Enzyme Activity Of Glucose Oxidase Research Articles

Evaluation of vat blue removal by Aspergillus niger using scanning electron microscopy and glucose oxidase enzyme activity

AbstractThe use of fungi to reduce dye removal in water from diverse industries can be an efficient option for solving environmental problems. However, the mycoremediation processes involved in color removal are unknown. In this article, Aspergillus niger CDBB‐H‐175 was applied for vat blue discoloration (DC). Hence, inactive biomass by two methods (metabolic inhibitor and heat) and active biomass were studied by scanning electron microscopy and enzyme activities to determine the vat blue DC. When using active biomass, 62% of DC, glucose oxidase (GOX) enzyme activity, 1.19 U/mL and 1.43 mg/L of H2O2 were obtained. It was observed that 68% of DC was due to the degradation process and 32% was due to biomass sorption. When the biomass was inactivated with sodium azide, DC increased by 90% (1.45‐fold), low GOX activity (0.138 U/mL), and H2O2 (0.25 mg/L) were detected. High DC might be due to a high dye sorption in hyphae, since an increase in the thickness and morphology of the hyphae were observed by SEM. Inactivated biomass through heating, showed a 69.5% of DC, where 0.01 U/mL GOX and 0.25 mg/L H2O2 were reached, demonstrating that inactivation of A. niger biomass by sodium azide can be considered as an alternative treatment for colored wastewater.

Metallic Copper-Based Dual-Enzyme Biomimetic Nanoplatform for Mild Photothermal Enhancement of Anticancer Catalytic Activity.

Background: Chemodynamic therapy (CDT) is recognized as a promising cancer treatment. Recently, copper sulfide nanostructures have been extensively employed as Fenton-like reagents that catalyze the formation of acutely toxic hydroxyl radicals (·OH) from hydrogen peroxide (H2O2). However, CDT therapeutic potency is restricted by the tumor microenvironment (TME), such as insufficient amounts of hydrogen peroxide, excessive glutathione levels, etc. To address these disadvantages, glucose oxidase (GOx) or catalase (CAT) can be utilized to enhance CDT, while low therapeutic efficacy still inhibits their future applications. Our previous study revealed that mild photothermal effect could boost the CDT catalytic effectiveness as well as GOx enzyme activity over a range. Results: We engineered and constructed a hollow CuS nanoplatform loaded with GOx and CAT, coating with macrophage membranes (M@GOx-CAT@CuS NPs). The nanoplatforms allowed enhancement of the reactive oxygen species creation rate and GOx catalytic activeness of CDT through mild phototherapy directed by photoacoustic imaging. After actively targeting vascular cell adhesion molecule-1 (VCAM-1) in cancer cells mediated by macrophage membrane coating, M@GOx-CAT@CuS NPs released GOx and CAT under near-infrared irradiation. GOx catalyzed the formation of H2O2 and gluconic acid with glucose, creating a better catalytic environment for CDT. Meanwhile, CAT-catalyzed H2O2 decomposition to generate sufficient oxygen, appropriately alleviating the oxygen shortage in the TME. In addition, starvation effects decreased adenosine triphosphate levels and further underregulated heat shock protein expression to reduce the heat resistance of tumor cells, resulting in a better mild phototherapy outcome. Both invitro and invivo experiments demonstrated that the newly developed M@GOx-CAT@CuS nanoplatform has remarkable synergistic anticancer therapeutic effects. Conclusion: The cascade reaction-enhanced biomimetic nanoplatform opens up a new avenue for precision tumor diagnostic and therapeutic research.

Glucose oxidase virus-based nanoreactors for smart breast cancer therapy.

