A pH-Sensitive Glucose Oxidase and Hemin Coordination Micelle for Multi-Enzyme Cascade and Amplified Cancer Chemodynamic Therapy.

Chemodynamic therapy (CDT) is an emerging therapeutic paradigm for cancer treatment that utilizes reactive oxygen species (ROS) to induce apoptosis of cancer cells but few biomaterials have been developed to differentiate the cancer cells and normal cells to achieve precise and targeted CDT. Herein, a simple cascade enzyme system is developed, termed hemin-micelles-GOx, based on hemin and glucose oxidase (GOx)-encapsulated Pluronic F127 (F127) micelles with pH-sensitive enzymatic activities. Histidine-tagged GOx can be easily chelated to hemin-F127 micelles via the coordination of histidine and ferrous ions in the center of hemin by simple admixture in an aqueous solution. In tumor microenvironment (TME), hemin-micelles-GOx exhibits enhanced peroxidase (POD)-like activities to generate toxic hydroxyl radicals due to the acidic condition, whereas in normal cells the catalase (CAT)-like, but not POD-like activity is amplified, resulting in the elimination of hydrogen peroxide to generate oxygen. In a murine melanoma model, hemin-micelles-GOx significantly suppresses tumor growth, demonstrating its great potential as a pH-mediated enzymatic switch for tumor management by CDT.

Devastating the Supply Wagons: Multifaceted Liposomes Capable of Exhausting Tumor to Death via Triple Energy Depletion.

The anabolism of tumor cells can not only support their proliferation, but also endow them with a steady influx of exogenous nutrients. Therefore, consuming metabolic substrates or limiting access to energy supply can be an effective strategy to impede tumor growth. Herein, a novel treatment paradigm of starving-like therapy-triple energy-depleting therapy-is illustrated by glucose oxidase (GOx)/dc-IR825/sorafenib liposomes (termed GISLs), and such a triple energy-depleting therapy exhibits a more effective tumor-killing effect than conventional starvation therapy that only cuts off one of the energy supplies. Specifically, GOx can continuously consume glucose and generate toxic H2O2 in the tumor microenvironment (including tumor cells). After endocytosis, dc-IR825 (a near-infrared cyanine dye) can precisely target mitochondria and exert photodynamic and photothermal activities upon laser irradiation to destroy mitochondria. The anti-angiogenesis effect of sorafenib can further block energy and nutrition supply from blood. This work exemplifies a facile and safe method to exhaust the energy in a tumor from three aspects and starve the tumor to death and also highlights the importance of energy depletion in tumor treatment. It is hoped that this work will inspire the development of more advanced platforms that can combine multiple energy depletion therapies to realize more effective tumor treatment.

In-Situ Generation of Hydrogen Polysulfides (H2Sn) with Thioglucose, Glucose Oxidase, and Chloroperoxidase.

Hydrogen polysulfides (H2Sn) have emerged as critical physiological mediators that are closely associated with hydrogen sulfide (H2S) signaling. H2Sn exhibit greater nucleophilicity than H2S while also having electrophilic characteristics, enabling unique activities such as protein S-persulfidation. Despite their physiological importance, mechanisms and reactivities of H2Sn remain inadequately explored due to their inherent instability in aqueous environments. Consequently, there is a need to develop biocompatible methods for controlled H2Sn generation to elucidate their behaviors in biological contexts. Herein, we present a dual enzyme system (containing glucose oxidase (GOx) and chloroperoxidase (CPO)) with thioglucose as the substrate to facilitate the controlled release of H2Sn. Fluorescence measurements with SSP4 and the trapping studies allowed us to confirm the production of H2Sn. Such a method may be useful in elucidating the reactivity of hydrogen polysulfides in biological systems as well as provide a potential delivery of H2Sn to target sites for biological applications.

Enzyme-functionalized alginate microparticles enable anaerobic culture under ambient oxygen.

