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PubMed1987

Application of high-performance chromatographic and electrophoretic methods to the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum.

The high resolving power of the preparative and analytical high-performance chromatographic and electrophoretic methods recently developed in this laboratory for the separation of biopolymers has been demonstrated by the purification and characterization of glucose oxidase and catalase from Penicillium chrysogenum. Crude glucose oxidase was purified to homogeneity in one step by high-performance hydrophobic-interaction chromatography (HIC) on a pentylagarose column. Crude catalase was purified by a combination of HIC and high-performance anion-exchange chromatography on 3-diethylamino-2-hydroxypropylagarose. The homogeneity of the enzymes was monitored by high-performance electrophoresis and free zone electrophoresis. The pI values of these two enzymes determined by isoelectric focusing in the high-performance electrophoresis apparatus were 4.2 and 6.5, respectively. Their molecular weights were determined by high-performance molecular sieve chromatography on an agarose column. Glucose oxidase has a molecular weight of 175,000 and probably consists of two identical subunits, as sodium dodecyl sulphate polyacrylamide gel electrophoresis gave a molecular weight of around 72,000. The molecular weight of catalase, which is probably composed of non-identical subunits, as indicated by sodium dodecyl sulphate electrophoresis, is around 320,000. Some other characteristics of these two enzymes were also investigated, e.g., electrophoretic mobility, pH stability and optimum pH.

Amino Acids↗

PubMed2004

Inactivation of cytochrome-c with glucose oxidase.

A novel reaction of cytochrome-c from the horse heart with the enzyme glucose oxidase from Aspergillus niger (EC 1.1.3.4), in acidic media is described. Glucose oxidase is able to induce a rapid, profound and irreversible physico-chemical change in cytochrome-c, under anaerobic conditions and in the presence of glucose. The initial rate of reaction is almost independent of the concentration of enzyme and glucose. The striking feature of this reaction is the fact that the reaction proceeds efficiently even below a concentration of 10 nM enzyme.

Aspergillus niger↗

PubMed2001

Fabrication of ultra-thin polypyrrole-glucose oxidase film from supporting electrolyte-free monomer solution for potentiometric biosensing of glucose.

A simple electropolymerisation process is described for the fabrication of an ultra-thin ( approximately 55 nm) polypyrrole (PPy)-glucose oxidase (GOD) film in a supporting electrolyte-free monomer solution for potentiometric biosensing of glucose. The optimum conditions for growing the ultra-thin film include 0.1 M pyrrole, 55-110 U/ml GOD, an applied current density of 0.05 mA/cm(2) and an electrical charge of 25 mC/cm(2). Long-term storage of the biosensor in acetate buffer improved the sensitivity of the biosensor by a factor of approximately two. The biosensor can also be used repeatedly for over 2 months with little or no loss in sensitivity. The interference effect of ascorbic acid was successfully reduced by inclusion of an outer PPy-Cl layer.

Biosensing Techniques↗

PubMed1999

Effect of ammonium and nitrate ratio on glucose oxidase activity during gluconic acid fermentation by a mutant strain of Aspergillus niger.

Of the factors tested, the source and concentration of carbon and nitrogen in the medium exerted maximum effect on growth and acid production. Glucose (15%) and urea (0.14%) induced glucose oxidase synthesis and optimum yield of calcium gluconate. Potassium dihydrogen phosphate (0.2%) and magnesium sulphate (0.06%) stimulated glucose oxidase activity and calcium gluconate production. Borax at a concentration of 1.5 g/L induced maximum glucose oxidase and calcium gluconate production with increased glucose utilization.

Aspergillus niger↗

PubMed2003

Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives.

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.

Aspergillus niger↗

PubMed2005

Activity of glucose oxidase functionalized onto magnetic nanoparticles.

BACKGROUND: Magnetic nanoparticles have been significantly used for coupling with biomolecules, due to their unique properties. METHODS: Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chloride using two different base solutions. Glucose oxidase was bound to the particles by direct attachment via carbodiimide activation or by thiophene acetylation of magnetic nanoparticles. Transmission electron microscopy was used to characterize the size and structure of the particles while the binding of glucose oxidase to the particles was confirmed using Fourier transform infrared spectroscopy. RESULTS: The direct binding of glucose oxidase via carbodiimide activity was found to be more effective, resulting in bound enzyme efficiencies between 94-100% while thiophene acetylation was 66-72% efficient. Kinetic and stability studies showed that the enzyme activity was more preserved upon binding onto the nanoparticles when subjected to thermal and various pH conditions. The overall activity of glucose oxidase was improved when bound to magnetic nanoparticles CONCLUSION: Binding of enzyme onto magnetic nanoparticles via carbodiimide activation is a very efficient method for developing bioconjugates for biological applications.