Breast cancer is the most common malignant tumor disease and the leading cause of female mortality. The evolution of nanomaterials science opens the opportunity to improve traditional cancer therapies, enhancing therapy efficiency and reducing side effects. Herein, protein cages conceived as enzymatic nanoreactors were designed and produced by using virus-like nanoparticles (VLPs) from Brome mosaic virus (BMV) and containing the catalytic activity of glucose oxidase (GOx) enzyme. The GOx enzyme was encapsulated into the BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The effect of the synthesized GOx nanoreactors on breast tumor cell lines was studied in vitro. Both nanoreactor preparations VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell cultures. Cytotoxicity for human embryonic kidney cells was also found. The monitoring of nanoreactor treatment on triple-negative breast cancer cells showed an evident production of oxygen by the catalase antioxidant enzyme induced by the high production of hydrogen peroxide from GOx activity. The nanoreactors containing GOx activity are entirely suitable for cytotoxicity generation in tumor cells. The HSA functionalization of the VLP-GOx nanoreactors, a strategy designed for selective cancer targeting, showed no improvement in the cytotoxic effect. The GOx containing enzymatic nanoreactors seems to be an interesting alternative to improve the current cancer therapy. In vivo studies are ongoing to reinforce the effectiveness of this treatment strategy.

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

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

Antineoplastic Enzyme as Drug Carrier with Activatable Catalytic Activity for Efficient Combined Therapy.

The delivery of glucose oxidase (GOx) requires extra carriers, which suffers from early leakage and exposure of GOx. These issues will be of less concern if GOx itself acts as a drug carrier. However, the catalytic activity of GOx in the blood still needs to be inhibited. Herein, we found that GOx could self-assemble with hydrophobic molecules into uniform and stable nanoparticles (NPs), including sorafenib, 4'-(amino-methyl phenyl)-2,2' : 6',2''-terpyridine modified cyanin and paclitaxel. The catalytic activity of GOx in NPs was significantly inhibited due to the binding of small molecules with its hydrophobic pockets. After dissociation in the tumor acidic microenvironment, the enzyme activity of GOx could be largely recovered. This acidity-triggered "OFF-to-ON" process ensured safe intravenous administration of GOx-based NPs. In vivo experiments showed that the combined starvation therapy and ferroptosis/photothermal therapy/chemotherapy effectively inhibited 4T1 breast tumor growth.

Antineoplastic Enzyme as Drug Carrier with Activatable Catalytic Activity for Efficient Combined Therapy

AbstractThe delivery of glucose oxidase (GOx) requires extra carriers, which suffers from early leakage and exposure of GOx. These issues will be of less concern if GOx itself acts as a drug carrier. However, the catalytic activity of GOx in the blood still needs to be inhibited. Herein, we found that GOx could self‐assemble with hydrophobic molecules into uniform and stable nanoparticles (NPs), including sorafenib, 4′‐(amino‐methyl phenyl)‐2,2′ : 6′,2′′‐terpyridine modified cyanin and paclitaxel. The catalytic activity of GOx in NPs was significantly inhibited due to the binding of small molecules with its hydrophobic pockets. After dissociation in the tumor acidic microenvironment, the enzyme activity of GOx could be largely recovered. This acidity‐triggered “OFF‐to‐ON” process ensured safe intravenous administration of GOx‐based NPs. In vivo experiments showed that the combined starvation therapy and ferroptosis/photothermal therapy/chemotherapy effectively inhibited 4T1 breast tumor growth.

Engineered Organosilica Hybrid Micelles for Photothermal-enhanced Starvation Cancer Therapy.

Glucose oxidase (GOD)-based starvation therapy (ST), which inhibits the growth and proliferation of cancer cells by consuming glucose, has attracted intensive attention as an emerging non-invasive method for fighting cancers. However, the enzyme activity of GOD is greatly limited in vivo because of its optimal catalytic activity in the temperature range of 43-60 °C. Herein, a photothermal-enhanced starvation strategy is developed based on our engineered organosilica hybrid micelles (TiO2-x @POMs-GOD), in which the fluoride-doped TiO2-x with photothermal properties is encapsulated in the cores of organosilica cross-linked micelles and GOD is immobilized on the carboxyl groups of PAA segments. With its internalization by cancer cells, the conjugated GOD can effectively deplete glucose to achieve the ST effect, which can be remarkably enhanced by the loaded fluoride-doped TiO2-x with NIR laser irradiation, thus cooperatively contributing to the efficient treatment of TiO2-x @POMs-GOD on various cancer cells. This suggests great potential for TiO2-x @POMs-GOD in photothermal-enhanced ST in vivo.

