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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.

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].

[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]

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]

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]

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]

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]

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]

[Display omitted] The design of enzymatic droplet-sized reactors constitutes an important challenge with many potential applications such as medical diagnostics, water purification, bioengineering, or food industry. Coacervates, which are all-aqueous droplets, afford a simple model for the investigation of enzymatic cascade reaction since the reactions occur in all-aqueous media, which preserve the enzymes integrity. However, the question relative to how the sequestration and the proximity of enzymes within the coacervates might affect their activity remains open. Herein, we report the construction of enzymatic reactors exploiting the simple coacervation of ampholyte polymer chains, stabilized with agar. We demonstrate that these coacervates have the ability to sequester enzymes such as glucose oxidase and catalase and preserve their catalytic activity. The study is carried out by analyzing the color variation induced by the reduction of resazurin. Usually, phenoxazine molecules acting as electron acceptors are used to characterize glucose oxidase activity. Resazurin (pink) undergoes a first reduction to resorufin (salmon) and then to dihydroresorufin (transparent) in presence of glucose oxidase and glucose. We have observed that resorufin is partially regenerated in the presence of catalase, which demonstrates the enzymatic cascade reaction. Studying this enzymatic cascade reaction within coacervates as reactors provide new insights into the role of the proximity, confinement towards enzymatic activity. [ABSTRACT FROM AUTHOR]

In this work, we report the green production of few-layer bio-Graphene (bG) through liquid exfoliation of graphite in the presence of bovine serum albumin. Microscopic characterization evaluated the quality of the produced nanomaterial, showing the presence of 3–4-layer graphene. Moreover, spectroscopic techniques also confirmed the quality of the resulted bG, as well as the presence of bovine serum albumin on the graphene sheets. Next, for the first time, bG was used as support for the simultaneous covalent co-immobilization of three enzymes, namely β-glucosidase, glucose oxidase, and horseradish peroxidase. The three enzymes were efficiently co-immobilized on bG, demonstrating high immobilization yields and activity recoveries (up to 98.5 and 90%, respectively). Co-immobilization on bG led to an increase of apparent KM values and a decrease of apparent Vmax values, while the stability of the nanobiocatalysts prevailed compared to the free forms of the enzymes. Co-immobilized enzymes exhibited high reusability, preserving a significant part of their activity (up to 72%) after four successive catalytic cycles at 30 °C. Finally, the tri-enzymatic nanobiocatalytic system was applied in three-step cascade reactions, involving, as the first step, the hydrolysis of p-Nitrophenyl-β-D-Glucopyranoside and cellobiose. [ABSTRACT FROM AUTHOR]

The presented work involves obtaining and characterising a two-enzymatic one-pot bioreactor, including encapsulated (co-immobilised) glucose oxidase and catalase. The enzymatic capsules were applied to produce unsweet, lactose-free milk during low-temperature catalysis. Furthermore, operational conditions, like pH and aeration, were selected in the paper, which sorts out discrepancies in literature reports. All experiments were carried out at 12 °C, corresponding to milk storage and transportation temperature. Preliminary studies (for reasons of analytical accuracy) were carried out in a buffer (pH, concentration of sugars mimicking conditions in the lactose-free milk, the initial glucose concentration 27.5 g/L) verified by processes carried out in milk in the final stage of the study. The presented results showed the need for regulating pH and the aeration of the reaction mixture in the continuous mode during the process. The procedure of co-immobilisation was performed in an alginate matrix with the cross-linking of glutaraldehyde or carbodiimide while carbodiimide showed better enzymes retention inside alginate capsules. Co-encapsulated enzymes could be used for nine cycles, preserving finally about 40% of the initial activity. [ABSTRACT FROM AUTHOR]

Neuroblastoma cells can acquire resistance mechanisms that make them invulnerable to chemotherapeutic agents. The use of nanoparticles as drug carriers provides the possibility to deliver several drugs simultaneously to specific tumoral cell populations, improving their therapeutic outcome. Herein, the development of a multifunctional nanoplatform based on the assembly of protocells (PC) and polymeric nanocapsules (PNC) is reported. PC provides the ability to transport and release cytotoxic drugs while PNC offers the capacity to transport enzymes to the tumoral tissues preserving their catalytic activity. Doxorubicin (Dox) and Glucose Oxidase (Gox) are housed within PC and PNC, respectively. The external surface of these nanoassemblies is decorated with synthetic targeting moieties, providing selectivity to neuroblastoma cells. Thus, the nanoplatform is endowed with the ability to generate multiple insults within neuroblastoma cells as cytotoxic drug release, glucose starvation, and oxidative damage. This nanoplatform exhibits significantly higher cytotoxic activity in comparison with only drug‐loaded protocells or empty protocells decorated with glucose oxidase nanocapsules, which points out the existence of a potent synergic effect between the action of both therapeutic agents: Dox and Gox. This strategy can be adapted to the production of multifunctional nanoassemblies, improving the arsenal against different types of tumors. [ABSTRACT FROM AUTHOR]

