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Due to differences in the chemical properties or optimal reaction conditions of the catalysts, the challenge in the design of bio-chemical hybrid catalysts is that the bio-catalysts or chemical catalysts usually cannot maintain the initial catalytic performance. Herein, we report a general bio-chemical hybrid catalyst synthesis method using a natural enzyme scaffold-confined metal nanocluster. A redox-active enzyme is a nanoreactor that allows access to and reduces metal ions into metal nanoclusters in situ, resulting in the enzyme-confined metal nanocluster hybrid catalyst with a synergistic effect to boost catalytic performance. Specifically, bilirubin oxidase-Ir nanoclusters (BOD-Ir NCs) with catalytic properties for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) are designed. The BOD-Ir NCs exhibit an approximately 2-fold ORR activity compared with pure BOD and a 4-fold OER activity compared with pure Ir NCs. BOD-Ir NCs exhibit stability for over 50,000 s, exceeding that of pure Ir NCs (22,000 s). The synergistic catalytic performance is attributed to the following: the mild preparation condition and matched sizes of BOD and the Ir NCs maintain the natural activity of BOD; the highly conductive Ir NCs improve the ORR activity of BOD; and the confining effect of BOD, which improves the stability and activity of the Ir NCs during the OER. In particular, BOD-Ir NCs exhibit a high half-wave potential of 0.97 V for the ORR and a low overpotential of 319 mV at 10 mA cm -2 for the OER, surpassing most of reported catalysts under neutral conditions. Furthermore, laccase-Ir NCs and glucose oxidase-Pd NCs with synergistic catalytic performances are fabricated, proving the universality of this synthetic method. This facile strategy for designing synergistic hybrid catalysts is expected to be applied to more complex chemical transformations.

The integration of chemo- and enzymatic catalysis for effective multistep cascades has presented critical challenges for decades. In this work, the biomimetic quasi NH 2 -MIL-101 (qNM) with highly efficient peroxidase-like activity was synthesized via a palmitic acid-induced strategy followed by pyrolysis. The effects of the amount of palmitic acid and calcination temperature on the synthesis of qNM were optimized. It was found that qNM was an excellent catalyst for oxidations of various peroxidase substrates, and a possible mechanism was proposed, i.e., the presence of Fe II species in qNM was responsible for its excellent activity, which facilitated the transition between Fe II and Fe III species to produce more hydroxyl radicals by H 2 O 2 decomposition. The qNM served as the potential matrix for enzyme immobilization through a cross-linking method, and kinetic studies revealed that the catalytic efficiency ( k cat / K m ) for the immobilized GOx (23.7 mM -1 s -1 ) is comparable to that of free GOx (26.9 mM -1 s -1 ). The immobilized GOx also showed improved stability against high temperatures and organic solvents compared to free GOx, and analysis of the secondary structure of GOx indicated that the improved stability resulted from enzyme rigidity by the intense covalent linkage with qNM. Furthermore, qNM contributed its biomimetic activity to cooperate with a single enzyme (GOx) or two enzymes (β-Gal and GOx) for the enzymatic cascade reactions. Compared with the mixture of each component in the solution, the combination of the single-enzyme system (GOx) or the two-enzyme system (β-Gal and GOx) in qNM achieved 2.67-fold and 1.83-fold enhancements in the activity of catalytic cascades, respectively. This study provides new insights into the construction of effective and synergistic cascade reactions by integrating biomimetic MOF with natural enzyme, which holds potential for applications in biotechnology and ecofriendly and biomimetic catalysis.

This paper investigates the potential of the enzymatic management of high pH in white juice and wine using a combination of enzymes-glucose oxidase coupled with catalase. Catazyme ® 25 L, a commercially available blend of the two enzymes, was added at different doses (0.2 g/L, 0.6 g/L, and 1g/L) to white grape juice and various parameters (glucose, gluconic acid, pH) were monitored over 24 h of treatment. Treated wines were fermented to dryness without any difficulty and the wines were chemically and sensorially evaluated. At the highest dose (1 g/L), pH was reduced from 3.9 to 3.2, with 20.5 g of gluconic acid produced, while at the lowest dose (0.2 g/L), pH decreased from 4.0 to 3.5 and 8.8 g of gluconic acid was produced. Flash profiling indicated that treated wines were lighter in color than the control and were described using terms such as floral, fruit, citrus, and sour while the control wine was described as being fermented, medicinal, pungent, and oxidized. In conclusion, glucose oxidase coupled with catalase was shown to be effective at significantly reducing juice and wine pH in a short amount of time and with a positive impact on the organoleptic profiles of the treated wines.

Numerous dough improvers are used alone or in combination to enhance the quality of baked goods such as breads. While modern consumers demand consistent quality, the expectations for ingredients have changed over the past few years, and reformulations have taken place to provide "clean label" options. However, the effects and mechanisms of blended dough conditioners suitable for such baked products have not been systematically summarized. In this review, dough and bread properties as affected by different improver combinations are examined, with a focus on additive or synergistic interactions between enzymes or between enzymes and ascorbic acid. The combination of enzymes that hydrolyze starch and cell wall polysaccharides has been shown to reduce textural hardness in fresh and stored bakes goods such as breads. Enzymes that hydrolyze arabinoxylans, the main nonstarch polysaccharide in wheat, have synergistic effects with enzymes that result in cross-linking of wheat flour biopolymers. In some studies, the effects of bread improvers varied for wheat flours of different strength. Overall, bread products in which wheat is used in whole grain form or in a blend with other flours especially benefit from multiple improvers that target different flour constituents in doughs., (© 2021 Institute of Food Technologists®.)