Methods for culturing oxygen-sensitive cells and organisms under anaerobic conditions are vital to biotechnology research. Here, we report a biomaterial-based platform for anaerobic culture that consists of glucose oxidase (GOX) functionalized alginate microparticles (ALG-GOX), which are designed to deplete dissolved [O2 ] through enzymatic activity. ALG-GOX microparticles were synthesized via a water-in-oil emulsion and had a size of 132.0 ± 51.4 µm. Despite having a low storage modulus, the microparticles remained stable under aqueous conditions due to covalent crosslinking through amide bonds. Enzyme activity was tunable based on the loaded GOX concentration, with a maximum activity of 3.6 ± 0.3 units/mg of microparticles being achieved at an initial loading concentration of 5 mg/mL of GOX in alginate precursor solution. High enzyme activity in ALG-GOX microparticles resulted in rapid oxygen depletion, producing a suitable environment for anaerobic culture. Microparticles loaded with both GOX and catalase (ALG-GOX-CAT) to reduce H2 O2 buildup exhibited sustained activity for potential long-term anaerobic culture. ALG-GOX-CAT microparticles were highly effective for the anaerobic culture of Bacteroides thetaiotaomicron, with 10 mg/mL of ALG-GOX-CAT microparticles supporting the same level of growth in an aerobic environment compared to an anaerobic chamber after 16 h (8.70 ± 0.96 and 10.03 ± 1.03 million CFU, respectively; N.S. p = 0.07). These microparticles could be a valuable tool for research and development in biotechnology.

Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion.

Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD-hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild-type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7-fold higher kcat /KM . Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high-producing strain, 8GM1, containing eight copies of AOX1 promoter-GAS1 signal peptide-MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co-expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2-fold. In the 5-L bioreactor, conventional fed-batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3-fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high-producing strains for other industrial enzymes.

Effective synthesis of high-content fructooligosaccharides in engineered Aspergillus niger.

BACKGROUND: Aspergillus niger ATCC 20611 is an industrially important fructooligosaccharides (FOS) producer since it produces the ß-fructofuranosidase with superior transglycosylation activity, which is responsible for the conversion of sucrose to FOS accompanied by the by-product (glucose) generation. This study aims to consume glucose to enhance the content of FOS by heterologously expressing glucose oxidase and peroxidase in engineered A. niger. RESULTS: Glucose oxidase was successfully expressed and co-localized with ß-fructofuranosidase in mycelia. These mycelia were applied to synthesis of FOS, which possessed an increased purity of 60.63% from 52.07%. Furthermore, peroxidase was expressed in A. niger and reached 7.70 U/g, which could remove the potential inhibitor of glucose oxidase to facilitate the FOS synthesis. Finally, the glucose oxidase-expressing strain and the peroxidase-expressing strain were jointly used to synthesize FOS, which content achieved 71.00%. CONCLUSIONS: This strategy allows for obtaining high-content FOS by the multiple enzymes expressed in the industrial fungus, avoiding additional purification processes used in the production of oligosaccharides. This study not only facilitated the high-purity FOS synthesis, but also demonstrated the potential of A. niger ATCC 20611 as an enzyme-producing cell factory.

The importance of factorial design on the optimization of biosensor performance: immobilization of glucose oxidase as a case study.

Conventionally, the optimization of glucose biosensors is achieved by varying the concentrations of the individual reagents used to immobilize the enzyme. In this work, the effect and interaction between glucose oxidase enzyme (GOx), ferrocene methanol (Fc), and multi-walled carbon nanotubes (MWCNTs) at different concentrations were investigated by a design of experiments (DoE). For this analysis, a factorial design with three factors and two levels each was used with the software RStudio for statistical analysis. The data were obtained by electrochemical experiments on the immobilization of GOx-Fc/MWCNT at different concentrations. The results showed that the factorial DoE method was confirmed by the non-normality of the residuals and the outliers of the experiment. When examining the effects of the variables, analyzing the half-normal distribution and the effects and contrasts for GOx-Fc/MWCNT, the factors that showed the greatest influence on the electrochemical response were GOx, MWCNT, Fc, and MWCNT:Fc, and there is a high correlation between the factors GOx, MWCNT, Fc, and MWCNT:Fc, as shown by the analysis of homoscedasticity and multicollinearity. With these statistical analyses and experimental designs, it was possible to find the optimal conditions for different factors: 10 mM mL-1 GOx, 2 mg mL-1 Fc, and 15 mg mL-1 MWCNT show a greater amperometric response in the glucose oxidation. This work contributes to advancing enzyme immobilization strategies for glucose biosensor applications. Systematic investigation of DoE leads to optimized immobilization for GOx, enables better performance as a glucose biosensor, and allows the prediction of some outcomes.