High performance microprobes for combined sensing of glucose and choline were fabricated using microcontact printing (μCP) to transfer choline oxidase (ChOx) and glucose oxidase (GOx) onto targeted sites on microelectrode arrays (MEAs). Most electroenzymatic sensing sites on MEAs for neuroscience applications are created by manual enzyme deposition, which becomes problematic when the array feature size is less than or equal to ∼100 μm. The μCP process used here relies on use of soft lithography to create features on a polydimethylsiloxane (PDMS) microstamp that correspond to the dimensions and array locations of targeted, microscale sites on a MEA. Precise alignment of the stamp with the MEA is also required to transfer enzyme only onto the specified microelectrode(s). The dual sensor fabrication process began with polyphenylenediamine (PPD) electrodeposition on all Pt microelectrodes to block common interferents (e.g., ascorbic acid and dopamine) found in brain extracellular fluid. Next, a chitosan film was electrodeposited to serve as an adhesive layer. The two enzymes, ChOx and GOx, were transferred onto different microelectrodes of 2 × 2 arrays using two different PDMS stamps and a microscope for stamp alignment. Using constant potential amperometry, the combined sensing microprobe was confirmed to have high sensitivity for choline and glucose (286 and 117 μA mM cm-2, respectively) accompanied by low detection limits (1 and 3 μM, respectively) and rapid response times (≤2 s). This work demonstrates the use of μCP for facile creation of multianalyte sensing microprobes by targeted deposition of enzymes onto preselected sites of a microelectrode array.

Enzymatic biofuel cells (EBCs) represent a promising technology for biosensors, biodevices, and sustainable green energy applications, thanks to enzymes' high specificity and catalytic efficiency. Nevertheless, drawbacks such as limited output power and short lifetime have to be solved. Nowadays, research is addressed to the use of 3D electrode structures, but the high cost and the industrialization difficulties of such electrodes represent a key issue. The purpose of the paper is thus to describe the use of a low-cost commercial conductive polymer (Sigracell® PV15) as support for the covalent immobilization of glucose oxidase and laccase, for bioanode and biocathode fabrication, respectively. Efficient immobilization protocols were determined for the immobilized enzymes in terms of employed linkers and enzyme concentrations, resulting in significant enzymatic activities for units of area. The analysis focuses specifically on the optimization of the challenging immobilization of laccase and assessing its stability over time. In particular, an optimum activity of 23 mU/cm2 was found by immobilizing 0.18 mg/cm2 of laccase, allowing better performances, as for voltage output and electrochemical stability, and a direct electron transfer mechanism to be revealed for the fabricated biocathode. This study thus poses the basis for the viable development of low-cost functional EBC devices for biomedical applications. [ABSTRACT FROM AUTHOR]

This study was carried out on samples of honey that represented most of the governorates of Iraq from different plant sources and included the following: The protein content study of the different honey varieties was studied and it was noted that there were significant differences at the p<0.05 level in the percentages of protein. It was noted that the protein percentage ranged between 0.14-0.73% and the highest percentage was in the sample H12 and the lowest In the sample H15. The results of tests to determine the values of the enzyme activity of honey enzymes (diastase, invertase and glucose-oxidase) showed that there were significant differences for these values between types of honey, as the highest value of diastase in sample H12 was 21 unit and the lowest value reached 8 units in samples H4 and H8, in While the highest activity of invertase was recorded in the H7 sample which amounted to 284.3 U/kg and the lowest activity in the H11 sample, which amounted to 107.5 U/kg, while the activity of glucose-oxidase was the highest in the H12 sample amounting to 133.5 micrograms H2O2/g/h and the least effective in the H11 sample reaching 72.2 micrograms H2O2/g/h. [ABSTRACT FROM AUTHOR]

Mesoporous silica, alumina and zirconia, were utilized as appropriate support for the formation of magnetite nanoparticles by in-situ crystallization and GOx immobilization. Magnetic and nonmagnetic supports were functionalized with amino groups and using glutaraldehyde as a linker. A comparative study showed that the nature of the carrier and the presence of magnetite nanoparticles all affect the catalytic properties of biocatalysts and their stability. Biocatalysts based on immobilized glucose oxidase demonstrated high catalytic activity (preservation of the activity of the free enzyme up to 98 %) and stability in D-glucose oxidation to Dgluconic acid at pH 4-8 and temperature of 30-60 °C. [ABSTRACT FROM AUTHOR]

The general objective of Analytical Chemistry, nowadays, is to obtain best-quality information in the shortest time to contribute to the resolution of real problems. In this regard, electrochemical biosensors are interesting alternatives to conventional methods thanks to their great characteristics, both those intrinsically analytical (precision, sensitivity, selectivity, etc.) and those more related to productivity (simplicity, low costs, and fast response, among others). For many years, the scientific community has made continuous progress in improving glucose biosensors, being this analyte the most important in the biosensor market, due to the large amount of people who suffer from diabetes mellitus. The sensitivity of the electrochemical techniques combined with the selectivity of the enzymatic methodologies have positioned electrochemical enzymatic sensors as the first option. This review, focusing on the electrochemical determination of glucose using paper-based analytical devices, shows recent approaches in the use of paper as a substrate for low-cost biosensing. General considerations on the principles of enzymatic detection and the design of paper-based analytical devices are given. Finally, the use of paper in enzymatic electrochemical biosensors for glucose detection, including analytical characteristics of the methodologies reported in relevant articles over the last years, is also covered. [ABSTRACT FROM AUTHOR]

