently. We have now developed a new methodology utilizing an immobilized arylsulfatase and an immobilized ß-glucuronidase to magnetic beads for treatment of human urine samples. The enzyme activities remained the same compared to the enzyme in solution. The separate mass spectrometric investigation of each metabolite class in a single sample was successfully applied to obtain the dietary glucuronidation and sulfation profile of 116 compounds. Our new chemical biology strategy provides a new tool for the investigation of metabolites in biological samples with the potential for broad-scale application in metabolomics, nutrition, and microbiome studies.

Design of enzyme@metal organic framework composites with thermo-responsivity for colourimetric detection of glucose.

Enzyme immobilization on metal-organic frameworks (MOFs) has interested researchers in recent decades due to the outstanding characteristics of MOFs. However, despite some enzyme@MOF composites exhibiting better tolerance, stability and catalysis than free enzymes, boosting the catalytic performance of stimuli-responsive polymer-grafted MOFs composites remains a challenging task. Herein, a glucose oxidase (GOx)-horseradish peroxidase (HRP)@MOF (UiO-66-NH2, U)@polymer composite with tunable catalytic ability was constructed by modification with thermo-responsive poly(N-isopropylacrylamide) (PN) via a surface-selective post-synthetic protocol. Temperature increases changed the PN-based soft armour from a "stretch" to a "coil" conformation on the MOF surface, resulting in the confinement effect and boosting the catalytic performance of the GOx-HRP@U@PN composites. Compared with its maximum catalytic reaction rate at 25 °C, the proposed composites showed 18-fold improvement in catalytic performance at 37 °C. Additionally, a colourimetric method for serum glucose analysis was developed using a GOx-HRP-based catalytic cascade reaction with a linear range from 0.1 to 2.0 mM and a low detection limit of 0.03 mM. Remarkably, the surface PN-shell-based soft armour proved to be the key factor for enhancing the catalytic performance of the as-designed composites. The co-immobilization of GOx-HRP onto the thermo-responsive U@PN surface provides a new approach for the development of highly sensitive colourimetric glucose sensing protocols.

The screening of lipase inhibitors based on the metal-organic framework Zeolitic Imidazolate Framework-8-immobilized enzyme microreactor.

An online capillary electrophoresis method based-lipase immobilized enzyme microreactor was developed for lipase kinetic study and inhibitor screening from compounds from natural products. Zeolitic Imidazolate Framework-8 (ZIF-8) has the advantages of large pore size, mild synthesis conditions and good biocompatibility. Lipase was immobilized on the inner wall of capillary with the help of the metal-organic framework ZIF-8. The results of electron microscopy showed that lipase could be aggregated and fixed on the inner wall of capillary by ZIF-8. After the experimental conditions including electrophoretic separation and enzymatic reaction were optimized, the baseline separation of substrate p-nitrophenyl acetate (pNPA) and product p-nitrophenol (pNP) was achieved within 3 min. The immobilized enzyme microreactor showed good repeatability and stability, and the determined Michaelis-Menten constant (Km) of lipase was 2.75 mM, which was lower than the kinetic constant determined in off-line reaction, indicating that the immobilized enzyme had a high affinity with the substrate. In addition, the IC50 value of the positive control compound orlistat on lipase inhibition was 7.26 nM, which was consistent with the literature. Then the inhibitory activity of 10 compounds from natural products on lipase was evaluated by the ZIF-8-IMER. Among them, 7 compounds including baicalein, luteolin, epicatechin gallic acid, and chlorogenic acid, had a certain inhibitory effect on lipase. The molecular docking technology proved the interaction between the enzyme and the screened inhibitor, which provides a new method for the screening of lipase inhibitors.

Orientation-Controllable Enzyme Cascade on Electrode for Bioelectrocatalytic Chain Reaction.