For many years, the baking industry has been using chemical improvers as a way for compensating for flour quality variation due to growing conditions or wheat cultivar. However, the replacement of chemical dough improvers with natural ingredients or processing aids (i.e. enzymes) allows for the production of 'cleaner label' products. In the present research, dough and bread properties (mixing time, oven rise, loaf volume, crumb firmness and C-cell parameters) were analysed as a function of wheat cultivar (Glenn, Harvest, Lillian, CDC Plentiful and Stettler), additive-type (ascorbic acid, azodicarbonamide, glucose oxidase and fungal xylanase) and concentration. Overall, the cultivar Glenn appeared to have improved baking performance relative to the other cultivars, regardless of the additive and additive concentration. On the other hand, Stettler showed poorer baking quality and performance even with the addition of oxidizers and enzymes in relation to the control. The concentration of additive was found to have little or no effect on improving baking properties within each cultivar. Enzymes had similar or better performance than oxidizers in most cases.

The flavin cofactor performs many functions in the cell based on the ability of the isoalloxazine ring to undergo one- or two-electron reduction and form covalent adducts with reactants such as amino acids. In addition, the strong visible absorption of the cofactor is also the basis for flavin-dependent photoreceptors. Vibrational spectroscopy is uniquely suited to studying the mechanism of flavoproteins since the frequency of the vibrational modes is very sensitive to the electronic structure and environment of the isoalloxazine ring. This chapter describes the mechanistic information that can be gained using vibrational spectroscopy as well experimental challenges and approaches that are used to obtain and interpret the complex data contained in a vibrational spectrum., (© 2019 Elsevier Inc. All rights reserved.)

Here we report immobilization of glucose oxidase (GOx) on magnetic silica (Fe 3 O 4 -SiO 2 ) and alumina (Fe 3 O 4 -Al 2 O 3 ) functionalized with amino groups using glutaraldehyde as a linker. Magnetic support based biocatalysts demonstrate high catalytic activity in d-glucose oxidation to D-gluconic acid at pH 5-7.5 and temperature of 30-50 °C with the best activities of 95% and 91% for magnetic silica and alumina, respectively. A comparison of magnetic and non-magnetic alumina and silica shows a significant enhancement of the relative catalytic activity for magnetic supports, while the silica based biocatalysts show a higher activity than the biocatalysts based on alumina. A noticeably higher activity of GOx immobilized on magnetic supports is explained by synergy of the GOx inherent activity and enzyme-like activity of iron oxide nanoparticles, while the enhancement with silica based catalysts is most likely due to a larger pore size and stronger Brønsted acid sites. Excellent relative activity of Fe 3 O 4 -SiO 2 -GOx (95% of native GOx) in a tolerant pH and temperature range as well as high stability in a repeated use (6% relative activity loss after five catalytic cycles) makes this catalyst promising for practical applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Intracellular reactive oxygen species (iROS) induction by HOCl was used as a novel strategy to improve enzyme productivities in Aspergillus niger growing in a bioreactor. With induced iROS, the specific intracellular activities of alpha-amylase, protease, catalase, and glucose oxidase were increased by about 170%, 250%, 320%, and 260%, respectively. The optimum specific iROS level for achieving maximum cell concentration and enzyme production was about 15 mmol g cell-1. The type of iROS inducing the enzyme production was identified to be a derivative of the superoxide radical.

The technology for fluorescence protein-sensing is advancing rapidly owing to the continued introduction of new concepts, new fluorophores, and proteins engineered for sensing-specific analytes. Concerns about the reversibility and selectivity of engineered proteins are being addressed by developing biosensors that are based on the utilisation of coenzyme-depleted enzymes. Such biomolecules do not consume the substrate and can exhibit conformational changes upon the binding of the analyte, which can be easily detected as fluorescence change. In addition, concerns about the stability of biosensors can be overcome by using thermostable enzymes isolated from thermophilic microorganisms. Finally, the development of new techniques such as polarization-based sensing, anisotropy-based sensing and lifetime-based sensing, all of which can be accomplished with light-emitting diodes as the light source, is prompting the design of a new class of specific and stable biosensors, as has occurred with blood glucose measurement. These biosensors represent a valid alternative to the conventional clinical chemistry diagnostics.

The direct monitoring of sugars such as lactose, maltose, saccharose is not only useful at the applied point of view but also at the fundamental point of view for studying enzymology, especially in microbiology and fermentation. Benzyme systems were extensively used in solution for analytical applications in industry and medicine. The progress in the field of immobilization of bienzyme systems [1-3], especially within membranes [4-5], makes possible the production of new analytical devices. From the studies dealing with concentration profiles in artificial enzyme membranes [14], evidence was obtained for a well defined relationship between the local concentration of a metabolite and concentration of the first substrate in the bulk solution. In the described systems a substrate is transformed into glucose within a membrane, the glucose is then transformed in gluconic acid with a local oxygen consumption. The local pO2 level is linked to the glucose oxidase velocity, which is only linked to the glucose production, that is to say to the concentration of the first substrate. The enzyme electrode is based on the transformation of kinetic phenomena (reaction rates) into absolute values (local concentrations) through the diffusion-reaction coupling process. The manufacture of magnetic enzyme electrodes [6] allows convenient use of the active sensors. The pO2 electrode has some adventages, namely the specificity based on the selectivity of the gas permeable membrane and the linear relationship between the oxygen and the output of the electrode. pCO2, pH, ion electrodes give a logarithmic response as a function of the concentration. The grafting of a multienzyme system on a sensor allows a study of sequential systems in a defined context with a measurement of the local concentration of the metabolites. The tool is useful for both kinetics [4] and regulation studies [5].