The activity of honey enzymes are affected by floral and geographical origins, climate conditions, honeybee species, health and nutrition. This article investigated the effect of floral and geographical origins on the activity of glucose oxidase (GOx) and catalase (CAT) enzymes in honey samples from the southwestern region of Saudi Arabia. Moreover, the moisture, total sugars, pH and conductivity were measured as quality parameters. The floral origin of the honey samples was determined microscopically while the quality parameters were measured according to the methods of international honey commission. The activity of the honey enzyme was determined following the instructions of the Megazyme International kits. The obtained results were statistically analyzed by the statistical Package for Social Sciences (SPSS, v.20). The GOx activity of the Acacia, Ziziphus and polyfloral honey samples of the Asir region were (5.19 ± 2.33 U/g), (4.01 ± 1.17 U/g) and (5.69 ± 1.67 U/g), respectively. The Acacia, Ziziphus and polyfloral honey samples from the Jazan region had GOx activities of (6.85 ± 0.47 U/g), (10.48 ± 9.22 U/g) and (5.31 ± 2.7 U/g), respectively. The geographical origin significantly affected the GOx activity of Ziziphus honey (p-value = 0.005) and the GOx activity of the Ziziphus honey was significantly more than that of the polyfloral honey of the Jazan region (p-value = 0.009). With regard to the CAT activity in Asir region honey samples, the mean values of the Acacia, Ziziphus and polyfloral honeys were (2.89 ± 1.08 U/g), (3.58 ± 1.59 U/g) and (2.84 ± 1.24 U/g), respectively. The mean values of the CAT activity in the Jazan honey samples were Acacia (4.35 ± 1.01 U/g), Ziziphus (3.94 ± 0.04 U/g) and polyfloral (3.43 ± 0.67 U/g). The geographical origin significantly affected the CAT activity in Acacia honey (p-value = 0.014). The geographical and floral origins had significant effects on the activity of the honey GOx and CAT enzymes. [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 Trichoderma reesei strain AR-352 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism, but the absence of its recombinant DNA could not be established. The food enzyme is intended to be used in four food manufacturing processes, namely baking processes, cereal-based processes, grain treatment for the production of starch and gluten fractions, and egg processing. Since residual amounts of total organic solids (TOS) are removed by repeated washing during the production of starch and gluten, dietary exposure was calculated only for the remaining three processes. Dietary exposure to the food enzyme-TOS was estimated to be up to 0.13 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of more than 7,800. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Cancer is one of the most serious human diseases, causing millions of deaths worldwide annually, and, therefore, it is one of the most investigated research disciplines. Developing efficient anticancer tools includes studying the effects of different natural enzymes of plant and microbial origin on tumor cells. The development of various smart delivery systems based on enzyme drugs has been conducted for more than two decades. Some of these delivery systems have been developed to the point that they have reached clinical stages, and a few have even found application in selected cancer treatments. Various biological, chemical, and physical approaches have been utilized to enhance their efficiencies by improving their delivery and targeting. In this paper, we review advanced delivery systems for enzyme drugs for use in cancer therapy. Their structure-based functions, mechanisms of action, fused forms with other peptides in terms of targeting and penetration, and other main results from in vivo and clinical studies of these advanced delivery systems are highlighted. [ABSTRACT FROM AUTHOR]

Silica nanoparticles hold tremendous potential for the encapsulation of enzymes. However, aqueous alcohol solutions and catalysts are prerequisites for the production of silica nanoparticles, which are too harsh for maintaining the enzyme activity. Herein, a procedure without any organic solvents and catalysts (acidic or alkaline) is developed for the synthesis of silica-encapsulated glucose-oxidase-coated magnetic nanoparticles by a facile self-assembly route, avoiding damage of the enzyme structure in the reaction system. The encapsulated enzyme was characterized by scanning electron microscopy, transmission electron microscopy, energy-dispersive spectrometry, and a vibrating sample magnetometer. Finally, a colorimetric sensing method was developed for the detection of glucose in urine samples based on the encapsulated glucose oxidase and a hydrogen peroxide test strip. The method exhibited a good linear performance in the concentration range of 20~160 μg mL−1 and good recoveries ranging from 94.3 to 118.0%. This work proves that the self-assembly method could be employed to encapsulate glucose oxidase into silica-coated magnetic particles. The developed colorimetric sensing method shows high sensitivity, which will provide a promising tool for the detection of glucose and the monitoring of diabetes. [ABSTRACT FROM AUTHOR]