The accomplishment of concurrent interenzyme chain reaction and direct electric communication in a multienzyme-electrode is challenging since the required condition of multienzymatic binding conformation is quite complex. In this study, an enzyme cascade-induced bioelectrocatalytic system has been constructed using solid binding peptide (SBP) as a molecular binder that coimmobilizes the invertase (INV) and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) cascade system on a single electrode surface. The SBP-fused enzyme cascade was strategically designed to induce diverse relative orientations of coupling enzymes while enabling efficient direct electron transfer (DET) at the FAD cofactor of GDHγα and the electrode interface. The interenzyme relative orientation was found to determine the intermediate delivery route and affect overall chain reaction efficiency. Moreover, interfacial DET between the fusion GDHγα and the electrode was altered by the binding conformation of the coimmobilized enzyme and fusion INVs. Collectively, this work emphasizes the importance of interenzyme orientation when incorporating enzymatic cascade in an electrocatalytic system and demonstrates the efficacy of SBP fusion technology as a generic tool for developing cascade-induced direct bioelectrocatalytic systems. The proposed approach is applicable to enzyme cascade-based bioelectronics such as biofuel cells, biosensors, and bioeletrosynthetic systems utilizing or producing complex biomolecules.

Enzyme-immobilized hierarchically porous covalent organic framework biocomposite for catalytic degradation of broad-range emerging pollutants in water.

Efficient enzyme immobilization is crucial for the successful commercialization of large-scale enzymatic water treatment. However, issues such as lack of high enzyme loading coupled with enzyme leaching present challenges for the widespread adoption of immobilized enzyme systems. The present study describes the development and bioremediation application of an enzyme biocomposite employing a cationic macrocycle-based covalent organic framework (COF) with hierarchical porosity for the immobilization of horseradish peroxidase (HRP). The intrinsic hierarchical porous features of the azacalix[4]arene-based COF (ACA-COF) allowed for a maximum HRP loading capacity of 0.76 mg/mg COF with low enzyme leaching (<5.0 %). The biocomposite, HRP@ACA-COF, exhibited exceptional thermal stability (∼200 % higher relative activity than the free enzyme), and maintained ∼60 % enzyme activity after five cycles. LCMSMS analyses confirmed that the HRP@ACA-COF system was able to achieve > 99 % degradation of seven diverse types of emerging pollutants (2-mercaptobenzothiazole, paracetamol, caffeic acid, methylparaben, furosemide, sulfamethoxazole, and salicylic acid)in under an hour. The described enzyme-COF system offers promise for efficient wastewater bioremediation applications.

The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production.

The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into D-allulose. Additionally, the process can also utilise D-tagatose 3-epimerase (DTEase). However, the process is not efficient due to the poor thermotolerance of the enzymes and low conversion rates between the sugars. This review describes three newly identified DAEases that possess desirable properties for the industrial-scale manufacturing of D-allulose. Other methods used to enhance process efficiency include the engineering of DAEases for improved thermotolerance or acid resistance, the utilization of Bacillus subtilis for the biosynthesis of D-allulose, and the immobilization of DAEases to enhance its activity, half-life, and stability. All these research advancements improve the yield of D-allulose, hence closing the gap between the small-scale production and industrial-scale manufacturing of D-allulose.

Nucleoside hydrolase immobilized on magnetic particles as a tool for onflow screening and characterization of inhibitors.

Nucleoside Hydrolases (NH) are considered a target for the development of new antiprotozoal agents. The development of new and automated screening assays for the identification of NH inhibitors can accelerate the first stages of the drug discovery process. In this work, NH from Leishmania donovani (LdNH) was covalently immobilized onto magnetic particles (LdNH-MPs) and trapped by magnets into a TFE tube to yield an immobilized enzyme reactor (IMER). For an automated assay, the LdNH-MP-IMER was connected in-line to an analytical column in an HPLC-DAD system to monitor the enzyme activity through quantification of the product hypoxanthine. Kinetic studies provided a KM value of 2079 ± 87 µmol.L-1 for the inosine substrate. Validation of the LdNH-MP-IMER for onflow screening purposes was performed with a library containing 12 quinolone ribonucleosides. Among them, three were identified as new competitive LdNH inhibitors, with Ki values between 83.5 and 169.4 µmol.L-1. This novel in-line screening assay has proven to be reliable, fast, low cost, and applicable to large libraries of compounds.

Interfacial Hyperactivation of Candida rugosa Lipase onto Ca2Fe2O5 Nanoparticles: pH and Ionic Strength Fine-Tuning to Modulate Protein-Support Interactions.

