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PubMed1999

The fluorescence emission of the apo-glucose oxidase from Aspergillus niger as probe to estimate glucose concentrations.

We developed a new method of glucose sensing using an inactive form of glucose oxidase from Aspergillus niger. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting apo-glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8-Anilino-1-naphthalenesulfonic acid (ANS) was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the K(D) of holo-glucose oxidase. These results suggest that apo-glucose oxidase can be used as a reversible nonconsuming sensor for glucose.

Anilino Naphthalenesulfonates↗
PubMed1990

Optimization of a polypyrrole glucose oxidase biosensor.

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of +0.65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentiostating the enzyme electrode at a potential of +0.7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determine the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0.3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0.01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0.17 microns (75 mC/cm2) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1.0 mM to 7.5 mM glucose and kinetic parameters determined for the optimized biosensors were 33.4 mM for the Km and 7.2 microA for the Imax. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.

Biosensing Techniques↗
PubMed1991

Effects of carbohydrate depletion on the structure, stability and activity of glucose oxidase from Aspergillus niger.

Glucose oxidase from Aspergillus niger was purified to homogeneity by hydrophobic interaction and ion-exchange chromatography. Approx. 95% of the carbohydrate moiety was cleaved from the protein by incubation of glucose oxidase with endoglycosidase H and alpha-mannosidase. Cleavage of the carbohydrate moiety effected a 24-30% decrease in the molecular weight and a reduction in the number of isoforms of glucose oxidase. No significant changes were observed in the circular dichroism spectra of the deglycosylated enzyme. Other properties, such as thermal stability, pH and temperature optima of glucose oxidase activity and substrate specificity were not affected. However, removal of the carbohydrate moiety marginally affected the kinetics of glucose oxidation and stability at low pH. From these results it appears that the carbohydrate chain of glucose oxidase does not contribute significantly to the structure, stability and activity of glucose oxidase.

Amino Acid Sequence↗
PubMed1995

The characterization of liposomal glucose oxidase electrodes for the measurement of glucose.

Many biosensors have been described for the measurement of glucose in order to monitor diabetic patients. Glucose oxidase has been used commonly in the construction of glucose sensors but the performance of this enzyme is limited by enzyme saturation kinetics, which restrict the measurement of clinically relevant glucose concentrations (0 to 25 mM). Diffusion limiting membranes have been described that result in the exposure of the enzyme to lower concentrations of glucose than are present in the bulk test solution. Recently a liposomal enzyme electrode was reported whereby glucose oxidase was encapsulated within liposomes so that the lipid bilayer was the diffusion limiting membrane. It was shown that the electrode response was defined by the lipid constituents of the liposome, and that a linear response to glucose could be achieved up to 40 mM. This paper describes research undertaken to improve the methods of production of a liposomal enzyme electrode. Improved immobilization of liposomes is demonstrated with the use of poly-L-lysine solution. The variation in electrode response with respect to the amount of glucose oxidase liposomally encapsulated is reported. The new method allows a greater number of sensors to be produced from a single batch of liposomes. Studies also show the biofouling effects of the lipid constituents of ruptured liposomes on the response of the electrode to glucose over time.

Biosensing Techniques↗
PubMed1997

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense.

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not-been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH4)2SO4, which stabilised glucose oxidase 100- to 300-fold at 50 degrees C and pH 7-8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 degrees C and pH 6. In sodium acetate buffer, changes in pH (4-6) affected the affinity for glucose but had no effect on the Vmax of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in Vmax and a 2-fold decrease in K(m) were observed.

Amino Acid Sequence↗
PubMed1982

New procedure for detecting antinuclear antibodies using glucose oxidase immunoenzyme technic.

Enzyme glucose oxidase was used as the label on the secondary antibody in an indirect ANA-test procedure using rat kidney tissue as the antigen-source. Glucose oxidase is not endogenously present in mammalian tissues and, therefore, produces no background staining as obtained with flurochrome and peroxidase labels, which are commonly used to detect tissue antigens. Pseudoperoxidase-like activity of the haptoglobin-hemoglobin complexes in mammalian tissues is responsible for the interfering background stain when peroxidase label is used. Unlike Diaminobenzidine, the staining reagent commonly used with peroxidase, chromogenic reagents used with glucose oxidase are not carcinogenic. Glucose oxidase as a label for antibodies has advantages over flurochrome labels in being permanently stable, producing no background stain, and increasing the sensitivity of the method. Comparative evaluation of the glucose oxidase procedure and the commonly used flurochrome-ANA and peroxidase-ANA methods is reported. Results with 150 serum samples show the potential of this new method as a routine ANA-testing procedure in clinical laboratories.