Enzymes/Nanoparticles (NPs) bioconjugates are massively used nowadays to develop thin films for optical and electrochemical biosensors. Nevertheless, their full characterization as a thin coating onto electrodes remains little discussed, in particular the influence of NPs size and enzyme/NPs ratio used in the electrodeposition solution. In this study, GOx (160 kDa) and HRP (44 kDa) were used in association with tannic acid capped gold NPs (a series with sizes from 7 to 40 nm) to electrodeposit biosensor coatings, sensitive towards glucose and H2O2, respectively. The electrodeposition process was based on a mussel-inspired electro-crosslinking between gallol moieties of tannic acid (at the surface of NPs) and amine moieties of the enzymes. On one hand, the sensitivity of the GOx/NPs coatings depends strongly on the NP size and the enzyme/NPs molar ratio of the electrodeposition solution. An optimal sensitivity was obtained by electrodeposition of 11 nm NPs at a GOx/NPs molar ratio close to the theoretical value of the enzyme monolayer. On the other hand, a modest influence of the NPs size was found on the sensitivity in the case of the electrodeposited HRP/NPs coatings, reaching a plateau at the HRP/NPs molar ratio close to the value of the theoretical enzyme monolayer. In both cases, the enzyme/NPs molar ratio played a role in the sensitivity. To fully understand the parameters driving the biosensor sensitivity, a comprehensive evaluation of the colloidal state of the bioconjugates is proposed here. [ABSTRACT FROM AUTHOR]

Enzymatic biosensors based on glucose oxidase has been proven to be one of the effective strategies for the detection of glucose and contributed to health improvements. Therefore, research and debates to date are ongoing in an attempt to find the most effective way to detect this analyte using this enzyme as the recognition center. The 3rd generation biosensors using direct electron transfer (DET) type enzymes are a great way towards practical devices. In this work, we developed a simple method for the functionalization of glucose oxidase with redoxable ferrocene groups in chloroform. The enzyme retained its activity after storage in this organic solvent and after the functionalization procedures. This enzyme functionalization strategy was employed to develop the biosensing monolayer-based platforms for the detection of glucose utilizing the quasi-DET mechanism. As a result of an electrochemical regeneration of the catalytic center, the formation of harmful H2O2 is minimized during enzymatic electrocatalysis. [ABSTRACT FROM AUTHOR]

Developing a rapid and sensitive photo-electrochemical (PEC) sensing platform for glucose detection is highly desirable. Herein, a visible light-driven PEC enzyme biosensor for sensitive detection of glucose was proposed, employing reduced graphene oxide/titania (TiO2) nanotubes (RGO/TNTs) as photoactive materials and glucose oxidase (GOD) as the identification element. The RGO/TNTs composites were synthesized via a facile hydrothermal process. Notably, the unique nanostructure system significantly increases the absorption in the visible region and effectively separates the photo-generated electron–hole pairs, which is essential for promoting the PEC activity. In the presence of dissolved oxygen, the GOD immobilized on the modified electrode could specifically catalyze the electro-oxidation of glucose to produce H2O2, resulting in an increase of photocurrent intensity. Based on the variation of the photocurrent response, the constructed PEC enzyme biosensor exhibited high sensitivity with a wide linear range from 10–2 to 10 mM and a low detection limit of 5.0 μΜ. Meanwhile, the potential applicability of the PEC enzyme biosensor was evaluated by detecting glucose in human serum samples, and satisfactory results were obtained. The PEC enzyme biosensor based on RGO/TNTs composites successfully realized the sensitive detection of glucose and displayed good selectivity, high stability and satisfactory repeatability. [ABSTRACT FROM AUTHOR]

Enzymes are one of the main biocatalysts with various applications in the food industry. Stabilization of enzymes on insoluble carriers is important due to the low reuse, low operational stability, and high cost in applications. The immobility and the type of carrier affect the activity of the immobile enzyme. Hydrogels are three-dimensionally cross-linked macromolecular network structures designed from various polymers. Hydrogels can provide a matrix for an immobile enzyme due to their extraordinary properties such as high water absorbing capacity, carrier of bioactive substances and enzymes, biocompatibility, safety, and biodegradability. Therefore, this study mainly focuses on some enzymes (lactase, lipases, amylases, pectinase, protease, glucose oxidase) that are of special importance in the food industry. These enzymes could be immobilized in the hydrogels constructed of macromolecules such as kappacarrageenan, chitosan, Arabic gum, pectin, alginate, and cellulose. At last, in the preparation of these hydrogels, different enzyme immobilization methods in macromolecular hydrogels, and effect of hydrogels on enzyme activity were discussed. [ABSTRACT FROM AUTHOR]