Hydrogen‐bonded organic frameworks (HOF) represent an emerging category of organic structures with high crystallinity and metal‐free, which are not commonly observed in alternative porous organic frameworks. These needle‐like porous structure can help in stabilizing enzymes and allow transfer of molecules between enzymes participating in cascade reactions for enhanced substrate channelling. Herein, we systematically synthesized and investigated the stability of HOF at extreme conditions followed by one‐pot encapsulation of single and bi‐enzyme systems. Firstly, we observed HOF to be stable at pH 1 to 14 and at high temperatures (up to 115 °C). Secondly, the encapsulated glucose oxidase enzyme (GOX) showed 80 % and 90 % of its original activity at 70 °C and pH 11, respectively. Thirdly, transient time close to 0 seconds was observed for HOF encapsulated bi‐enzyme cascade reaction system demonstrating a 4.25‐fold improvement in catalytic activity when compared to free enzymes with enhanced substrate channelling. Our findings showcase a facile system synthesized under ambient conditions to encapsulate and stabilize enzymes at extreme conditions. [ABSTRACT FROM AUTHOR]

[Display omitted] Starvation therapy has shown promise as a cancer treatment, but its efficacy is often limited when used alone. In this work, a multifunctional nanoscale cascade enzyme system, named CaCO 3 @MnO 2 -NH 2 @GOx@PVP (CMGP), was fabricated for enhanced starvation/chemodynamic combination cancer therapy. CMGP is composed of CaCO 3 nanoparticles wrapped in a MnO 2 shell, with glucose oxidase (GOx) adsorbed and modified with polyvinylpyrrolidone (PVP). MnO 2 decomposes H 2 O 2 in cancer cells into O 2 , which enhances the efficiency of GOx-mediated starvation therapy. CaCO 3 can be decomposed in the acidic cancer cell environment, causing Ca2+ overload in cancer cells and inhibiting mitochondrial metabolism. This synergizes with GOx to achieve more efficient starvation therapy. Additionally, the H 2 O 2 and gluconic acid produced during glucose consumption by GOx are utilized by MnO 2 with catalase-like activity to enhance O 2 production and Mn2+ release. This process accelerates glucose consumption, reactive oxygen species (ROS) generation, and CaCO 3 decomposition, promoting the Ca2+ release. CMGP can alleviate tumor hypoxia by cycling the enzymatic cascade reaction, which increases enzyme activity and combines with Ca2+ overload to achieve enhanced combined starvation/chemodynamic therapy. In vitro and in vivo studies demonstrate that CMGP has effective anticancer abilities and good biosafety. It represents a new strategy with great potential for combined cancer therapy. [ABSTRACT FROM AUTHOR]

The inorganic content and the catalytic performance pose metal‐loaded enzyme nanoflowers as promising candidates for developing bioelectrodes capable of functioning without the external addition of a redox mediator. However, these protein‐inorganic hybrids have yet to be successfully applied in combination with electrode materials. Herein, the synthesis procedure of these bionanomaterials is reproposed to precisely control the morphology, composition, and performance of this particular protein‐mineral hybrid, formed by glucose oxidase and cobalt phosphate. This approach aims to enhance the adherence and electron mobility between the enzyme and a carbon electrode. The strategy relies on dressing the protein in a tailored thin nanogel with multivalent chemical motifs. The functional groups of the polymer facilitate the fast protein sequence‐independent biomineralization. Furthermore, the engineered enzymes enable the fabrication of robust cobalt‐loaded enzyme inorganic hybrids with exceptional protein loads, exceeding 90% immobilization yields. Notably, these engineered biohybrids can be readily deposited onto flat electrode surfaces without requiring chemical pre‐treatment. The resulting bioelectrodes are robust and exhibit electrochemical responses even without the addition of a redox mediator, suggesting that cobalt complexes promote electron wiring between the active site of the enzyme and the electrode. [ABSTRACT FROM AUTHOR]

Fish gelatin (FG)-based wound dressings exhibit superior water absorption capacity, thermal stability, and gelation properties, which enhance the performance of these dressings. In this study, our objective was to investigate the conditions underlying the enzymatic hydrolysis of FG and subsequent cross-linking to prepare high-performance gels. A two-step enzymatic method of protease-catalyzed hydrolysis followed by glutamine transglutaminase (TGase)-catalyzed cross-linking was used to prepare novel high-performance fish gelatin derivatives with more stable dispersion characteristics than those of natural gelatin derivatives. Compared with conventional TGase cross-linked derivatives, the novel derivatives were characterized by an average pore size of 150 μm and increased water solubility (423.06% to 915.55%), water retention (by 3.6-fold to 43.89%), thermal stability (from 313 °C to 323 °C), and water vapor transmission rate, which reached 486.72 g·m−2·24 h−1. In addition, loading glucose oxidase onto the fish gelatin derivatives increased their antibacterial efficacy to >99% against Escherichia coli and Staphylococcus aureus. [ABSTRACT FROM AUTHOR]

Background: Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily. Results: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold. Conclusions: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions. [ABSTRACT FROM AUTHOR]