In situ Forming Hyperbranched PEG-Thiolated Hyaluronic Acid Hydrogels With Honey-Mimetic Antibacterial Properties.

The rapidly increasing resistance of bacteria to currently approved antibiotic drugs makes surgical interventions and the treatment of bacterial infections increasingly difficult. In recent years, complementary strategies to classical antibiotic therapy have, therefore, gained importance. One of these strategies is the use of medicinal honey in the treatment of bacterially colonized wounds. One of the several bactericidal effects of honey is based on the in situ generation of hydrogen peroxide through the activity of the enzyme glucose oxidase. The strategy underlying this work is to mimic this antibacterial redox effect of honey in an injectable, biocompatible, and rapidly forming hydrogel. The hydrogel was obtained by thiol–ene click reaction between hyperbranched polyethylene glycol diacrylate (HB PEGDA), synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization, and thiolated hyaluronic acid (HA-SH). After mixing 500 µL HB PEGDA (10%, w/w) and 500 µL HA-SH (1%, w/w) solutions, hydrogels formed in ∼60 s (HB PEGDA/HA-SH 10.0–1.0), as assessed by the tube inverting test. The HB PEGDA/HA-SH 10.0–1.0 hydrogel (200 µL) was resistant to in vitro dissolution in water for at least 64 days, absorbing up to 130 wt% of water. Varying glucose oxidase (GO) amounts (0–500 U/L) and constant glucose content (2.5 wt%) were loaded into HB PEGDA and HA-SH solutions, respectively, before hydrogel formation. Then, the release of H2O2 was evaluated through a colorimetric pertitanic acid assay. The GO content of 250 U/L was selected, allowing the formation of 10.8 ± 1.4 mmol H2O2/L hydrogel in 24 h, under static conditions. The cytocompatibility of HB PEGDA/HA-SH 10.0–1.0 hydrogels loaded with different GO activities (≤ 500 U/L) at a constant glucose amount (2.5 wt%) was investigated by in vitro assays at 24 h with L929 and HaCaT cell lines, according to DIN EN ISO 10993-5. The tests showed cytocompatibility for GO enzyme activity up to 250 U/L for both cell lines. The antibacterial activity of HB PEGDA/HA-SH 10.0–1.0 hydrogels loaded with increasing amounts of GO was demonstrated against various gram-positive bacteria (S. aureus and S. epidermidis), antibiotic-resistant gram-positive bacteria (MRSA and MRSE), gram-negative bacteria (P. aeruginosa, E. coli, and A. baumanii), and antibiotic-resistant gram-negative strains (P. aeruginosa and E. coli) using agar diffusion tests. For all gram-positive bacterial strains, increasing efficacy was measured with increasing GO activity. For the two P. aeruginosa strains, efficacy was shown only from an enzyme activity of 125 U/L and for E. coli and A. baumanii, efficacy was shown only from 250 U/L enzyme activity. HB PEGDA/HA-SH 10.0–1.0 hydrogels loaded with ≤250 U/L GO and 2.5 wt% glucose are promising formulations due to their fast-forming properties, cytocompatibility, and ability to produce antibacterial H2O2, warranting future investigations for bacterial infection treatment, such as wound care.