Tumor tissues need vast supply of nutrients and energy to sustain the rapid proliferation of cancer cells. Cutting off the glucose supply represents a promising cancer therapy approach. Herein, a tumor tissue-targeted enzyme nanogel (rGCP nanogel) with self-supply oxygen capability was developed. The enzyme nanogel synergistically enhanced starvation therapy and photodynamic therapy (PDT) to mitigate the rapid proliferation of cancer cells. The rGCP nanogel was fabricated by copolymerizing two monomers, porphyrin and cancer cells-targeted, Arg-Gly-Asp (RGD), onto the glucose oxidase (GOX) and catalase (CAT) surfaces. The cascade reaction within the rGCP nanogel could efficiently consume intracellular glucose catalyzed by GOX. Concurrently, CAT safely decomposed the produced H 2 O 2 with systemic toxicity to promote oxygen generation and achieved low toxicity starvation therapy. The produced oxygen subsequently facilitated the glucose oxidation reaction and significantly enhanced the generation of cytotoxic singlet oxygen (1O 2) in the presence of 660 nm light irradiation. Combining starvation therapy and PDT, the designed enzyme nanogel system presented an amplified synergic cancer therapy effect. This approach potentially paved a new way to fabricate a combinatorial therapy approach by employing cascaded catalytic nanomedicines with good tumor selectivity and efficient anti-cancer effect. The performance of starvation and photodynamic therapy (PDT) is usually suppressed by intrinsic tumorous hypoxia. Herein, an oxygen self-supplied and tumor tissue-targeted enzyme nanogel was created by copolymerization of two monomers, porphyrin and cancer cell-targeted Arg-Gly-Asp (RGD), onto the surface of glucose oxidase (GOX) and catalase (CAT), which synergistically enhanced starvation therapy and PDT. Moreover, the enzyme nanogels possessed high stability and could be synthesized straightforwardly. This anti-cancer system provides an approach for constructing a combinatorial therapy approach by employing cascaded catalytic nanomedicine with good tumor selectivity and therapeutic efficacy. [Display omitted] [ABSTRACT FROM AUTHOR]

In the work, the possibility of using nanoparticles of gold (AuNPs) to upgrade bioselective elements of biosensors in order to improve their analytical characteristics is considered. The bioselective elements of biosensors based on acetylcholinesterase (AChE), butyryl cholinesterase (BuChE) and glucose oxidase (GOD) were used as an experimental model. Immobilization of enzymes on the surfaces of conductometric transducers was performed by the crosslinking of corresponding enzymes using glutaraldehyde. The conditions of immobilization of AChE with gold nanoparticles were optimized. Thus, we determined the optimal values of concentration of crosslinking agent (glutaraldehyde), duration of immobilization, the enzyme to AuNPs ratio, the AuNPs concentration and size. The performance characteristics of the biosensors based on enzymes and AuNPs were investigated and compared with the characteristics of biosensors based on enzymes only. It was also examined how the addition of AuNPs to the bioselective element of biosensors affects the biosensor stability. In particular, the reproducibility of preparation and continuous operation of biosensors was tested as well as their stability at storage. It was shown that the presence of AuNPs in the composition of bioselective elements can improve some characteristics of biosensors, which may be promising for further study and use. [ABSTRACT FROM AUTHOR]

Biotechnology is an ecological advantageous and moreover economically beneficial technology. The most established application of biotechnology in textiles has been in the field of enzymatic pretreatment. Glucose oxidase (GOD) catalyse the oxidation of b-D-glucose into gluconic acid by utilizing molecular oxygen as an electron acceptor with a simultaneous production of hydrogen peroxide (HP). Glucose oxidases are commercially gaining a lot of attention in textile technology. In an enzymatic pretreatment, the textile substrate is less damaged when compared to a classical pretreatment. Enzymatic pre-treatments of cellulose fabrics often save large amounts of raw materials, chemicals, energy and water. Bleaching with glucose oxidase thus represents an economic and ecological potential when compared to the classical process with added hydrogen peroxide. This review represents the basic properties and production of glucose oxidases and their applications in textile technology.

Our work provides new insight into self-stabilizing monodispersed metal–organic frameworks by glucose oxidase without any chemical modifications, and constructs an unprecedented double enzyme delivering system with excellent in vivo and in vitro cancer synergistic therapy effect at very low doses without using any chemotherapeutic drugs. [Display omitted] • A self-stabilizing strategy to functionalize MOFs without chemical modifications is designed. • Long-time stable mono-dispersity in water are unprecedented obtained. • Controlled loading mixed enzymes allows efficient synergetic therapy without any chemical drugs. • Apoptosis, anti-metastasis mechanism and immunofluorescence pathways are fully discovered. Controlled loading more than one active cargos on metal–organic frameworks (MOFs) and keeping monodispersity have long been a great challenge due to easy agglomeration and collapse of MOFs in water. Herein, we develop an emerging self-stablized double enzyme delivering system with long-time monodispersion, and achieve synergistic cancer starvation and photothermal therapy (PTT) without any chemotherapeutics. Loading of glucose oxidase (GOx) confers strong coordination interactions with MOFs, which not only endows robust self-stablized monodispersion without any surface modification, but also guarantees uniform deposition of GOx-like nanoenzyme of Au nanoparticles with catalytic and photothermal conversion property. Our design establishes an effective synergistic therapy platform to generate reactive oxygen species, decreases mitochondrial membrane potential, and induces cell apoptosis. Immunofluorescence study confirms activation of Caspase 3 protein of apoptosis pathway, and significantly inhibits proliferation of cancer cells. Our work provides new insight into stabilizing MOFs by GOx without any chemical modifications, and constructs an unprecedented double enzyme delivering system with excellent in vivo and in vitro cancer synergistic therapy effect at very low doses without using any chemotherapeutic drugs. [ABSTRACT FROM AUTHOR]