Metal-ligand cross-link strategy engineered iron-doped dopamine-based superstructure as peroxidase-like nanozymes for detection of glucose.

Nanozymes are nanomaterials with mimetic enzyme properties and the related research has attracted much attention. It is of great value to develop methods to construct nanozymes and to study their application in bioanalysis. Herein, the metal-ligand cross-linking strategy was developed to fabricate superstructure nanozymes. This strategy takes advantage of being easy to operate, adjustable, cheap, and universal. The fabricated superstructure nanozymes possess efficient peroxidase-like catalytic activity. The enzyme reaction kinetic tests demonstrated that for TMB and H2O2, the Km is 0.229 and 1.308 mM, respectively. Furthermore, these superstructure nanozymes are applied to highly efficient and sensitive detection of glucose. The linear range for detecting glucose is 20-2000 µM, and the limit of detection is 17.5 µM. Furthermore, mechanistic research illustrated that this integrated system oxidizes glucose to produce hydrogen peroxide and further catalyzes the production of ·OH and O2·-, which results in a chromogenic reaction of oxidized TMB for the detection of glucose. This work could not only contribute to the development of efficient nanozymes but also inspire research in the highly sensitive detection of other biomarkers.

Fenton-like system of UV/Glucose-oxidase@Kaolin coupled with organic green rust: UV-enhanced enzyme activity and the mechanism of UV synergistic degradation of photosensitive pollutants.

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 H2O2, 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 •O2-, 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.

Improving glucose oxidase catalysis in Aspergillus niger via Vitreoscilla hemoglobin fusion protein.

Oxygen is crucial for converting glucose to gluconic acid catalyzed by glucose oxidase (Gox). However, industrial gluconic acid production faces oxygen supply limitations. To enhance Gox efficiency, Vitreoscilla hemoglobin (VHb) has been considered as an efficient oxygen transfer carrier. This study identified GoxA, a specific isoform of Gox in the industrial gluconic acid-producing strain of Aspergillus niger. Various forms of VHb expression in A. niger were tested to improve GoxA's catalytic efficiency. Surprisingly, the expression of free VHb, both intracellularly and extracellularly, did not promote gluconic acid production during shake flask fermentation. Then, five fusion proteins were constructed by linking Gox and VHb using various methods. Among these, VHb-GS1-GoxA, where VHb's C-terminus connected to GoxA's N-terminus via the flexible linker GS1, demonstrated a significantly higher Kcat/Km value (96% higher) than GoxA. Unfortunately, the expression of VHb-GS1-GoxA in A. niger was limited, resulting in a low gluconic acid production of 3.0 g/L. To overcome the low expression problem, single- and dual-strain systems were designed with tools of SpyCatcher/SpyTag and SnoopCatcher/SnoopTag. In these systems, Gox and VHb were separately expressed and then self-assembled into complex proteins. Impressively, the single-strain system outperformed the GoxA overexpression strain S1971, resulting in 23% and 9% higher gluconic acid production under 0.6 vvm and 1.2 vvm aeration conditions in the bioreactor fermentation, respectively. The successful construction of Gox and VHb fusion or complex proteins, as proposed in this study, presents promising approaches to enhance Gox catalytic efficiency and lower aerodynamic costs in gluconic acid production. KEY POINTS: • Overexpressing free VHb in A. niger did not improve the catalytic efficiency of Gox • The VHb-GS1-GoxA showed an increased Kcat/Km value by 96% than GoxA • The single-strain system worked better in the gluconic acid bioreactor fermentation.

Blocking the utilization of carbon sources via two pathways to induce tumor starvation for cancer treatment.