Photo-controlled liquid metal nanoparticle-enzyme for starvation/photothermal therapy of tumor by win-win cooperation

Here, a highly cooperative liquid metal nanoparticle-enzyme (LM@GOX) was constructed for combinational starvation/photothermal therapy of tumor. It was found that the enzyme activity of glucose oxidase (GOX) could be strengthened along with the increased temperature within a given range and its optimal activity is around about 43–60 °C. Utilizing the photothermal conversion ability of liquid metal (LM), the GOX catalytic efficiency could be photo-controlled with improved starvation therapeutic efficiency. Furthermore, due to the accelerating blood flow during the photothermal therapy (PTT), the hypoxic situation in tumor tissues could also be relieved, which would contribute to conquering the hypoxia-suppressed GOX catalysis. In the meanwhile, the severe thermo-resistance of tumor cells during PTT process could be overcome by GOX induced decrease of adenosine triphosphate (ATP) and heat shock proteins (HSPs) level, eventually leading to an improved therapeutic effect of PTT. Both in vitro and in vivo studies proved that LM@GOX could significantly inhibit the growth of solid tumor under NIR illumination by a win-win cooperative starvation/photothermal therapy.

Layer-By-Layer Assembly of Enzymes and Nanoparticles onto Cellulose Support

Multilayered films of cellulose nanoparticles (NFC’s) and modified multi-walled carbon nanotubes (MWCNT’s) were assembled by means of alternate electrostatic adsorption with positively charged poly(ethyleneimine) (PEI) onto cellulose support. The free carboxylic groups of NFC’s and MWCNT’s were coupled with ethylenediamine. Glucose oxidase and laccase were immobilized by means of Schiff base reaction between aldehyde groups of glutaraldehyde and the free amino sites of the proteins. The immobilized enzymes on the surface of nanoparticles have higher value for the specific activity compared to the enzymes immobilized directly on the cellulose surface indicating the stabilization of the proteins by the nanoparticles. The kinetics of the enzymes, catalyzed reactions and reusability of the enzymes were investigated and were showing better properties for enzymes cross-linked with glutaraldehyde. After 5 days the initial enzyme activity of glucose oxidase was around 85%, but the initial enzyme activity of laccase was 60%. The kinetic investigations of the immobilized enzymes showed no significant difference in Michaelis constant but the maximum reaction rate is decreased.

Ultra-Sensitive Nano Optical Sensor Samarium-Doxycycline Doped in Sol Gel Matrix for Assessment of Glucose Oxidase Activity in Diabetics Disease

A low cost and very sensitive method for the determination of the activity of glucose oxidase enzyme in different diabetics serum samples was developed. The method based on the assessment of the H2O2 concentration produced from the reaction of the glucose oxidase (GOx) enzyme with glucose as substrate in the serum of diabetics patients by nano optical sensor Sm-doxycycline doped in sol gel matrix. H2O2 enhances the luminescence intensity of all bands of the nano Sm-doxycycline complex [Sm-(DC)2]+ doped in sol-gel matrix, especially the 645nm band at λex=400nm and pH7.0 in water. The influence of the different analytical parameters that affect the luminescence intensity of the nano optical sensor, e.g. pH, H2O2 concentration and foreign ions concentrations were studied. The remarkable enhancement of the luminescence intensity of nano optical sensor [Sm-(DC)2]+ complex in water at 645nm by the addition of various concentrations of H2O2 was successfully used as an optical sensor for the assessment of the activity of the glucose oxidase enzyme in different diabetics serum samples. The calibration plot was achieved over the activity range 0.1-240U/L with a correlation coefficient of 0.999 and a detection limit of 0.05U/L.

Tailor-made resealable micro(bio)reactors providing easy integration of in situ sensors

Flow microreactors utilizing immobilized enzymes are of great interest in biocatalysis development. Most of the common devices are permanently closed, single-use systems, which allow limited physical and chemical surface modifications and evaluation methods. In this paper we will present resealable flowcells that overcome these limitations and moreover allow a quick and easy integration of sensor systems, because of the use of modular building blocks. The devices were utilized to study the enzyme activity of glucose oxidase immobilized on chemically modified glass surfaces under flow conditions, employing integrated optical oxygen sensors for on-line monitoring.