To alleviate the discomfort associated with frequent blood glucose detection in diabetic patients, a novel non-invasive tear glucose biosensor has been developed. This involved the design and preparation of a photoelectrochemical probe based on an optical fiber and biological enzymes. One end of the optical fiber connects to a light source, acting as an energy source and imparting, self-powered capability to the biosensor. The opposite end is loaded with nanomaterials and glucose oxidase, designed for insertion into the sample to realize photoelectrochemical sensing. This innovative configuration not only improves the integration of the biosensor but is also suitable for analyzing minuscule voluminal samples. The results show that the proposed biosensor exhibits a linear range from 10 nM to 100 μM, possesses a low detection limit of 4.1 nM and a short response time of 0.7 s. Benefiting from the high selectivity of the enzyme, the proposed biosensor demonstrates excellent resistance to the interference of common tear components. In summary, this work provides a more effective method for non-invasive glucose detection and affords valuable ideas for the design and fabrication of non-invasive and self-powered biosensors. [ABSTRACT FROM AUTHOR]

This study introduces the UV/glucose-oxidase@Kaolin (GOD@Kaolin) coupled organic green rust (OGR) system (UV/OGR/GOD@Kaolin) to investigate the promotion of glucose oxidase activity by UV light and its synergistic degradation mechanism for photosensitive pollutants, specifically targeting the efficient degradation of 4-chlorophenol (4-CP). The enzyme system demonstrates its ability to overcome drawbacks associated with traditional Fenton systems, including a narrow pH range and high localized concentration of H 2 O 2 , by gradually releasing hydrogen peroxide in situ within a neutral environment. In the presence of UV radiation under specific conditions, enhanced enzyme activity is observed, resulting in increased efficiency in pollutant removal. The gradual release of hydrogen peroxide plays a crucial role in preventing unwanted reactions among active substances. These unique features facilitate the generation of highly reactive species, such as Fe(IV) O, •OH, and •O 2 −, tailored to efficiently target the organic components of interest. Additionally, the system establishes a positive iron cycle, ensuring a sustained reactive capability throughout the degradation process. The results highlight the UV/OGR/GOD@Kaolin system as an effective and environmentally friendly approach for the degradation of 4-CP, and the resilience of the enzyme extends the system's applicability to a broader range of scenarios. [Display omitted] • UV irradiation enhances the enzymatic activity through irritating GOD photosensitivity. • UV irradiation reduces Fe3+ to Fe2+, promoting the iron cycle. • The in-situ release of hydrogen peroxide improves its utilization efficiency. • The in-situ release of hydrogen peroxide improves its utilization efficiency. • GOD loading on kaolin enhances enzyme structure and activity and can also be recycled. [ABSTRACT FROM AUTHOR]

Enzymatic Biofuel Cell (EBC) represents a promising green source since it is capable of harvesting electricity from renewable and abundantly available biofuels using enzymes as catalysts. Nevertheless, nowadays long-term stability and low power output are currently the main concerns. To this end, several research studies focus on using complex tridimensional and highly expensive nanostructures as electrode support for enzymes. This increases cell performance whilst drastically reducing the economic feasibility needed for industrial viability. Thus, this paper analyzes a novel flow-based EBC consisting of covalent immobilized GOx (bioanode) and Laccase (biocathode) on a commercial flat conductive polymer. A suitable immobilization technique based on covalent ligands is carried out to enhance EBC durability. The experimental characterization demonstrates that the cell generates power over three weeks, reaching 590 mV and 2.41 µW cm−2 as maximum open circuit voltage and power density, respectively. The most significant contributions of this configuration are definitely ease of implementation, low cost, high scalability, and reproducibility. Therefore, such a design can be considered a step forward in the viable EBC industrialization process for a wide range of applications. [ABSTRACT FROM AUTHOR]

Glucose oxidase (GOx) is an important oxidoreductase enzyme with many important roles in biological processes. It is considered an "ideal enzyme" and is often called an oxidase "Ferrari" because of its fast mechanism of action, high stability and specificity. Glucose oxidase catalyzes the oxidation of β -d-glucose to d-glucono- δ -lactone and hydrogen peroxide in the presence of molecular oxygen. d-glucono- δ -lactone is sequentially hydrolyzed by lactonase to d-gluconic acid, and the resulting hydrogen peroxide is hydrolyzed by catalase to oxygen and water. GOx is presently known to be produced only by fungi and insects. The current main industrial producers of glucose oxidase are Aspergillus and Penicillium. An important property of GOx is its antimicrobial effect against various pathogens and its use in many industrial and medical areas. The aim of this review is to summarize the structure, function, production strains and biophysical and biochemical properties of GOx in light of its various industrial, biotechnological and medical applications. [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 Aspergillus niger strain DP‐Aze23 by Danisco US, Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in baking processes, cereal‐based processes and egg processing. Based on the maximum use levels, dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.05 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 19.55 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure above 380. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and one match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Increasing antibiotic resistance becomes a serious threat to public health. Photothermal therapy (PTT) and antibacterial enzyme‐based therapy are promising nonresistant strategies for efficiently killing drug‐resistant bacteria. However, the poor thermostability of enzymes in PTT hinders their synergistic therapy. Herein, antibacterial glucose oxidase (GOx) is embedded in a Ag graphitic nanocapsule (Ag@G) arrayed silk film to fabricate a GOx‐synergistic PTT system (named silk‐GOx‐Ag@G, SGA). The SGA system can stabilize GOx by a vitrification process through the restriction of hydrogen bond and rigid β‐sheet, and keep the antibacterial activity in the hyperthermal PTT environment. Moreover, the arrayed Ag@G possesses excellent chemical stability due to the protection of graphitic shell, providing stable plasmonic effect for integrating PTT and surface enhanced Raman scattering (SERS) analysis even in the GOx‐produced H2O2 environment. With in situ SERS identification of bacterial intrinsic signals in the mouse wound model, such SGA realizes superior synergistic antibacterial effect on the infected Escherichia coli, Staphylococcus aureus, and methicillin‐resistant Staphylococcus aureus in vivo, while without causing significant biotoxicity. This system provides a therapeutic method with low resistance and in situ diagnosis capability for efficiently eliminating bacteria. [ABSTRACT FROM AUTHOR]