Animals↗
PubMed1989

Ferrocene modified polypyrrole with immobilised glucose oxidase and its application in amperometric glucose microbiosensors.

Gold microelectrodes were modified with electropolymerised polypyrrole in both organic and aqueous electrolytes. Cyclic voltammetry studies confirmed that electron conduction through the films in aqueous electrolytes is greatly reduced and is inefficient at mediating the oxidation/reduction of solution redox species. Films formed in aqueous electrolyte were also found to be more heterogenous in structure compared to those formed in acetonitrile. Aqueous polymerised films were used as an immobilisation matrix for glucose oxidase, and modified gold micro electrodes were used in the analysis of glucose using hydrogen peroxide detection. It was found that glucose oxidase was strongly absorbed into the films and that the sensors remained stable for several weeks. An improved linear range and oxygen insensitivity was achieved by incorporating the "water-insoluble" electron transfer mediator, dimethyl ferrocene, into the polypyrrole films by absorption. Good currents and response times to glucose addition were obtained from these electrodes, however, the electrodes possessed poor stability caused by the apparent loss of mediator from the films. Greater operational stability was achieved by covalently functionalising the films with the ferrocene derivative, ferrocenecarbonyl chloride. Covalently bound ferrocence was found to be an efficient oxidant of reduced glucose oxidase. Electrodes constructed in this manner possessed a good linear range, oxygen insensitivity and better stability. Electron transfer mechanisms are discussed.

Biosensing Techniques↗
PubMed2005

Glucose oxidase multilayer modified microcantilevers for glucose measurement.

We report a novel enzyme-based microcantilever sensor by using layer-by-layer nanoassembly modification. A glucose oxidase (GOx) functionalized microcantilever underwent bending when it was exposed to glucose solutions. The magnitudes of bending were proportional to the concentrations of glucose. The cantilever bending was specific toward glucose, but not to other sugars such as mannose, fructose, or galactose. The flow rate effect on the cantilever bending response is also discussed. The bending mechanism was investigated, and the kinetic and thermodynamic analysis and experimental results showed that the conformational change of GOx and gluconic formation were the origin of cantilever deflection.

Enzymes, Immobilized↗
PubMedDate not supplied

[Thermal stability of Penicillium adametzii glucose oxidase].

The thermal stability of glucose oxidase was studied at temperatures between 50 and 70 degrees C by kinetic and spectroscopic (circular dichroism) methods. The stability of glucose oxidase was shown to depend on the medium pH, protein concentration, and the presence of protectors in the solution. At low protein concentrations (< 15 micrograms/ml) and pH > 5.5, the rate constants kin (s-1) for thermal inactivation of glucose oxidase were high. Circular dichroic spectra suggested an essential role of beta structures in stabilizing the protein globule. At a concentration of 15 micrograms protein/ml, the activation energy Ea of thermal inactivation of glucose oxidase in aqueous solution was estimated at 79.1 kcal/mol. Other thermodynamic activation parameters estimated at 60 degrees C had the following values: delta H = 78.4 kcal/mol, delta G = 25.5 kcal/mol, and delta S = 161.9 entropy units. The thermal inactivation of glucose oxidase was inhibited by KCl, polyethylene glycols, and polyols. Among polyols, the best was sorbitol, which stabilized glucose oxidase without affecting its activity. Ethanol, phenol, and citrate exerted destabilizing effects.

Circular Dichroism↗
PubMed2005

Biocomposites of covalently linked glucose oxidase on carbon nanotubes for glucose biosensor.

The formation of covalently linked composites of multi-walled carbon nanotubes (MWCNT) and glucose oxidase (GOD) with high-function density for use as a biosensing interface is described. The reaction intermediates and the final product were characterized by using FT-IR spectroscopy, and the MWCNT-coated GOD nanocomposites were examined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Interestingly, it was found that the GOD-MWCNT composites are highly water soluble. Electrochemical characterization of the GOD-MWCNT composites that were modified on a glassy carbon electrode shows that the covalently linked GOD retains its bioactivity and can specifically catalyze the oxidation of glucose. The oxidation current shows a linear dependence on the glucose concentration in the solution in the range of 0.5-40 mM with a detection limit of 30 microM and a detection sensitivity of 11.3 microA/mMcm2. The present method may provide a way to synthesize MWCNT related composites with other biomolecules and for the construction of enzymatic reaction-based biofuel cells and biosensors.

