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PubMed1991

Rotating disc electrode characterization of immobilized glucose oxidase.

The kinetic properties of glucose oxidase (EC 1.1.3.4) which has been covalently immobilized to a rotating glassy carbon electrode surface have been investigated. Analysis of the rotation rate dependence of the hydrogen peroxide-derived current suggests that oxygen mass transport to the enzyme-electrode surface is rate controlling at low rotation rates. Only as the diffusion layer approaches zero thickness (i.e., infinitely fast rotation rate) does mass transport become unimportant. A diffusion-free glucose Km for air-saturated buffer is found to be 66 mM using this methodology. The importance of mass transport restrictions in two-substrate enzymes such as glucose oxidase is discussed in the context of biosensor design.

Electrochemistry↗

PubMed1985

Enhanced detection of anti-tissue antibody by anti-immunoglobulin bound glucose oxidase.

An immunohistochemical procedure, using glucose oxidase (GO) linked to anti-immunoglobulin serum, has been adapted to detect anti-tissue antibody to smooth muscle, parietal cells, and mitochondria. With this procedure antibody to smooth muscle was detected in a high percentage of normal sera. Anti-tissue antibodies were frequently detected in sera of patients clinically suspected of having an autoimmune disease when an immunofluorescence (IF) test to detect the antibodies was negative. The glucose oxidase immunohistochemical procedure to detect anti-tissue antibodies is feasible and highly sensitive, and produces permanent results. The clinical usefulness of this procedure will have to be carefully evaluated. The detection of anti-tissue antibody in normal sera may indicate that such antibody is normally present in low concentration and that it may possibly have physiologic functions.

Adult↗

PubMed1994

The effect of the partial pressure of oxygen on blood glucose concentration examined using glucose oxidase with ferricyan ion.

Glucose oxidase with ferricyan ion (GOD-F) is widely applied in clinical settings as a glucose sensor. However, blood oxygen concentration affects this blood glucose value because oxygen, at increased concentrations, consumes blood glucose, which cannot then be measured by this sensor. We investigated the effect of PO2 on blood glucose concentration in 48 patients who were breathing high concentrations of oxygen. Arterial and pulmonary arterial blood glucose values were analyzed using the GOD-F method and, as a control, the hexokinase method. The respective PO2 values were also measured. The blood glucose concentrations measured by the GOD-F method show a significant linear relation with that measured by the hexokinase method in both arterial (y = -24.4 + 1.01x, r = 0.99) and pulmonary arterial blood (y = -3.4 + 1.01x, r = 0.96). The difference of intercepts is statistically significant, but because of the relatively large limits of agreement indicating any hidden extraneous variabilities, the error of the GOD-F method could not be assessed just by the difference. The equation defining the effect of PO2 on the percent change between blood glucose measured by the GOD-F method and that measured by the hexokinase method is -19.8/(1 + 203900/PO2(2.68) (r = 0.62). This formula generally follows our measured materials and introduces the relationship among blood glucose value, PO2, and the error of the GOD-F method. We hesitate to suggest that the arterial blood glucose concentration when measured by the GOD-F method could be underestimated by as much as 20% in patients with high arterial oxygen pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesiology↗

PubMed2004

The chemical mechanism of action of glucose oxidase from Aspergillus niger.

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of beta-D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V1/K(B), for reactions of these substrates were collected from pH 2.5-8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of beta-D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid. Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pKa of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pKa of His516 in the free reduced enzyme is 6.9.

Aspergillus niger↗

PubMed1982

The dissociation of glucose oxidase by sodium n-dodecyl sulphate.