We describe the creation of a conductive microcavity based on the assembly of two pieces of carbon nanotube buckypaper for the entrapment of two enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx), as well as a redox mediator: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS). The hollow electrode, employing GOx, HRP, and the mediator, as an electrochemical enzyme cascade model, is utilized for glucose sensing at a potential of 50 mV vs. Ag/AgCl. This bienzyme electrode demonstrates the ability to oxidize glucose by GOx and subsequently convert H2O2 to water via the electrical wiring of HRP by ABTS. Different redox mediators (ABTS, potassium hexacyanoferrate (III), and hydroquinone) are tested for HRP wiring, with ABTS being the best candidate for the electroenzymatic reduction of H2O2. To demonstrate the possibility to optimize the enzyme cascade configuration, the enzyme ratio is studied with 1 mg HRP combined with variable amounts of GOx (1–4 mg) and 2 mg GOx combined with variable amounts of HRP (0.5–2 mg). The bienzyme electrode shows continuous operational stability for over a week and an excellent storage stability in phosphate buffer, with a decay of catalytic current by only 29% for 1 mM glucose after 100 days. [ABSTRACT FROM AUTHOR]

Wearable metabolite sensors are often limited by easily denaturable enzymes that only allow short‐duration monitoring. Although encapsulating enzymes in metal–organic frameworks (MOFs) shows promise of long‐term enzyme protection, it is typically accompanied by significantly decreased activity due to increased diffusion barrier, steric hindrance for enzyme‐substrate binding, and poor enzyme‐electronic interface. Herein, the co‐encapsulation of enzymes and ultrasmall arginine‐derived carbon dots (Argdot) into a mesoporous Zeolitic Imidazolate Framework‐8 (mZIF‐8) matrix and the enhancement effect of Argdot on enzyme stability and activity, which consequently improves the electrochemical sensor's long‐term sensitivity are investigated. Specifically, the glucose oxidase (GOx)‐Argdot@mZIF‐8 nanocomposite consistently exhibits 40% higher electrochemical sensitivity compared to control GOx@mZIF‐8, an improvement similarly demonstrated with another model enzyme lactate oxidase (LOx). Furthermore, GOx‐Argdot@mZIF‐8 displays excellent stability, retaining 100% of initial sensitivity over 30 days of repeated testing at 37 °C. A touch‐based glucose sensor prototype is demonstrated as an excellent reusable sensor to monitor finger‐tip sweat glucose levels over one month at room temperature. This enzyme encapsulation strategy is not only useful for developing reusable sweat sensors with long‐term monitoring capability, but also promising to expand the industry use of enzymes under harsh conditions. [ABSTRACT FROM AUTHOR]

Glucose oxidase (GOx)-based enzyme therapeutics are potential alternatives for colorectal cancer (CRC) treatment via glucose consumption and accumulation of hydrogen peroxide (H 2 O 2). Given that H 2 O 2 can be eliminated by cytoprotective autophagy, autophagy inhibitors that can interrupt autolysosome-induced H 2 O 2 elimination are promising combination drugs of GOx. Here, we developed a multifunctional biomimetic nanocarrier for effective co-delivery of an autophagy inhibitor-chloroquine phosphate (CQP) and GOx to exert their synergistic effect by irreversibly upregulating intracellular reactive oxygen species (ROS) levels. Poly (D, l -lactide- co -glycolide) (PLGA) nanoparticles (NPs) were used to encapsulate both GOx and CQP using a W/O/W multi-emulsion method. Calcium phosphate (CaP) was used to "fix" CQP to GOx in the internal water phase, where it served as a pH-sensitive unit to facilitate intracellular drug release. Folic acid-modified red blood cell membranes (FR) were used to camouflage the GOx/CQP/CaP encapsulated PLGA NPs (referred to as PLGA/GCC@FR). In an AOM/DSS-induced CRC mouse model, PLGA/GCC@FR exhibited improved antitumor effects, in which the number of tumor nodes were only a quarter of that in the free drug combination group. The enhanced therapeutic effects of PLGA/GCC@FR were attributed to the prolonged tumor retention which was verified by both dynamic in vivo imaging and drug biodistribution. This multifunctional biomimetic nanocarrier facilitated combined enzyme therapeutics by depleting glucose and augmenting intracellular ROS levels in tumor cells, which exerted a synergistic inhibitory effect on tumor growth. Therefore, this study proposed a novel strategy for the enhancement of combined enzyme therapeutics, which provided a promising method for effective CRC treatment. [Display omitted] • CQP was fixed to GOx via CaP formation to facilitate co-encapsulation by PLGA NPs. • PBA-PEG-FA was inserted on RBCm to realize "right-side-out" coating and CRC targeting. • Glucose consumption and ROS accumulation mediated CRC tumor regression. [ABSTRACT FROM AUTHOR]