Study of direct electron transfer and enzyme activity of glucose oxidase on graphene surface

In recent years, graphene has been widely used as a high performance two-dimensional material in the development of biosensors and biofuel cells for facilitating direct electron transfer (DET) of glucose oxidase (GOx). However, almost all of these reports perform experiments in the presence of oxygen (a natural mediator of oxidase) and whether the GOx with DET property retained their catalytic activity in the absence of mediators has not been studied in detail so far. In this paper, we investigated the DET property and enzyme activity of GOx on graphene surface without and with mediators. Experimental results showed that the biosensor had no response to glucose in mediator-free solutions, even though the DET of GOx was observed, indicating that the GOx with DET property lacked enzymatically catalytic activity. However, in the presence of mediators, the biosensor showed sensitive response to glucose, illustrating that the mediated enzymatic oxidation of glucose occurred, which can be attributed to the catalytically active GOx without DET capability. These results suggest that DET property and enzyme catalytic activity cannot occur on the same GOx simultaneously. Therefore, keeping enzyme activity and DET of GOx at the same time is still a major challenge for biosensor and biofuel cell researches.

A droplet-based microfluidic electrochemical sensor using platinum-black microelectrode and its application in high sensitive glucose sensing

We describe a droplet-based microfluidic electrochemical sensor using platinum-black (Pt-black) microelectrode. Pt-black microelectrode was generated by electrodeposition of Pt nanoparticles on bare Pt microelectrode. Scanning electron microscope (SEM) image displays a flower-like microstructure of Pt nanoparticels. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) indicate that the Pt-black efficiently decreased the charge transfer resistance and improved the electrocatalytic activity towards oxidation of hydrogen peroxide (H2O2). Compared with bare Pt microelectrode, the current response on Pt-black microelectrode increased 10.2 folds. The effect of applied potential and electrodeposition time has been investigated in detail. The proposed sensor was validated by performing enzyme activity assay in flowing droplets. For demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of β-d-glucose to the product H2O2. The enzyme activity of GOx was evaluated by measuring the electrochemical current responding to various glucose concentrations. And the results indicate that this microfluidic sensor holds great potential in fabricating novel glucose sensors with linear response up to 43.5mM. The analytical applications of the droplet-based microfluidic sensor were tested by using human blood serum samples. Reproducibility, interferences, and long-term stability of the modified electrode were also investigated. The present approach shows the feasibility and great potentials in constructing highly sensitive and low-consumption sensors in the field of droplet microfluidics.

Convenient Partial Purification of Glucose Oxidase from <i>Aspergillus niger</i> A9 Fermentation Broth by Reversed Micelles

Aim of this work was to establish the optimum conditions for the extraction and recovery by cationic reversed micelles of glucose oxidase (GOX) from Aspergillus niger A9, The influence of pH, temperature, solvent/co-solvents ratio on the extraction was investigated by experiment, using the residual enzyme activity to evaluate the results. The best condition for GOX extraction were ensured using iso-octane as solvent and butanol and n-hexanol co-solvent at 76/18/6 volume ratio, pH 4.80, 200mM cetyl-trimethyl ammonium bromide (CTAB) as cationic surfactant, The enzyme activity of GOX is measured by DNS method (3,5-dinitro salicylic acid method). In the extraction process, ultrasonic oscillation was adopted to mix organic solvent and water, ultrasonic oscillation temperature is 45 °C. Protein activity recovery of GOX can reach 88.2% in extraction.