1. The enzymic activity of glucose oxidase was determined as a function of pH and sodium n-dodecyl sulphate (SDS) concentration. 2. Glucose oxidase is not deactivated by SDS at pH 6 even after prolonged incubation, but is deactivated at pH 4.3 and 3.65. 3. Sedimentation-rate analysis showed that glucose oxidase dissociates into its two subunits at pH 5 and below, and sedimentation-equilibrium experiments in the presence of SDS gave a subunit molecular weight of 73,500. 4. SDS binds to glucose oxidase in acid solutions; specific binding occurs ap pH 3.65, but at pH 6 only co-operative binding was observed. 5. Glucose oxidases in which some of the carboxy groups were blocked with glycine methyl ester were deactivated by SDS at pH 6.0; the rate of deactivation increased with the extent of esterification. 6. Deactivation of esterified glucose oxidases correlated with thermal analysis of the initial SDS interaction, the exothermicity of the interaction increasing with the extent of esterification. 7. The results show that carboxy groups confer resistance to deactivation by SDS on glucose oxidase by screening cationic residues and inhibiting specific interactions that facilitate dissociation into subunits.

Amino Acids↗

PubMed1991

Amperometric glucose microelectrodes prepared through immobilization of glucose oxidase in redox hydrogels.

Glucose microelectrodes have been formed with glucose oxidase immobilized in poly[(vinylpyridine)Os(bipyridine)2Cl] derivative-based redox hydrogels on beveled carbon-fiber microdisk (7 microns diameter) electrodes. In the resulting microelectrode, the steady-state glucose electrooxidation current density is 0.3 mA cm-2 and the sensitivity is 20 mA cm-2 M-1. The current density and sensitivity are 10 times higher than in macroelectrodes made with the same hydrogel. Furthermore, the current is less affected by a change in the partial pressure of oxygen. The higher current density and lower oxygen sensitivity point to the efficient collection of electrons through their diffusion in the redox hydrogel to the electrode surface. These results contrast with those observed for enzyme electrodes based on diffusing mediators, where loss of the enzyme-reduced mediator by radial diffusion to the solution decreases the current densities of microelectrodes relative to similar macroelectrodes.

Enzymes, Immobilized↗

PubMed2000

One-step electropolymeric co-immobilization of glucose oxidase and heparin for amperometric biosensing of glucose.

Electropolymerization was used for the co-deposition of glucose oxidase and heparin onto metal electrode transducers. Such electropolymeric co-entrapment within a non-conducting poly(1,2-phenylenediamine) (PPD) film imparts both biocatalytic and anticoagulation activities onto the transducer, and greatly improves the performance of the sensor after exposure to whole blood. Essentially identical glucose signals are observed before and after exposure to blood samples. Scanning electron micrographs after such exposure reveal no platelet deposition or formation of a fibrin "clot". The effect of the heparin co-immobilization on the glucose response is examined. Improved biocompatibility is reported also in connection with a needle-type carbon paste biosensor configuration. The simultaneous localization of the enzyme and heparin offers great promise for simplifying the preparation of enzyme electrodes and designing biocompatible implantable glucose biosensors.

Biosensing Techniques↗

PubMed2002

Genetic modification of glucose oxidase for improving performance of an amperometric glucose biosensor.

Glucose oxidase (GOD) was genetically modified by adding a poly-lysine chain at the C-terminal with a peptide linker inserted between the enzyme and poly-lysine chain. The poly-lysine chain was added in order to anchor more electron transfer mediator, ferrocenecarboxylic acid, to GOD for the purpose of improving sensitivity and stability of glucose biosensors. The modified GOD had similar K(m) and K(cat) to those of the wild type enzyme. After interacted with the electron transfer mediator, the modified enzyme retained 90.01% of its native activity, while the commercial GOD and the wild type GOD (Aspergillus niger) retained only 22.43 and 22.17%, respectively. Screen-printed electrodes coated with the modified GOD, wild type yeast-derived GOD or the commercial GOD were tested in glucose solution of different concentrations. Experimental results showed that the biosensor based on the modified GOD gave the largest signal among the three. In addition, the linear range of the biosensor prepared by the modified GOD could extend to 45 mM, while they were about 20 mM for the biosensors based on the wild type yeast-derived enzyme and the commercial enzyme.

Biosensing Techniques↗

PubMed2006

Electrodeposition of polypyrrole-multiwalled carbon nanotube-glucose oxidase nanobiocomposite film for the detection of glucose.