The current study provides a comprehensive look of the adsorption process of Candida rugosa lipase (CRL) on Ca2Fe2O5 iron oxide nanoparticles (NPs). Protein-support interactions were identified across a broad range of pH and ionic strengths (mM) through a response surface methodology, surface charge determination, and spectroscopic and in silico analyses. The maximum quantity of immobilized protein was achieved at an ionic strength of 50 mM and pH 4. However, this condition did not allow for the greatest hydrolytic activity to be obtained. Indeed, it was recorded at acidic pH, but at 150 mM, where evaluation of the recovered activity revealed hyperactivation of the enzyme. These findings were supported by adsorption isotherms performed under different conditions. Based on zeta potential measurements, electrostatic interactions contributed differently to protein-support binding under the conditions tested, showing a strong correlation with experimentally determined immobilization parameters. Raman spectra revealed an increase in hydrophobicity around tryptophan residues, whereas the enzyme immobilization significantly reduced the phenylalanine signal in CRL. This suggests that this residue was involved in the interaction with Ca2Fe2O2 and molecular docking analysis confirmed these findings. Fluorescence spectroscopy showed distinct behaviors in the CRL emission patterns with the addition of Ca2Fe2O5 at pH 4 and 7. The calculated thermodynamic parameters indicated that the contact would be mediated by hydrophobic interactions at both pHs, as well as by ionic ones at pH 4. In this approach, this work adds to our understanding of the design of biocatalysts immobilized in iron oxide NPs.

[Development of enzyme immobilization systems for CO2 bioconversion: advances and challenges].

Enzyme-catalyzed CO2 reduction to value-added commodities is important for alleviating the global environmental issues and energy crises due to high selectivity and mild conditions. Owing to high energy density, formic acid or methanol produced from CO2 using formate dehydrogenase (FDH) or multi-enzyme cascades are promising target chemicals for CO2 utilization. However, the low activity, poor stability and low reusability of key enzymes involved in such process hampered its large-scale application. Enzyme immobilization provides an effective solution to these problems and significant progress have been made in immobilization carriers. Moreover, integration of enzyme immobilization with other catalysis techniques have been explored extensively. This review summarized the recent advances in the immobilization of enzymes using membranes, inorganic materials, metal-organic frameworks, covalent organic frameworks and other carriers, and illustrated the characteristics and advantages of different immobilization materials and immobilization methods. The synergistic effects and applications of immobilized enzymes and electrocatalytic or photocatalytic coupling reaction systems for CO2 reduction were further summarized. Finally, the current challenges of enzyme immobilization technology and coupling reaction systems were pointed out and their development prospects were presented.

Immobilization of horseradish peroxidase with zwitterionic polymer material for industrial phenolic removal.

Horseradish peroxidase (HRP) is a hemoglobin composed of a single peptide chain that catalyzes the oxidation of various substrates such as phenol and aniline in the presence of hydrogen peroxide via its iron-porphyrin catalytic center. This enzyme is widely used in industrial phenol removal, food additives, biomedicine, and clinical test reagents due to its rapid reaction rate and obvious reaction outcomes. However, the large-scale use of HRP in industrial applications still faces numerous challenges, including activity, stability, and sustainability. This study demonstrates that when peroxidase is immobilized in zwitterionic polymer hydrogels, polycarboxybetaine (PCB) and polysulfobetaine (PSB), the properties of the enzyme are improved. PCB and PSB-embedded HRP exhibit a 6.11 and 1.53 times increase in Kcat/Km value, respectively, compared to the free enzyme. The immobilized enzyme also experiences increased activity over a range of temperatures and better tolerance to extreme pH and organic solvents, including formaldehyde. In addition, immobilized HRP exhibits excellent performance in storage and reproducibility. Remarkably, PCB-HRP still retains 80% of the initial activity after a 6-week storage period and can still attain the initial catalytic level of the free enzyme after six repeated cycles. It also removes 90% of phenol within 12 min, surpassing the current pharmacy on the market. These experimental results indicated that we have successfully designed a set of stable and efficient support substrates for horseradish peroxidase, which enhances its suitability for deployment in industrial applications.

Construction of Immobilized Enzymes with Yeast and Metal-Organic Frameworks for Enhanced Biocatalytic Activities.