Because the transition from oxidative phosphorylation to anaerobic glycolytic metabolism is a hallmark of cancer progression, approaches to identify single living cancer cells by their unique glucose metabolic signature would be useful. Here, we present nanopipettes specifically developed to measure glucose levels in single cells with temporal and spatial resolution, and we use this technology to verify the hypothesis that individual cancer cells can indeed display higher intracellular glucose levels. The nanopipettes were functionalized as glucose nanosensors by immobilizing glucose oxidase (GOx) covalently to the tip so that the interaction of glucose with GOx resulted in a catalytic oxidation of β-d-glucose to d-gluconic acid, which was measured as a change in impedance due to drop in pH of the medium at the nanopipette tip. Calibration studies showed a direct relationship between impedance changes at the tip and glucose concentration in solution. The glucose nanosensor quantified single cell intracellular glucose levels in human fibroblasts and the metastatic breast cancer lines MDA-MB-231 and MCF7 and revealed that the cancer cells expressed reproducible and reliable increases in glucose levels compared to the nonmalignant cells. Nanopipettes allow repeated sampling of the same cell, as cells remain viable during and after measurements. Therefore, nanopipette-based glucose sensors provide an approach to compare changes in glucose levels with changes in proliferative or metastatic state. The platform has great promise for mechanistic investigations, as a diagnostic tool to distinguish cancer cells from nonmalignant cells in heterogeneous tissue biopsies, as well as a tool for monitoring cancer progression in situ.

Use of enzyme for extraction of nanocellulose from sugarcane bagasse is greener alternative. Literature indicates that effectiveness of these enzymes can be improved by auxiliary enzymes or mediators. In the current study, extraction of nanocellulose using laccase with these moderators, auxiliary enzyme glucose oxidase and mediator molecule, ABTS [2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate)] individually was done. Cellulose and lignin content, FT-IR, TGA and DSC analysis, XRD, SEM and PSA were done. Enzyme moderators improved the performance of laccase in lignin degradation. Lignin and cellulose content, crystallinity were used as parameters to optimize the concentrations, which was found to be ABTS (at 1.4 mM) and glucose oxidase (at 0.15 mg ml−1). At the optimal concentration, nanocellulose was extracted. Properties of nanocellulose obtained from both routes were compared. Size analysis revealed 339 nm and 636 nm for nanocellulose obtained with glucose oxidase and ABTS, respectively. Defibrillation was better in the case of the former one as seen from SEM. [ABSTRACT FROM AUTHOR]

Experiment-based guidelines for analysis of metal surface effect to overall catalytic process was suggested. [Display omitted] • Catalytic input of nanoparticle surface in enzyme-gold nanoparticle catalysts was investigated. • Kinetic behavior of catalysts with four coating scenarios was analyzed. • No actual enzymatic acceleration on gold nanoparticles was observed. In recent years, many papers have reported a catalytic "acceleration" of enzymes when immobilized on gold nanoparticles. Concordantly, gold nanoparticles are often considered as an inert and safe nanomaterial, and are widely used for various purposes, e.g., experiments with humans are being conducted in vivo. In this work we have carried out an in-detail study of catalytic properties of citrate-capped gold nanoparticles and gold nanoparticle-protein conjugates using three model proteins – enzymes glucose oxidase and catalase, and catalytically inactive protein bovine serum albumin. Catalytic properties were studied at different protein-nanoparticle ratios. UV–Vis, DLS, AFM and ζ potential measurements confirmed protein-nanoparticle conjugate formation. Catalytic activity measurements were conducted using oxygen electrode and the data were analyzed by modeling the activity of conjugates. The designed experiments demonstrated that gold nanoparticles form stable conjugates with all the investigated proteins, yet they do not increase catalytic activity of the investigated enzymes – in certain conditions gold nanoparticles mimic enzymatic reactions, which may be misattributed to accelerated enzymatic catalysis. Additionally, we present specific key points demonstrating why it may be difficult to differentiate between enzyme- and gold nanoparticle-catalyzed reactions, as well as suggest specific measurements enabling better differentiation. We do not claim that enzymes cannot be accelerated on nanoparticles in general, but rather emphasize, that experimental results demonstrating atypical catalytic performance of enzymes on nanoparticles should be interpreted with additional care, and a widely propagated view of "inert gold nanoparticles" should probably be reconsidered. [ABSTRACT FROM AUTHOR]