A nanobiocomposite film consisted of polypyrrole (PPy), functionalized multiwalled carbon nanotubes (cMWNTs), and glucose oxidase (GOx) were electrochemically synthesized by electrooxidation of 0.1M pyrrole in aqueous solution containing appropriate amounts of cMWNTs and GOx. Potentiostatic growth profiles indicate that the anionic cMWNTs is incorporated within the growing PPy-cMWNTs nanocomposite for maintaining its electrical neutrality. The morphology of the PPy-cMWNTs nanocomposite was characterized by scanning electron microscopy (SEM). The PPy-cMWNTs nanocomposite was deposited homogeneously onto glassy carbon electrode. The amperometric responses vary proportionately to the concentration of hydrogen peroxide at the PPy-cMWNTs nanocomposite modified electrode at an operating potential of 0.7V versus Ag/AgCl (3M). The results indicate that the electroanalytical PPy-cMWNTs-GOx nanobiocomposite film was highly sensitive and suitable for glucose biosensor based on GOx function. The GOx concentration within the PPy-cMWNTs-GOx nanobiocomposite and the film thickness are crucial for the performance of the glucose biosensor. The amperometric responses of the optimized PPy-cMWNTs-GOx glucose biosensor (1.5 mgmL(-1) GOx, 141 mCcm(-2) total charge) displayed a sensitivity of 95 nAmM(-1), a linear range up to 4mM, and a response time of about 8s.

Biosensing Techniques↗

PubMed2002

Hydrogen peroxide generation with immobilized glucose oxidase for textile bleaching.

Glucose oxidase was covalently immobilized on commercially available alumina and glass supports, with a high level of protein recovery. The operational stability of the alumina carrier was an advantage over the glass support, though the rate of generation of hydrogen peroxide in the case of the latter was higher. The immobilization technique provided repeated application of the enzyme even in low concentration, and the hydrogen peroxide generated in the enzymatic reaction was successively used for textile bleaching.

Aluminum Oxide↗

PubMed1996

In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae.

Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pABGO-1, raised against purified glucose oxidase from T. flavus was highly specific for glucose oxidase. Only one protein band in culture filtrates (from glucose medium), migrating at 71 kDa, was detected in Western blots (immunoblots) with this antiserum. This band comigrated with purified glucose oxidase. No bands were detected in culture filtrates from the xylan medium. Glucose oxidase was removed via immunoprecipitation from culture filtrates of T. flavus grown in glucose medium, resulting in filtrates which no longer inhibited in vitro microsclerotial germination. When glucose oxidase-depleted filtrates were amended with purified glucose oxidase from T. flavus, the ability to kill microsclerotia in vitro was restored to original levels. We conclude that glucose oxidase is the only protein in culture filtrates of T. flavus responsible for inhibition of germination of microsclerotia of V. dahliae.

Animals↗

PubMed2005

Preparation of a very stable immobilized biocatalyst of glucose oxidase from Aspergillus niger.

Glucose oxidase (GOX) has been immobilized on different activated supports, including glyoxyl agarose, epoxy sepabeads and glutaraldehyde-activated supports. Immobilization onto supports pre-activated with glutaraldehyde rendered the most thermo-stable preparation of GOX. Therefore, as the glutaraldehyde chemistry gave a high stabilization of the enzyme, we proposed another technique for improving the multipoint attachment through glutaraldehyde: the enzyme was ionically adsorbed on cationic supports with primary amino groups and then the immobilized preparation was treated with a glutaraldehyde solution. The decrease on enzyme activity was <20%. Following this methodology, we achieved the highest stability of all the immobilization systems analyzed, showing a half-life 100 times higher than the soluble enzyme. Moreover, this derivative showed a higher stability in the presence of organic solvents (for instance methanol) or hydrogen epoxide than the ionically adsorbed enzyme or the soluble one. Therefore, the adsorption of GOX on aminated cationic support and subsequent treatment with glutaraldehyde was presented as a very successful methodology for achieving a very stable biocatalyst.

Aspergillus niger↗

PubMed2004

Preparation of a highly stable, very active and high-yield multilayered assembly of glucose oxidase using carbohydrate-specific polyclonal antibodies.