Metal-organic frameworks (MOFs) have become promising host materials for enzyme immobilization and protection. Herein, ZIF-8 nanocubes were successfully self-assembled onto yeast as a biological template to obtain hybrid Y@ZIF-8. The size, morphology, and loading efficiency of ZIF-8 nanoparticles assembled on yeast templates can be well-regulated by adjusting the various synthetic parameters. Particularly, the amount of water significantly affected the particle size of ZIF-8 assembled on yeast. Through using a cross-linking agent, the relative enzyme activity of Y@ZIF-8@t-CAT could be greatly enhanced and remained the highest even after seven consecutive cycles, with improved cycling stability, as compared to that of Y@ZIF-8@CAT. In addition to the effect of the physicochemical properties of Y@ZIF-8 on the loading efficiency, the temperature tolerance, pH tolerance, and storage stability of Y@ZIF-8@t-CAT were also systematically investigated. Importantly, the catalytic activity of free catalase was decreased to 72% by 45 days, while the activity of the immobilized catalase remained above 99%, suggesting good storage stability. The present work demonstrates that yeast-templated ZIF-8 nanoparticles have a high potential to be used as biocompatible immobilization materials and are promising candidates for the preparation of effective biocatalysts in biomedicine applications.

Enhancing Enzyme Activity Using Hydrophilic Hollow Layered Double Hydroxides as Encapsulation Carriers.

Enzyme immobilization enables the fabrication of flexible and powerful biocatalytic systems that can meet the needs of green and efficient development in various fields. However, restricted electron and mass transfer during enzymatic reactions and disruption of the enzyme structure during encapsulation limit the wide application of the immobilized enzyme systems. Herein, we report an encapsulation strategy based on hollow-shell-layered double hydroxides (LDHs; ZnCo-LDH) for green and nondestructive enzyme immobilization. Benefiting from the protective and enzyme-friendly microenvironment provided by the hydrophilic hollow structure of ZnCo-LDH, the encapsulated enzyme maintains a nearly natural enzyme biostructure and enhanced stability. Notably, mesoporous ZnCo-LDH with excellent electrical properties considerably facilitates electron and mass transport during enzymatic reactions, exhibiting 5.56 times the catalytic efficiency of free enzymes or traditional enzyme encapsulation systems. The current study broadens the family of encapsulated carriers and alleviates the trade-off between enzyme stability and catalytic activity in the encapsulated state, presenting a promising avenue for the industrial application of the enzyme.

The Development and Evaluation of Reagentless Glucose Biosensors Using Dendritic Gold Nanostructures as a Promising Sensing Platform.

Reagentless electrochemical glucose biosensors were developed and investigated. A graphite rod (GR) electrode modified with electrochemically synthesized dendritic gold nanostructures (DGNs) and redox mediators (Med) such as ferrocenecarboxylic acid (FCA), 1,10-phenathroline-5,6-dione (PD), N,N,N',N'-tetramethylbenzidine (TMB) or tetrathiafulvalene (TTF) in combination with glucose oxidase (GOx) (GR/DGNs/FCA/GOx, GR/DGNs/PD/GOx, GR/DGNs/TMB/GOx, or GR/DGNs/TTF/GOx) were developed and electrochemically investigated. A biosensor based on threefold-layer-by-layer-deposited PD and GOx (GR/DGNs/(PD/GOx)3) was found to be the most suitable for the determination of glucose. To improve the performance of the developed biosensor, the surface of the GR/DGNs/(PD/GOx)3 electrode was modified with polypyrrole (Ppy) for 5 h. A glucose biosensor based on a GR/DGNs/(PD/GOx)3/Ppy(5 h) electrode was characterized using a wide linear dynamic range of up to 39.0 mmol L-1 of glucose, sensitivity of 3.03 µA mM-1 cm-2, limit of detection of 0.683 mmol L-1, and repeatability of 9.03% for a 29.4 mmol L-1 glucose concentration. The Ppy-based glucose biosensor was characterized by a good storage stability (τ1/2 = 9.0 days). Additionally, the performance of the developed biosensor in blood serum was investigated.

Fabrication of Biomimetic Cascade Nanoreactor Based on Covalent Organic Framework Capsule for Biosensing.