Objective: Enzyme cascade reaction typically involves the synergistic action of two or more enzymes. It is an efficient tool in the field of biosensing. In this research, with the combination of natural enzyme and nanozyme, a high‐performance enzyme cascade bio‐platform is constructed for sensing of H2O2 and glucose. Methods: The enzyme cascade bio‐platform consisting of natural enzyme glucose oxidase (GOX) and nanozyme (hemin@ZIF‐8) was constructed through one‐step co‐precipitation method. The morphology and structure of the composites were characterized by using scanning electron microscopy, x‐ray diffraction and Fourier transform infrared spectrometer. UV‐vis spectrophotometer was used to research the catalytic activity and sensing performances of enzyme cascade bio‐platform. Results: The synergistic catalysis between hemin and ZIF‐8 in hemin@ZIF‐8 nanozyme resulted in highly efficient peroxidase‐like activity, which could be used for H2O2 sensing. The porous structure of the metal‐organic framework provided a good microenvironment for loading the natural enzyme, guaranteeing the enzyme activity and spatial proximity of enzymes. The bio‐platform achieved the sensitive detection of glucose with low detection limit of 7.15 μM. Due to the color change in the catalytic reaction, this bio‐platform can be used for detecting glucose qualitatively and semi‐quantitatively with the naked eye. Conclusions: The constructed efficient enzyme cascade bio‐platform combined the advantages of natural enzyme and nanozyme. This research not only expanded the application possibilities of nanozyme‐based enzyme cascade system, but also offered an effective strategy for the development of simple, rapid and reliable sensing methods for real application. [ABSTRACT FROM AUTHOR]

A novel strategy based on gradient porous hollow fiber membrane (GPF) is proposed for the modular assembly of enzyme-nanozyme cascade systems. The porous structure of GPF provided sufficient specific surface area, while the gradient structure effectively minimized the leaching of enzymes and nanozymes. To enhance stability, we prepared and immobilized metal–organic framework (MOF) nanozymes, resulting in the fabrication of GPF-MOF with excellent stability and reusability for colorimetric H2O2 detection. To improve specificity and expand the detection range, micro-crosslinked natural enzymes were modularly assembled, using glucose oxidase as the model enzyme. The assembled system, GPF-mGOx@MOF, achieved a low detection limit of 0.009 mM and a linear range of 0.2 to 11 mM. The sensor retained 87.2% and 80.7% of initial activity after being stored for 49 days and 9 recycles, respectively. Additionally, the reliability of the biosensor was validated through glucose determination of human blood and urine samples, yielding comparable results to a commercial glucose meter. [ABSTRACT FROM AUTHOR]

Electrophoretic deposition is a powerful tool for depositing materials onto a substrate by using an electric field; its application in biotechnological areas, namely, electrophoretic protein deposition (EPD), is the most promising for, e.g., fabricating novel amperometric biosensors. Unfortunately, EPD suffers from several drawbacks due to coupled parasite electrochemical processes damaging the deposit; moreover, the nature of the deposition process, the deposit, and its stability are still controversial and unknown. The present research presents a deep investigation of the EPD processes conducted by using several electroanalytical techniques and an electrochemical quartz crystal microbalance (EQCM); notably, EPD was used here as a novel tool for performing an electrophoretically assisted, classical enzyme immobilization technique like co-crosslinking, thus permitting the immobilization of the desired protein in situ, i.e., exclusively onto the deposition electrode. An electrochemical study permitted the acquisition of useful insights about electrophoresis processes as well as solvent discharge and gas evolution at the deposition electrode; further, the use of appropriate current or potential pulse sequences, as investigated and improved in this study, together with fine-tuned chemical conditions, allowed the optimization of this novel EPD approach. Moreover, an EQCM study gave useful insights into the kinetics of the process, permitting a quantitative estimate of the deposit. [ABSTRACT FROM AUTHOR]

This research investigated the possibility of using the method of adding an additional enzyme to a monoenzyme oxidase‐based biosensor to improve the biosensor's analytical characteristics. Namely, the catalase (CAT) was used to better saturate the biomembrane with oxygen for the best glucose oxidase (GOx) performance. For this purpose, two different biosensors were created – based on one enzyme (GOx) and two (GOx and CAT). To create a bioselective membrane, the method of covalent binding in vapors of glutaraldehyde was used. The method of coimmobilization of catalase (CAT) and glucose oxidase (GOx) enzymes, as well as the concentration of enzymes in the mixture, was chosen in the work. The stability of the biosensor functioning, i. e. the repeatability during measurements and the reproducibility of the biosensors' preparation, were investigated. Analytical characteristics of the proposed biosensor were studied in comparison with a monoenzyme biosensor based on glucose oxidase only. It was shown that the addition of catalase as an auxiliary enzyme significantly improves the analytical characteristics of the biosensor, in particular, the sensitivity, the repeatability of the measurement results, and the limits of the linear and dynamic ranges of operation. The developed method can be successfully used to optimize conductometric enzyme biosensors based on oxidases. [ABSTRACT FROM AUTHOR]