Glucose oxidase (GOx) is often used to starvation therapy. However, only consuming glucose cannot completely block the energy metabolism of tumor cells. Lactate can support tumor cell survival in the absence of glucose. Here, we constructed a nanoplatform (Met@HMnO2-GOx/HA) that can deplete glucose while inhibiting the compensatory use of lactate by cells to enhance the effect of tumor starvation therapy. GOx can catalyze glucose into gluconic acid and H2O2, and then HMnO2 catalyzes H2O2 into O2 to compensate for the oxygen consumed by GOx, allowing the reaction to proceed sustainably. Furthermore, metformin (Met) can inhibit the conversion of lactate to pyruvate in a redox-dependent manner and reduce the utilization of lactate by tumor cells. Met@HMnO2-GOx/HA nanoparticles maximize the efficacy of tumor starvation therapy by simultaneously inhibiting cellular utilization of two carbon sources. Therefore, this platform is expected to provide new strategies for tumor treatment.

Multi-walled carbon nanotubes functionalized with a new Schiff base containing phenylboronic acid residues: application to the development of a bienzymatic glucose biosensor using a response surface methodology approach.

An innovative supramolecular architecture is reported for bienzymatic glucose biosensing based on the use of a nanohybrid made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with a Schiff base modified with two phenylboronic acid residues (SB-dBA) as platform for the site-specific immobilization of the glycoproteins glucose oxidase (GOx) and horseradish peroxidase (HRP). The analytical signal was obtained from amperometric experiments at - 0.050 V in the presence of 5.0 × 10-4 M hydroquinone as redox mediator. The concentration of GOx and HRP and the interaction time between the enzymes and the nanohybrid MWCNT-SB-dBA deposited at glassy carbon electrodes (GCEs) were optimized through a central composite design (CCD)/response surface methodology (RSM). The optimal concentrations of GOx and HRP were 3.0 mg mL-1 and 1.50 mg mL-1, respectively, while the optimum interaction time was 3.0 min. The bienzymatic biosensor presented a sensitivity of (24 ± 2) × 102 µA dL mg-1 ((44 ± 4) × 102 µA M-1), a linear range between 0.06 mg dL-1 and 21.6 mg dL-1 (3.1 µM-1.2 mM) (R2 = 0.9991), and detection and quantification limits of 0.02 mg dL-1 (1.0 µM) and 0.06 mg dL-1 (3.1 µM), respectively. The reproducibility for five sensors prepared with the same MWCNT-SB-dBA nanohybrid was 6.3%, while the reproducibility for sensors prepared with five different nanohybrids and five electrodes each was 7.9%. The GCE/MWCNT-SB-dBA/GOx-HRP was successfully used for the quantification of glucose in artificial human urine and commercial human serum samples.

A catalytic membrane approach as a way to obtain sweet and unsweet lactose-free milk.

The growing need in the current market for innovative solutions to obtain lactose-free (L-F) milk is caused by the annual increase in the prevalence of lactose intolerance inside as well as the newborn, children, and adults. Various configurations of enzymes can yield two distinct L-F products: sweet (ß-galactosidase) and unsweet (ß-galactosidase and glucose oxidase) L-F milk. In addition, the reduction of sweetness through glucose decomposition should be performed in a one-pot mode with catalase to eliminate product inhibition caused by H2O2. Both L-F products enjoy popularity among a rapidly expanding group of consumers. Although enzyme immobilization techniques are well known in industrial processes, new carriers and economic strategies are still being searched. Polymeric carriers, due to the variety of functional groups and non-toxicity, are attractive propositions for individual and co-immobilization of food enzymes. In the presented work, two strategies (with free and immobilized enzymes; ß-galactosidase NOLA, glucose oxidase from Aspergillus niger, and catalase from Serratia sp.) for obtaining sweet and unsweet L-F milk under low-temperature conditions were proposed. For free enzymes, achieving the critical assumption, lactose hydrolysis and glucose decomposition occurred after 1 and 4.3 h, respectively. The tested catalytic membranes were created on regenerated cellulose and polyamide. In both cases, the time required for lactose and glucose bioconversion was extended compared to free enzymes. However, these preparations could be reused for up to five (ß-galactosidase) and ten cycles (glucose oxidase with catalase).

A Small Highly Sensitive Glucose Sensor Based on a Glucose Oxidase-Modified U-Shaped Microfiber.