The NiFe2O4 magnetic nanoparticles (NF‐MNPs) were prepared for one‐step selective affinity purification and immobilization of His‐tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier‐transform infrared spectrophotometer and microscopy. The immobilization and purification of His‐tagged GluDH on NF‐MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris‐HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 μg mg–1 support, respectively. The immobilized GluDH exhibited high thermostability, pH‐stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni‐NTA resin, the NF‐MNPs displayed a higher specific affinity with His‐tagged recombinant GluDH. [ABSTRACT FROM AUTHOR]

Two-dimensional (2D) layered materials that integrate metallic conductivity, catalytic activity and the ability to stabilize biological receptors provide unique capabilities for designing electrochemical biosensors for large-scale detection and diagnostic applications. Herein, we report a multifunctional MXene-based 2D nanostructure decorated with enzyme mimetic cerium oxide nanoparticle (MXCeO 2) as a novel platform and catalytic amplifier for electrochemical biosensors, specifically targeting the detection of oxidase enzyme substrates. We demonstrate enhanced catalytic efficiency of the MXCeO 2 for the reduction of hydrogen peroxide (H 2 O 2) and its ability to immobilize oxidase enzymes, such as glucose oxidase, lactate oxidase and xanthine oxidase. The designed biosensors exhibit high selectivity, stability, and sensitivity, achieving detection limits of 0.8 μM H 2 O 2 , 0.49 μM glucose, 3.6 μM lactate and 1.7 μM hypoxanthine, when the MXCeO 2 and their respective enzymes were used. The MXCeO 2 was successfully incorporated into a wearable fabric demonstrating high sensitivity for lactate measurements in sweat. The unique combination of MXenes with CeO 2 offers excellent conductivity, catalytic efficiency and enhanced enzyme loading, demonstrating potential of the MXCeO 2 as a catalytically active material to boost efficiency of oxidase enzyme reactions. This design can be used as a general platform for increasing the sensitivity of enzyme based biosensors and advance the development of electrochemical biosensors for a variety of applications. [Display omitted] [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effects of supplementing glucose oxidase (GOX; 1000 unit/g) to the diet of growing pigs on growth performance, apparent nutrient digestibility, faecal bacteria counts, and serum antioxidant enzyme parameters. A total of 200 growing pigs [(Yorkshire × Landrace) × Duroc] with an initial body weight of 23.3 ± 0.33 kg were used in a 42-day trial. Pigs were randomly allotted to four dietary treatments with 10 replicate pens per treatment and five pigs (mixed sex) per pen based on the initial body weight. The dietary treatments consisted of the basal diet (control), control + 0.01% GOX, control + 0.02% GOX, or control + 0.03% GOX. One unit of GOX concentration is defined as the amount of enzyme which oxidises 1 μmol β-D-glucose per minute to D-gluconic acid and hydrogen peroxide at 37 °C and pH5.5. Body weight on day 42 (p =.035), average daily gain during days 22–42 (p =.001) and 1-42 (p =.001), average daily feed intake during days 22–42 (p =.008) and 1–42 (p =.020), apparent nitrogen digestibility on day 42 (p =.029), apparent energy retention on day 42 (p =.032), and the concentrations of serum glutathione peroxidase (p =.044) and glutathione (p =.032) on day 42 increased linearly at graduated doses of GOX increased in the diet. Therefore, supplementing GOX to the diet of growing pigs could improve the apparent nutrient digestibility and serum antioxidant enzyme parameters in a dose-dependent manner, thus improving growth performance. In this study, the suitable dosage of GOX used in the diet of growing pigs was 0.03%. Feeding growing pigs with GOX containing diet has beneficial effects on the growth performance Dietary supplementation of GOX could improve the apparent nutrient digestibility in growing pigs The antioxidant status of growing pigs has improved by GOX supplementation. [ABSTRACT FROM AUTHOR]

Enzymatic Biofuel Cell (EBC) represents a promising green source since it is capable of harvesting electricity from renewable and abundantly available biofuels using enzymes as catalysts. Nevertheless, nowadays long-term stability and low power output are currently the main concerns. To this end, several research studies focus on using complex tridimensional and highly expensive nanostructures as electrode support for enzymes. This increases cell performance whilst drastically reducing the economic feasibility needed for industrial viability. Thus, this paper analyzes a novel flow-based EBC consisting of covalent immobilized GOx (bioanode) and Laccase (biocathode) on a commercial flat conductive polymer. A suitable immobilization technique based on covalent ligands is carried out to enhance EBC durability. The experimental characterization demonstrates that the cell generates power over three weeks, reaching 590 mV and 2.41 µW cm−2 as maximum open circuit voltage and power density, respectively. The most significant contributions of this configuration are definitely ease of implementation, low cost, high scalability, and reproducibility. Therefore, such a design can be considered a step forward in the viable EBC industrialization process for a wide range of applications.