The food enzyme glucose oxidase (β‐d‐glucose:oxygen 1‐oxidoreductase; EC 1.1.3.4) is produced with the genetically modified Saccharomyces cerevisiae strain LALL‐GO by Lallemand Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism, but not from recombinant DNA. It is intended to be used in baking processes. Dietary exposure was estimated to be up to 0.233 mg total organic solids/kg body weight per day in European populations. Since the production strain meets the requirements for the Qualified Presumption of Safety approach and no issues of concern arising from the production process of the food enzyme were identified, the Panel considered that no toxicological studies were necessary. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded. The glucose oxidase under assessment shows sequence homology with an allergen from almond, which is listed in the Annex II of the Regulation (EU) No 1169/2011. Other than potential allergic reactions, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Aim. Among the methods of improving biosensor systems, the technique of using multi-enzyme bioselective elements is actively used. Its main idea is that, in addition to the main enzyme, auxiliary enzymes are added to the biosensor membrane, which can improve analytical characteristics or enhance the response of the biosensor to the target analyte. As a model biosensor that we intended to improve, we chose a conductometric biosensor for glucose determination based on glucose oxidase. The main enzyme of this biosensor was glucose oxidase (GOx), which is an enzyme of the oxidase class and catalyzes the reaction of splitting glucose into glucono-1,5-lactone and hydrogen peroxide using oxygen during the reaction. As an auxiliary enzyme, we additionally added catalase (CAT) to improve the characteristics of the sensor. It decomposes the peroxide formed during the first reaction with the participation of GOx into water and O2. This enables to increase the concentration of oxygen in the near-electrode layer, which improves the work of the main enzyme — GOx, and, accordingly, the entire biosensor. Methods. Two enzyme gels, glucose oxidase (GOx) and catalase (CAT) were mixed in a 2:1 ratio and applied to the first pair of electrodes for enzyme immobilization. A BSA protein gel was applied to the second pair of electrodes for reference. Immobilization occurred in glutaraldehyde vapors for 20 minutes, followed by air-drying for 10 minutes. The sensor was then washed for 5 minutes to remove residual GA and other unbound elements. Measurements were conducted in a 2 ml open cell with a 5 mM phosphate buffer solution at pH 6.2. The substrate concentration was adjusted by adding aliquotes of 500 mM D-glucose to working solution. Response time was 2–3 minutes with a 5-minute wash between responses. Response measurement could be done by comparing baseline conductance before and after exposure to the target molecule. Results and Discussion. The initial step in developing of the GOx-CAT bienzyme biosensor was to identify the most effective enzyme immobilization method on the transducer surface. We tested five biosensor variants based on different coimmobilization techniques of GOx and CAT and assessed their key analytical properties. Calibration curves were also generated for each variant to guide the selection process. Ultimately, experiments revealed that immobilization in glutaraldehyde vapors, combining GOx and CAT in a 2:1 ratio within a single-layer membrane, offered the highest sensitivity and wide linear operating range. This method was thus chosen for further development. The next stage of the work was the selection of the optimal concentration of catalase in the membrane. For this, a number of biosensors with different concentrations of catalase in the composition of the bioselective element were made and the sensitivity of these biosensors to the target analyte was compared. Thus, it was shown that the biosensor based on 5% CAT was characterized by the greatest response to the substrate. The difference between mono- and bienzyme biosensors lies in their sensitivity to glucose and their linear detection range. The sensitivity of the bienzyme biosensor (CAT-GOx mixture) was 378 μS/mM, compared to 161 μS/mM for the monoenzyme biosensor (GOx alone). Adding CAT to the biosensor extended its linear range to 1700 μM, compared to 800 μM for the monoenzyme biosensor. Additionally, the minimum detection limit decreased from 16 μM to 8 μM glucose for the monoenzyme and bienzyme biosensors, respectively. Conclusions. The study showed that the addition of catalase as an auxiliary enzyme significantly improved the analytical characteristics of the biosensor based on GOx, in particular, sensitivity, repeatability of measurement results, limits of linear and dynamic ranges of operation. It has been proven that the technology of adding an additional enzyme to the biomembrane of a biosensor can be used to improve the analytical characteristics of biosensors based on oxidases, which in turn will allow their more effective use. [ABSTRACT FROM AUTHOR]

Various cellular functions are successfully mimicked, opening the door to the next generation of therapeutic approaches and systems biology. Herein, the first steps are taken toward the construction of artificial organelles for mimicking cell communication by docking and undocking of cargo in the membrane of swollen artificial organelles. Stimuli‐responsive and crosslinked polymeric vesicles are used to allow docking processes at acidic pH at which ferrocene units in the swollen membrane state can undergo desired specific host–guest interaction using β‐cyclodextrin as model cargo. The release of the cargo mediated by two different enzymes, glucose oxidase and α‐amylase, is investigated, triggered by distinct enzymatic undocking mechanisms. Different release times for a useful transport are shown that can be adapted to different communication pathways. In addition, Förster resonance energy transfer (FRET) experiments further support the hypotheses of host–guest inclusion complexation formation and their time‐dependent breakdown. This work paves a way to a platform based on polymeric vesicles for synthetic biology, cell functions mimicking, and the construction of multifunctional cargo delivery system. [ABSTRACT FROM AUTHOR]

The unique catalytic properties of enzymes have led to the production of useful medicinal intermediates, foods, and biofuels from sustainable sources. However, the instability of soluble/free enzymes under several challenging conditions (e.g., pH, proteolysis, temperature, ionic potential, chemical denaturants) restricts the use of enzyme-based biocatalysts. Encapsulation of enzymes on suitable carriers would mitigate the instability issues faced in robust biocatalysis. An "ideal" carrier material employed for protein immobilization should be nontoxic, scalable, biocompatible, and should not compromise the biological activity and structure of proteins/enzymes. Thus, biodegradable and renewable biomass-derived functional materials (BDFMs) are envisaged as promising carriers for enzymes. BDFMs have in-built chemical functionalities and desirable physicochemical properties that enable their use in enzyme catalysis at the industrial scale. Numerous BDFMs have been used as immobilization matrices to improve the biocatalytic activity and stability of various enzymes. These solid materials are renewable and environmentally friendly compared with synthetic polymers. This review highlights the advancements, challenges and prospects in the emerging field of BDFMs (cellulose, silk protein, chitin, chitosan, lignocellulose, and a combination of biopolymers such as chitin/lignin and chitosan/alginate) for immobilization of enzymes (e.g., α-chymotrypsin, cytochrome c, carbonic anhydrase, glucose oxidase, ribonuclease, cholesterol oxidase, alkaline phosphatase, β-glucosidase, lipase, horseradish peroxidase, catalase, tyrosinase, acetylcholinesterase, amylase, invertase, protease, laccase, β-galactosidase, and several others) for biocatalytic processes. This review also describes the relationship between the structural properties and functionality of several enzymes immobilized in BDFMs, and profiles the impact of pH, temperature, reusability, stability of storage, and the activity of these enzymes. Future perspectives in this promising field, as well as potential difficulties, are discussed. This review will help in refining biocatalysis technologies whereby biomass-derived, environmentally friendly materials are employed as enzyme supports. [ABSTRACT FROM AUTHOR]