The purified oligosaccharide chains of Aspergillus niger glucose oxidase were coupled to BSA with the help of the cross-linking reagent glutaraldehyde. The neoglycoconjugate thus obtained was purified by concanavalin A-Sepharose chromatography and characterized by SDS/PAGE. Immunization of rabbits with the neoglycoprotein raised the glycosyl-specific anti-(glucose oxidase) polyclonal antibodies. Antibodies were purified by (NH(4))(2)SO(4) precipitation, followed by DEAE-cellulose chromatography. The IgG-Sepharose was prepared by covalently coupling the purified polyclonal antibodies to the CNBr-activated Sepharose 4B. The large assembly of glucose oxidase was made on the IgG-Sepharose by alternate incubation of glucose oxidase and glycosyl-specific anti-(glucose oxidase) polyclonal IgG. The immunoaffinity-layered assembled preparations were highly active and, after six alternate binding cycles with enzyme and glycosyl-specific IgG, the amount of enzyme immobilized could be raised 30-fold. The K (m) value of immunoaffinity-layered glucose oxidase preparations remained unaltered, while the V (max) slightly decreased as compared with the soluble enzyme. A layer-by-layer immobilization of glucose oxidase resulted in significant improvement in stability against high temperature, 4.0 M urea and very high concentrations of water-miscible organic solvents such as acetone, dimethylformamide, dioxan and tetrahydrofuran.

Adsorption↗

PubMedDate not supplied

[Immobilization of Penicillium vitale glucose-oxidase on aminosilochrome and properties of immobilized enzyme].

Penicillium vitale glucose-oxidase modified by means of the carbohydrate component oxidation is added covalently to aminoorganosylochrome. The activity of the immobilized preparations is 20-38% depending on the protein-carrier ration in immobilization. Comparison of some properties of native and immobilized glucose-oxidase showed that the rH optimum of the immobilized glucose-oxidase is slightly widened towards the alkaline regions; the immobilized glucose-oxidase possesses a considerably higher pH-stability at pH alkaline values; the immobilized glucose-oxidase preparations are characterized by a significantly greater thermostability: their thermoinactivation constant at 65 degrees C is 8-10 times lower than that of the native enzyme.

Drug Stability↗

PubMed2005

The host plant as a factor in the synthesis and secretion of salivary glucose oxidase in larval Helicoverpa zea.

We investigated the effect of the host plant on the synthesis and secretion of the elicitor glucose oxidase in the salivary glands of larval Helicoverpa zea. Glucose oxidase catalyses the oxidation of d-glucose to produce d-gluconic acid and hydrogen peroxide. Previous studies have found that the product hydrogen peroxide is primarily responsible for suppressing the wound-inducible defenses of the host plant. Using an antibody specific for glucose oxidase, we determined the effect of the host plant on the rate of secretion of glucose oxidase. Larval H. zea secrete microgram amounts of the enzyme glucose oxidase from their principal salivary glands, the labial glands. Larvae reared on different host plants produce varying amounts of glucose oxidase in their labial glands. We used a tissue printing procedure with our antibody to determine if larvae secrete glucose oxidase directly at the feeding or wound sites. Significant amounts of the enzyme are deposited at the feeding site, although some is deposited outside the feeding margins.

Animals↗

PubMed1996

A new glucose oxidase from Aspergillus niger: characterization and regulation studies of enzyme and gene.

A new glucose oxidase from Aspergillus niger was isolated and characterized. The enzyme showed different kinetic and stability characteristics when compared to a commercially available batch of A. niger glucose oxidase. The gene encoding the new glucose oxidase was isolated and DNA sequence analysis of the coding region showed 80% identity to the sequence of a glucose oxidase gene previously published. However, the similarity of the non-coding sequences up- and downstream of the open reading frame was much less, showing only 66% and 50% identity respectively. Despite the low degree of similarity between the promotor region of the new gene and the previously published one, the new glucose oxidase was likewise induced by calcium carbonate. In addition, we showed that this induction occurred on the transcriptional level. Observations concerning the effect of gluconolactone and the levels of glucose-6 phosphate isomerase upon calcium carbonate induction suggested that the enhancement of glucose oxidase biosynthesis by calcium carbonate was accompanied by a metabolic shift from glycolysis to the pentose phosphate pathway.