Surface modification of electrospun spherical activated carbon for a high-performance biosensor electrode

A glucose sensor electrode was prepared from electrospun spherical-type carbon materials. The glucose oxidase (GOD) enzyme was immobilized on a prepared electrode for efficient glucose sensing. The GOD immobilization was maximized by enlarged sites of carbon electrode and improved interfacial affinity between the carbon surface and the GOD achieved via physical activation and oxyfluorination, respectively. The specific surface area was enlarged significantly, by over 42 fold, through physical activation. In addition, the hydrophobic carbon surface was modified with hydrophilic functional groups by direct oxyfluorination for improved interfacial affinity between the carbon and GOD. Accordingly, the GOD immobilization improved significantly by approximately 9 fold. The glucose sensor was evaluated by amperometric measurements and cyclic voltammetry. The measured current increased with higher glucose concentrations based on the effects of the developed pore structure and the introduced hydrophilic functional groups. The enzymatic kinetics were also studied using the Lineweaver–Burk equation. The sensitivity of the glucose sensor was improved by approximately 3 fold with increased maximum current, whereas the GOD enzyme activity was diminished by efficient GOD immobilization. Conclusively, a high-performance glucose sensor was obtained using an electrospun spherical-type carbon material due to efficient GOD enzyme immobilization.

Characteristics of Enzyme-Based ZnO/Zn0.7Mg0.3O Heterojunction Field-Effect Transistor as Glucose Sensor

The characteristics of an enzyme-based ZnO/Zn0.7Mg0.3O heterojunction field-effect transistor (HFET) for glucose sensing are reported. The enzyme glucose oxidase (GOD) was immobilized on an amine-modified gate electrode of the ZnO/Zn0.7Mg0.3O HFET, and glucose sensing was performed by detecting biocatalytically yielded protons using an ion-sensitive function of the gate electrode. The chemical bonding states of the amine-modified and GOD-immobilized surfaces were analyzed by X-ray photoemission spectroscopy, and the enzyme activity of GOD was examined by a colorimetric method. In agreement with the promising results of these experiments, this enzyme-based HFET exhibited stable sensing performance with a linear response in a wide range of glucose concentrations from 0 to 4 mg·cm-3 and at a short time constant less than 20 s.

Polyion Complex Nanocomposite Electrode Incorporating Enzyme and Carbon Nanotube for Biofuel Cells

A nanocomposite electrode of enzyme, mediator, and carbon nanotube was fabricated for the application to an enzyme electrode in biofuel cells. The nanocomposite for the anode consisted of polyion complex matrix where glucose oxidase, tetrathiafulvalene as a mediator, and carbon nanotube as an electron transport enhancer were immobilized on the glassy carbon electrode. From electrochemical measurements, the enzyme electrode possessed an ability of electro-oxidizing glucose by utilizing the enzyme activity of glucose oxidase via the redox of tetrathiafulvalene in a buffer solution (pH=7.0) at 37°C. This nanocomposite anode demonstrated a higher current density of 500 µA cm−2 in a glucose solution at 250 mV vs. Ag/AgCl for the application of glucose/oxygen biofuel cells.

Glucoseoxidase from Aspergillus niger in reverse micelles: pH and wo dependence

The well known enzymatic method for the determination of glucose in aqueous solution with the system glucose oxidase/peroxidase/o-dianisidine was modified in a way that enables the quantitative determination of glucose in various reverse micellar systems. For instance in the 0.1 M sodium-bis-(2-ethylhexyl)-sulfosuccinate/n-octane system the useable glucose measuring range is between 10 and 70 micrograms glucose per 3 ml micellar solution. Investigations concerning the influence of the pH-value and the wo-value on the enzyme activity of glucose oxidase in the system 0.1 M sodium-bis-(2-ethylhexyl) sulfosuccinate/n-octane led to a shift of the pH-optimum from pH 5.5-6.0 in aqueous solution, to pH 4.0, in reverse micellar solution. The bell-shaped wo-dependence of the enzyme activity, typical for many other enzymes, was not found for glucose oxidase at pH 4.0, and was less clearly visible at the other investigated pH-values with an optimum at wo = 5.6.