Silver nanowires (AgNWs) are among the most promising materials for transparent conductive electrodes (TCE) owing to their stability and unique optoelectronic properties. However, the fact that they easily oxidize in chemical environments has limited their applications in devices such as electrochemical batteries, supercapacitors, and sensors. In this study, flexible Ni@AgNW/PET was prepared using the electrochemical deposition method. This electrode architecture prevented the delamination of the flexible electrodes during bending tests. Further, the Ni shell structure protected the AgNWs from oxidation and improved the stability of the conductive substrate. The Ni@AgNW/PET electrodes showed good electrochemical stability with a sheet resistance and optical transmittance of 6.8 Ω/sq and 64.1%, respectively. The electrocatalytic anodic currents correlated with the concentration of glucose, which had a detection limit of 40 μM (S/N = 3), a wide range of 0.01–5.0 mM (with a linear correlation coefficient of 0.998), and a high sensitivity of 915 μA mM−1 cm−2. The flexible Ni@AgNW/PET electrodes remained stable after 1000 bending cycles under different shapes. The excellent electrocatalytic performance for glucose showed that Ni@AgNWs/PET is a promising wearable electrode that can be used in glucose detection. [Display omitted] • The core-shell architecture prevented oxidation, corrosion, and delamination. • A flexible and transparent glucose sensor was fabricated based on Ni@AgNWs/PET. • The sensor exhibits high sensitivity, reasonable detection range, and excellent stability after bending 1000 times. [ABSTRACT FROM AUTHOR]

In this work, a sensitive coating based on Langmuir–Blodgett (LB) films containing monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) with an immobilized glucose oxidase (GOx) enzyme was created. The immobilization of the enzyme in the LB film occurred during the formation of the monolayer. The effect of the immobilization of GOx enzyme molecules on the surface properties of a Langmuir DPPE monolayer was investigated. The sensory properties of the resulting LB DPPE film with an immobilized GOx enzyme in a glucose solution of various concentrations were studied. It has shown that the immobilization of GOx enzyme molecules into the LB DPPE film leads to a rising LB film conductivity with an increasing glucose concentration. Such an effect made it possible to conclude that acoustic methods can be used to determine the concentration of glucose molecules in an aqueous solution. It was found that for an aqueous glucose solution in the concentration range from 0 to 0.8 mg/mL the phase response of the acoustic mode at a frequency of 42.7 MHz has a linear form, and its maximum change is 55°. The maximum change in the insertion loss for this mode was 18 dB for a glucose concentration in the working solution of 0.4 mg/mL. The range of glucose concentrations measured using this method, from 0 to 0.9 mg/mL, corresponds to the corresponding range in the blood. The possibility of changing the conductivity range of a glucose solution depending on the concentration of the GOx enzyme in the LB film will make it possible to develop glucose sensors for higher concentrations. Such technological sensors would be in demand in the food and pharmaceutical industries. The developed technology can become the basis for creating a new generation of acoustoelectronic biosensors in the case of using other enzymatic reactions. [ABSTRACT FROM AUTHOR]

Nanozymes are gaining attention as promising alternatives to natural enzymes due to their unique characteristics. In this study, CuO nanoparticles were decorated on three‐dimensional nitrogen‐doped graphene‐based frameworks (CuO/3D(N)GFs) were prepared through a facile hydrothermal process. The prepared nanocomposite was characterized using XRD, EDX, FTIR, and SEM to determine its structure, composition, and morphology. The CuO/3D(N)GFs showed excellent peroxidase mimetic activity, followed Michaelis‐Menten kinetics, and exhibited better kinetic parameters (Km and Vmax) for H2O2 substrate than the natural enzyme, making it a superior catalyst. A rapid and facile colorimetric platform was developed to diagnose diabetes with the naked eye by coupling the activities of the nanozyme and glucose oxidase. This technique can quantitatively measure the glucose with high precision in diabetic patients, with a linear range of 1–10 mM and good sensitivity/selectivity toward glucose. The highest peroxidase‐like activity of the nanozyme was observed at 30 °C, but it still retained over 60 % of its activity between 20–65 °C and showed long‐term storage stability. Therefore, the CuO/3D(N)GFs nanocomposite holds promise for catalysis, biosensing, and bioanalysis applications. [ABSTRACT FROM AUTHOR]

Hard‐healing diabetic wound brings burgeoning physical and mental burdens to patients. Current treatment strategies tend to achieve multistage promotion and real‐time reporting to facilitate wound management. Herein, a biomimetic enzyme cascade inverse opal microparticles system for wound healing, which is intergated with glucose oxidase (GOD) and copper peroxide (CP). Such microparticles are composed of biofriendly hyaluronic acid methacryloyl (HAMA) and pH‐responsive acrylic acid (AA), which provided abundant binding sites and spaces for chemical immobilizing and physical doping of enzymes and metal bioinorganics. When the cascade catalytic system is applied on wound sites, hyperglycemia environment would serve as a hydrogen peroxide (H2O2) generator through GOD catalysis, while acidic environment triggers the decomposition of CP, further catalyzing H2O2 to generate reactive oxygen species (ROS). Additionally, the distinctive structural color of the microparticles can visually reflect the wound pH and intelligently estimate the healing state. It is demonstrated that such microparticle systems exhibit excellent broad‐spectrum antibacterial and angiogenesis‐promoting properties, as well as significant real‐time reporting ability for wound healing. These features indicate that enzyme cascade structural color microparticles possess valuable potential in wound healing and related field. [ABSTRACT FROM AUTHOR]