Diabetes patients need to monitor blood glucose all year round. In this article, a novel scheme is proposed for blood glucose detection. The proposed sensor is based on a U-shaped microfiber prepared using hydrogen-oxygen flame-heating technology, and then 3-aminopropyltriethoxysilane (APTES) and glucose oxidase (GOD) are successively coated on the surface of the U-shaped microfiber via a coating technique. The glucose reacts with the GOD of the sensor surface to produce gluconic acid, which changes the effective refractive index and then shifts the interference wavelength. The structure and morphology of the sensor were characterized via scanning electron microscope (SEM) and confocal laser microscopy (CLM). The experimental results show that the sensitivity of the sensor is as high as 5.73 nm/(mg/mL). Compared with the glucose sensor composed of the same material, the sensitivity of the sensor increased by 329%. The proposed sensor has a broad application prospect in blood glucose detection of diabetic patients due to the advantages of miniaturization, high sensitivity, and good stability.

Glucose Oxidase Driven Hydrogen Sulfide-Releasing Nanocascade for Diabetic Infection Treatment.

Diabetic ulcers have received much attention in recent years due to their high incidence and mortality, motivating the scientific community to develop various strategies for such chronic disease treatments. However, the therapeutic outcome of these approaches is highly compromised by invasive bacteria and a severe inflammatory microenvironment. To overcome these dilemmas, microenvironment-responsive self-delivery glucose oxidase@manganese sulfide (GOx@MnS) nanoparticles (NPs) are developed by one-step biomineralization. When they encounter the high glucose level in the ulcer site, GOx particles catalyze glucose to decrease the local pH and trigger the steady release of both manganese ions (Mn2+) and hydrogen sulfide (H2S). Mn2+ reacts with hydrogen peroxide to generate hydroxyl radicals for the elimination of bacterial infection; meanwhile, H2S is able to suppress the inflammatory response and accelerate diabetic wound healing through macrophage polarization. The excellent biocompatibility, strong bactericidal activity, and considerable immunomodulatory effect promise GOx@MnS NPs have great therapeutic potential for diabetic wound treatment.

Molecular Photoswitching Unlocks Glucose Oxidase for Synergistically Reinforcing Fenton Reactions for Antitumor Chemodynamic Therapy.

We have developed a new type of nanoparticles with potent antitumor activity photoactivatable via the combination of molecular photoswitching of spiropyran (SP) and enzymatic reaction of glucose oxidase (GOx). As two key processes involved therein, Fe(III)-to-Fe(II) photoreduction in Fe(III) metal-organic frameworks (MOFs) brings about the release of free Fe2+/Fe3+ while the photoswitching of SP to merocyanine (MC) unlocks the enzymatic activity of GOx that was pre-passivated by SP. The release of free Fe3+ boosts its hydrolysis and therefore enables the acidification of microenvironment, which is further reinforced by one of the products of the GOx-mediated glucose oxidation reaction, gluconic acid (GlcA). Based on the generation of Fe2+ and acidic milieu together with another product of the oxidation reaction, hydrogen peroxide (H2O2), these two processes jointly present triple enabling factors for generating lethal hydroxyl radicals (•OH) species via Fenton reactions and therefore oxidative stress capable of inhibiting tumor. The antitumor potency of such nanoparticle is verified in tumor-bearing model mice in vivo, proclaiming its potential as a potent and safe agent based on the unique mechanism of optically manipulating enzyme activity for synergistic antitumor therapeutics with high spatial precision, enhanced efficacy and minimized side effects.

Engineered Glucose Oxidase-Carbon Nanotube Conjugates for Tissue-Translatable Glucose Nanosensors.

Continuous and non-invasive glucose monitoring and imaging is important for disease diagnosis, treatment, and management. However, glucose monitoring remains a technical challenge owing to the dearth of tissue-transparent glucose sensors. In this study, we present the development of near-infrared fluorescent single-walled carbon nanotube (SWCNT) based nanosensors directly functionalized with glucose oxidase (GOx) capable of immediate and reversible glucose imaging in biological fluids and tissues. We prepared GOx-SWCNT nanosensors by facile sonication of SWCNT with GOx in a manner that-surprisingly-does not compromise the ability of GOx to detect glucose. Importantly, we find by using denatured GOx that the fluorescence modulation of GOx-SWCNT is not associated with the catalytic oxidation of glucose but rather triggered by glucose-GOx binding. Leveraging the unique response mechanism of GOx-SWCNT nanosensors, we developed catalytically inactive apo-GOx-SWCNT that enables both sensitive and reversible glucose imaging, exhibiting a ΔF/F0 of up to 40 % within 1 s of exposure to glucose without consuming the glucose analyte. We finally demonstrate the potential applicability of apo-GOx-SWCNT in biomedical applications by glucose quantification in human plasma and glucose imaging in mouse brain slices.