Biosensing Techniques↗
PubMed2001

Involvement of protein dynamics in enzyme stability. The case of glucose oxidase.

Dynamics of glucose oxidase immobilized and in solution were compared through their tryptophan fluorescence spectra, decay times and quenching by acrylamide. Energy barrier for thermal inactivation and melting temperature of both soluble and immobilized enzyme were also measured. Data show that the fluctuation amplitude is at the origin of protein instability.

Enzyme Stability↗
PubMed1992

Enzyme immobilization on a low-cost magnetic support: kinetic studies on immobilized and coimmobilized glucose oxidase and glucoamylase.

Glucose oxidase (GOx) and glucoamylase (GA) were immobilized and coimmobilized through their carbohydrate moieties onto polyethyleneimine-coated magnetite crosslinked with glutaraldehyde and derivatized with adipic dihydrazide. The carbohydrates were oxidized with sodium periodate, and at optimal concentration, their Vm increased up to 18% for GOx and up to 16% for GA. After immobilization, a remaining activity as high as 88% and 70% for GA with maltose and maltodextrin respectively as substrates was obtained, independently of the particle loading. On the contrary, the remaining activity of GOx strongly decreased at high particle loading. Nevertheless, half of its initial activity was recovered at low loading and was not significantly affected when GA was coimmobilized by saturating the reactive groups left on the particle. The Vm of both immobilized enzymes was improved by crosslinking their carbohydrates with adipic dihydrazide, a treatment which allows further coimmobilization of the other enzyme on a second layer.

Enzymes, Immobilized↗
PubMed2004

Oxygen isotope effects on electron transfer to O2 probed using chemically modified flavins bound to glucose oxidase.

Apo-glucose oxidase has been reconstituted with flavins modified in the 7 and 8 positions and characterized with regard to the catalytic rate of O(2) reduction and oxygen-18 isotope effects on this process. Kinetic studies as a function of driving force indicate a reorganization energy for electron transfer to O(2) of lambda = 28 kcal mol(-)(1) at optimal pH, which is similar to the value obtained earlier from temperature dependencies of rates (Roth, J. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 62-67). For the various enzyme-bound flavins, competitive oxygen-18 kinetic isotope effects fall within the narrow range of 1.0266(5) to 1.0279(6), apparently because of the dominant contribution of outer-sphere reorganization to the activation barrier; within the context of semiclassical and quantum mechanical electron transfer theories, the magnitude of the isotope effects reveals the importance of nuclear tunneling.

Aspergillus niger↗
PubMed1976

Interaction of glucose oxidase with phospholipid vesicles.

The interactions between glucose oxidase and phospholipid vesicles were investigated. The investigations were carried on molecules adsorbed on the outer surfaces as well as entrapped in the interior of the vesicles . The adsorption of glucose oxidase on the surfaces of egg egg licithin vesicles, containing varying amounts of cholesterol and stearoylamine was measured by determining the free fraction of glucose oxidase detected in the filtrates. In general an enhancement of enzymic activity was observed upon interaction with the vesicles. The enhancement depends on the lipid composition of the vesicles and the surface concentration of the adsorbed glucose oxidase. It reached a maximal value at a surface concentration of 1.4-10(11) molecules/cm2 (approximately 7.1 - 10(4) A2/molecule) on pure phosphatidylcholine vesicles and about 6.5 - 10(10) molecules/cm2 (approximately 16 - 10(4) A2/molecule) when the vesicles contained cholesterol or cholesterol and stearoylamine. CD measurements indicated that the change in enzymic activity of the adsorbed glucose oxidase was accompanied by conformational modification of the enzyme. In order to entrap glucose oxidase into the vesicles, the lipid was sonicated in the presence of the enzyme. After removal of the free and adsorbed enzyme the amount of the entrapped enzyme was determined by measuring its activity after disintegration of the vesicles with Triton. The enzymic activity of the entrapped glucose oxidase served as a measure for the permeability of the bilayer membrane of the lipid vesicles to glucose. Addition of insulin to the suspension of vesicles containing the entrapped glucose oxidase increased the permeability of glucose by up to 9 - 10(-8) cm/s. This value is the lowest estimate based on the assumption that one glucose oxidase molecule was entrapped in every vesicle.

Binding Sites↗
PubMed1982

Hydrophobic-ionic chromatography: its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase.

Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH less than or equal to 4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobic-ionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.

Aspergillus niger↗