Fe–N-doped carbon nanomaterials (Fe–N/CMs) were designed as a novel biomimetic enzyme with excellent peroxidase-like activity to achieve high-efficient enzyme cascade catalytic amplification with the aid of glucose oxidase (GOx), which was further combined with target-induced DNA walker amplification to develop a sensitive electrochemical biosensor for thrombin detection. Impressively, massive output DNA was transformed from small amounts of target thrombin by highly effective DNA walker amplification as protein-converting strategy, which could then induce the immobilization of functionalized nanozyme on the electrode surface to achieve the high-efficient electrochemical biomimetic enzyme cascade amplification. As a result, an amplified enzyme cascade catalytic signal was measured for thrombin detection ranging from 0.01 pM to 1 nM with a low detection limit of 3 fM. Importantly, the new biomimetic enzyme cascade reaction coupled the advantages of natural enzyme and nanozyme, which paved an avenue to construct varied artificial multienzymes amplification systems for biosensing, bioanalysis, and disease diagnosis applications. [ABSTRACT FROM AUTHOR]

Glucose oxidase is a type of enzyme that converts glucose into hydrogen peroxide and gluconic acid by enzymatic reaction. Glucose oxidase is widely used in industry; however, in the textile industry, glucose oxidase has only received academic interest. Previously, wool was bleached by some reducing agents; however, currently in industry, hydrogen peroxide dominates the bleaching of wool fibres. In this study, the effect of glucose oxidase enzyme treatment on wool merino fibres and dyeability properties was investigated. Wool fibres were treated with glucose oxidase enzyme, after which the whiteness index (Stensby) and yellowness index (ASTM D 1925 and ASTM E 313) were investigated. Scanning electron microscopy and scanning electron microscopy with energy dispersive X‐ray spectroscopy were used to identify the morphological structure of wool fibres and their atomic content. The chemical damage caused by enzyme was investigated using a fluorescence and a light microscope, and the alkali solubility (ASTM D 1283) was determined. After enzymatic treatment, the wool fibres were dyed at a 2.0% concentration with reactive dyes. Dyeability (K/S) and CIELab values were assessed with a Minolta CM 3600 D spectrophotometer (D65, 10°). The washing fastness of wool fibres was investigated according to TS EN ISO 105‐C06 (A1S). [ABSTRACT FROM AUTHOR]

Dynamic interactions of enzymes, including programmable configuration and cycling of enzymes, play important roles in the regulation of cellular metabolism. Here, we constructed a super DNA‐enzymes molecule (SDEM) that comprises at least two cascade enzymes and multiple linked DNA strands to control and detect metabolism. We found that the programmable SDEM, which comprises glucose oxidase (GOx) and horseradish peroxidase (HRP), has a 20‐fold lower detection limit and a 1.6‐fold higher reaction rate than free enzymes. An SDEM can be assembled and disassembled using a hairpin structure and a displacement DNA strand to complete multiple cycles. An entropically driven catalytic assembly (catassembly) enables different SDEMs to switch from an SDEM with GOx and HRP cascades to an SDEM with sarcosine oxidase (SOX) and HRP cascades in over six orders of magnitude less time than without the catassembly to detect different metabolisms (GO and sarcosine) on demand. [ABSTRACT FROM AUTHOR]

Immobilized enzymes were prepared by using strongly alkaline macroporous resin, amino resin, and epoxy resin as immobilized carriers of glucose oxidase and catalase, respectively, and their effect on low-pH gluconic acid production was assessed. The results of the acid resistance test showed that all the three immobilized enzymes had stronger acid resistance than did the free enzyme. The free enzyme and the three immobilized enzymes were used to prepare gluconic acid under low pH conditions (pH value 3.5). The reaction times required for the strongly alkaline macroporous resin-immobilized enzyme, amino resin-immobilized enzyme, and epoxy resin-immobilized enzyme were 40, 24, and 27 h respectively. The enzymatic activity loss rates were 46.44%, 3.42%, and 21.84%, respectively. The free enzyme was completely inactivated during the reaction. The clarity and chromaticity of the three immobilized enzyme reaction solutions were significantly better than those of the free enzyme reaction solutions. The preparation process of amino resin-immobilized enzyme-after further optimization through orthogonal experiments - was as follows: the concentration of mixed enzyme solution was 10%, the ratio of glucose oxidase to catalase was 1.5:1, and the fixation temperature was 25 °C. Under these conditions, the recovery rate of amino resin-immobilized enzyme was as high as 93.15%, and the conversion time of the 100 g/L glucose solution was 23 h. At the end of the reaction, the ratio of gluconic acid to sodium gluconate had reached 0.899:1 (m/m), and the amino resin-immobilized enzyme was stable. Based on these results, we conclude that the amino resin immobilized enzyme can be used to establish a low-pH immobilized enzyme gluconic acid production process, with the goal of producing high-quality gluconic acid products. [ABSTRACT FROM AUTHOR]