Acidity-Responsive Nanoreactors Destructed "Warburg Effect" for Toxic-Acidosis and Starvation Synergistic Therapy.

"Warburg Effect" shows that most tumor cells rely on aerobic glycolysis for energy supply, leading to malignant energy deprivation and an "internal alkaline external acid" tumor microenvironment. Destructing the "Warburg Effect" is an effective approach to inhibit tumor progression. Herein, an acidity-responsive nanoreactor (Au@CaP-Flu@HA) is fabricated for toxic acidosis and starvation synergistic therapy. In the nanoreactor, the fluvastatin (Flu) could reduce lactate efflux by inhibiting the lactate-proton transporter (monocarboxylate transporters, MCT4), resulting in intracellular lactate accumulation. Meanwhile, the glucose oxidase-mimic Au-nanocomposite consumes glucose to induce cell starvation accompanied by gluconic acid production, coupling with lactate to exacerbate toxic acidosis. Also, the up-regulated autophagic energy supply of tumor cells under energy deprivation and hypoxia aggravation is blocked by autophagy inhibitor CaP. Cellular dysfunction under pHi acidification and impaired Adenosine Triphosphate (ATP) synthesis under starvation synergistically promote tumor cell apoptosis. Both in vitro and in vivo studies demonstrate that this combinational approach of toxic-acidosis/starvation therapy could effectively destruct the "Warburg Effect" to inhibit tumor growth and anti-metastatic effects.

A High-Performance Hybrid Biofuel Cell with a Honeycomb-Like Ti3 C2 Tx /MWCNT/AuNP Bioanode and a ZnCo2 @NCNT Cathode for Self-Powered Biosensing.

This work focusses on developing a hybrid enzyme biofuel cell-based self-powered biosensor with appreciable stability and durability using murine leukemia fusion gene fragments (tDNA) as a model analyte. The cell consists of a Ti3 C2 Tx /multiwalled carbon nanotube/gold nanoparticle/glucose oxidase bioanode and a Zn/Co-modified carbon nanotube cathode. The bioanode uniquely exhibits strong electron transfer ability and a high surface area for the loading of 1.14 × 10-9  mol cm-2 glucose oxidase to catalyze glucose oxidation. Meanwhile, the abiotic cathode with a high oxygen reduction reaction activity negates the use of conventional bioenzymes as catalysts, which aids in extending the stability and durability of the sensing system. The biosensor offers a 0.1 fm-1 nm linear range and a detection limit of 0.022 fm tDNA. Additionally, the biosensor demonstrates a reproducibility of ≈4.85% and retains ≈87.42% of the initial maximal power density after a 4-week storage at 4 °C, verifying a significantly improved long-term stability.

Review of glucose oxidase as a feed additive: production, engineering, applications, growth-promoting mechanisms, and outlook.

The regulation and prohibition of antibiotics used as growth promoters (AGP) in the feed field are increasing because they cause antimicrobial resistance and drug residue issues and threaten community health. Recently, glucose oxidase (GOx) has attracted increasing interest in the feed industry as an alternative to antibiotics. GOx specifically catalyzes the production of gluconic acid (GA) and hydrogen peroxide (H2O2) by consuming molecular oxygen, and plays an important role in relieving oxidative stress, preserving health, and promoting animal growth. To expand the application of GOx in the feed field, considerable efforts have been made to mine new genetic resources. Efforts have also been made to heterologously overexpress relevant genes to reduce production costs and to engineer proteins by modifying enzyme properties, both of which are bottleneck problems that limit industrial feed applications. Herein, the: different sources, diverse biochemical properties, distinct structural features, and various strategies of GOx engineering and heterologous overexpression are summarized. The mechanism through which GOx promotes growth in animal production, including the improvement of antioxidant capacity, maintenance of intestinal microbiota homeostasis, and enhancement of gut function, are also systematically addressed. Finally, a new perspective is provided for the future development of GOx applications in the feed field.