Amino Acid Sequence↗

PubMed1992

The immobilization of glucose oxidase onto radio-frequency plasma-modified poly(etherurethaneurea).

Glucose oxidase was covalently immobilized onto a radio-frequency plasma-modified poly(etherurethaneurea). Thin (90-100 nm) plasma-polymerized N-vinyl-2-pyrrolidone films were deposited onto poly(etherurethaneurea) films. Active sites for the immobilization were obtained via reduction with aqueous sodium borohydride and activation with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate. Modified poly(etherurethaneurea) films were assayed for binding and activity of the immobilized glucose oxidase layer. The results of a modified radioimmunoassay and an 'immunochemical stain' indicated that washing in 900 ml of continuously stirred 2% sodium dodecyl sulfate, 2% Triton X-100, and 20 mM sodium phosphate, pH 7.0, for 24 h each at 4 degrees C was necessary to remove physically adsorbed glucose oxidase from the solid supports. An amperometric activity determination in 9 ml of well-stirred 20 mM sodium phosphate-0.1 M sodium chloride, pH 7.4, gave a qualitative demonstration of the activity of the immobilized enzyme on 18.75 cm2 of modified poly(etherurethaneurea) film. A colorometric activity determination using the coupled reaction with o-dianisidine and peroxidase indicated that glucose oxidase covalently immobilized on approximately 2.4 cm2 of modified poly(etherurethaneurea) film had an activity approximately equal to that of 13.4 nM glucose oxidase in 50 mM sodium acetate, pH 5.1, with a specific activity of approximately 32.0 U/mg at pH 5.1 and room temperature.

Biocompatible Materials↗

PubMed1990

Cytotoxicity of glucose oxidase conjugated with antibodies to target cells: killing efficiency depends on the conjugate internalization.

The cytotoxic action of glucose oxidase conjugated with antibodies against the target cells has been examined in a culture of human endothelial cells. Internalizable (anti-endothelial, MoAb E25) and non-internalizable (anti-fibronectin, MoAb FN) monoclonal antibodies were employed as vectors. Anti-endothelial monoclonal antibody E78 (whether it can be internalized by endothelial cells is unclear) and polyclonal mouse antiserum to the human endothelium were also used. The conjugates were prepared by oxidation of the enzyme carbohydrate moiety with periodate. Free conjugates display similar enzyme activity in glucose solution. In contrast to glucose oxidase, conjugated with no-immune IgG, antibody-conjugated glucose oxidase binds specifically to target cells. The efficiency of targeting was different for various conjugates. Targeting via the anti-fibronectin antibody and anti-endothelial antiserum provided maximal quantitative binding of glucose oxidase to endothelial cells, while the conjugates with MoAb E25 and MoAb E78 monoclonal antibodies provided less effective binding. In the presence of glucose, targeted glucose oxidase generated H2O2. Hydrogen peroxide is relatively stable in buffer, but rapidly decays in the culture medium supplemented with 20% human serum. Though the quantitative binding of MoAb E25-conjugated glucose oxidase was minimal comparing to other conjugates, targeting via MoAb E25 produced the maximal cytotoxic effect as well as targeting via polyclonal antiserum. The killing efficiencies of MoAb FN-conjugated and MoAb E78-conjugated glucose oxidase were about 30-fold lower. The high efficiency of the MoAb E25-conjugated enzyme may be due to its internalization by target cells. Internalization can lead to unaccessibility of generated H2O2 for extracellular scavengers and pH optimization for glucose oxidase activity, which provides valuable advantages for the cytotoxicity of the conjugate. Thus, cytotoxicity of antibody-conjugated glucose oxidase depends not only on the efficiency of specific binding to the target cell, but also on the fate of cell-bound conjugate. Cytotoxicity is extremely effective in case of 'internalizable' conjugate and drastically less effective in case of 'non-internalizable' conjugate.

Animals↗