The cooperation of biocatalysis and chemocatalysis in a catalytic cascade reaction has received extensive attention in recent years, whereas its practical applications are still hampered due to the fragility of the enzymes, poor compatibility between the carriers and enzymes, and limited catalytic efficiency. Herein, a biomimetic cascade nanoreactor (GOx@COFs@Os) was presented by integrating glucose oxidase (GOx) and Os nanozyme with covalent organic framework (COF) capsule using metal-organic framework (ZIF-90) as a template. The obtained GOx@COFs@Os capsule provided a capacious microenvironment to retain the conformational freedom of GOx for maintaining its activity, wherein the enzyme activity of GOx in COF capsules was equal to 92.9% of the free enzyme and was 1.88-folds higher than that encapsulated in ZIF-90. Meanwhile, the COF capsule could protect the GOx against incompatible environments (high temperature, acid, and organic solvents), resulting in improved stability of the packaged enzymes. Moreover, the COF capsule with great pore structure significantly improved the affinity to substrates and facilitated efficient mass transfer, which achieved 2.19-folds improvement in catalytic efficiency than the free cascade system, displaying the great catalytic performance in the cascade reaction. More importantly, the biomimetic cascade capsule was successfully employed for glucose monitoring, glutathione sensing, and bisphenol S detection in the immunoassay as a proof-of-concept. Our strategy provided a new avenue in the improvement of biocatalytic cascade performance to encourage its wide applications in various fields.

Robust cross-linked cyclodextrin glucanotransferase from Bacillus lehensis G1 aggregates using an improved cross-linker and a new co-aggregant for the production of cyclodextrins.

One of the potentials of carrier-free cross-linked enzyme aggregates (CLEA) immobilization is the ability to be separated and reuse. Yet, it might be impeded by the poor mechanical stability resulting low recyclability. CLEA of CGTase from Bacillus lehensis G1 (CGTase G1-CLEA) using chitosan (CS) as a cross-linker demonstrated high activity recovery however, displayed poor reusability. Therefore, the relationship between mechanical strength and reusability is studied by enhancing the CS mechanical properties and applying a new co-aggregation approach. Herein, CS was chemically cross-linked with glutaraldehyde (GA) and GA was introduced as a co-aggregant (coGA). CGTase G1-CLEA developed using an improved synthesized chitosan-glutaraldehyde (CSGA) cross-linker and a new coGA technique showed to increase its mechanical stability which retained 63.4% and 52.2%, respectively compared to using CS that remained 33.1% of their initial activity after stirred at 500 rpm. The addition of GA impacted the morphology and interaction consequently stabilizing the CLEAs durability in production of cyclodextrins. As a result, the reusability of CGTase G1-CLEA with CSGA and coGA increased by 56.6% and 42.8%, respectively compared to previous CLEA after 5 cycles for 2 h of reaction. This verifies that the mechanical strength of immobilized enzyme influences the improvement of its operational stability.

Facile immobilization of cholesterol oxidase on Pt,Ru-C nanocomposite and ionic liquid-modified carbon paste electrode for an efficient amperometric free cholesterol biosensing.

In present work, the enzyme cholesterol oxidase (ChOx) was immobilized by Nafion® (Naf) on Pt,Ru-C nanocomposite and an ionic liquid (IL)-modified carbon paste electrode (CPE) in order to create cholesterol biosensor (Naf/ChOx/Pt,Ru-C/IL-CPE). The prepared working electrodes were characterized using scanning electron microscopy-energy-dispersive spectrometry, while their electrochemical performance was evaluated using electrochemical impedance spectroscopic, cyclic voltammetric, and amperometric techniques. Excellent synergism between IL 1-allyl-3-methylimidazolium dicyanamide ([AMIM][DCA]), Pt,Ru-C, and ChOx, as modifiers of CPE, offers the most pronounced analytical performance for improved cholesterol amperometric determination in phosphate buffer solution pH 7.50 at a working potential of 0.60 V. Under optimized experimental conditions, a linear relationship between oxidation current and cholesterol concentration was found for the range from 0.31 to 2.46 µM, with an estimated detection limit of 0.13 µM and relative standard deviation (RSD) below 5.5%. The optimized amperometric method in combination with the developed Naf/ChOx/Pt,Ru-C/IL-CPE biosensor showed good repeatability and high selectivity towards cholesterol biosensing. The proposed biosensor was successfully applied to determine free cholesterol in a human blood serum sample via its enzymatic reaction product hydrogen peroxide despite the presence of possible interferences. The percentage recovery ranged from 99.08 to 102.81%, while RSD was below 2.0% for the unspiked as well as the spiked human blood serum sample. The obtained results indicated excellent accuracy and precision of the method, concluding that the developed biosensor can be a promising alternative to existing commercial cholesterol tests used in medical practice.