Journal Article↗

PubMed1986

Purification and characterization of glucose oxidase from ligninolytic cultures of Phanerochaete chrysosporium.

Glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography. The enzyme is a flavoprotein with an apparent native molecular weight of 180,000 and a denatured molecular weight of 80,000. This enzyme does not appear to be a glycoprotein. It gives optimal activity with D-glucose, which is stoichiometrically oxidized to D-gluconate. The enzyme has a relatively broad pH optimum of 4 to 5. It is inhibited by Ag+ (10 mM) and o-phthalate (100 mM), but not by Cu2+, NaF, or KCN (each 10 mM).

Basidiomycota↗

PubMed2001

Monovalent cation-induced conformational change in glucose oxidase leading to stabilization of the enzyme.

Glucose oxidase (GOD) from Aspergillus niger is an acidic dimeric enzyme having a high degree of localization of negative charges on the enzyme surface and dimer interface. We have studied the effect of monovalent cations on the structure and stability of GOD using various optical spectroscopic techniques, limited proteolysis, size exclusion chromatography, differential scanning calorimetry, and enzymic activity measurements. The monovalent cations were found to influence the enzymic activity and tertiary structure of GOD, but no effect on the secondary structure of the enzyme was observed. The monovalent cation-stabilized GOD was found to have a more compact dimeric structure but lower enzymic activity than the native enzyme. The enzyme's K(m) for D-glucose was found to be slightly enhanced for the monovalent cation-stabilized enzyme (maximum enhancement of about 35% for LiCl) as compared to native GOD. Comparative denaturation studies on the native and monovalent cation-stabilized enzyme demonstrated a significant resistance of cation-stabilized GOD to urea (about 50% residual activity at 6.5 M urea) and thermal denaturation (Delta T(m) maximum of 10 degrees C compared to native enzyme). However, pH-induced denaturation showed a destabilization of monovalent cation-stabilized GOD as compared to the native enzyme. The effectiveness of monovalent cations in stabilizing GOD structure against urea and thermal denaturation was found to follow the Hofmeister series: K(+) > Na(+) > Li(+).

Aspergillus niger↗

PubMedDate not supplied

[Effect of the state of graphite surfaces on the activity of immobilized glucose oxidase].

The work deals with immobilization of glucose oxidase on modified graphite. The effect of the carrier surface composition on the major properties of the immobilized enzyme was examined. The level of activity of the immobilized enzyme is shown to depend on the quantity of Ti and Zr oxides applied on the graphite surface. The high activity of the enzyme immobilized on graphite will enable it to be used for construction of enzyme electrodes.

Adsorption↗

PubMed2001

Femtosecond dynamics of flavoproteins: charge separation and recombination in riboflavine (vitamin B2)-binding protein and in glucose oxidase enzyme.

Flavoproteins can function as hydrophobic sites for vitamin B(2) (riboflavin) or, in other structures, with cofactors for catalytic reactions such as glucose oxidation. In this contribution, we report direct observation of charge separation and recombination in two flavoproteins: riboflavin-binding protein and glucose oxidase. With femtosecond resolution, we observed the ultrafast electron transfer from tryptophan(s) to riboflavin in the riboflavin-binding protein, with two reaction times: approximately 100 fs (86% component) and 700 fs (14%). The charge recombination was observed to take place in 8 ps, as probed by the decay of the charge-separated state and the recovery of the ground state. The time scale for charge separation and recombination indicates the local structural tightness for the dynamics to occur that fast and with efficiency of more than 99%. In contrast, in glucose oxidase, electron transfer between flavin-adenine-dinucleotide and tryptophan(s)/tyrosine(s) takes much longer times, 1.8 ps (75%) and 10 ps (25%); the corresponding charge recombination occurs on two time scales, 30 ps and nanoseconds, and the efficiency is still more than 97%. The contrast in time scales for the two structurally different proteins (of the same family) correlates with the distinction in function: hydrophobic recognition of the vitamin in the former requires a tightly bound structure (ultrafast dynamics), and oxidation-reduction reactions in the latter prefer the formation of a charge-separated state that lives long enough for chemistry to occur efficiently. Finally, we also studied the influence on the dynamics of protein conformations at different ionic strengths and denaturant concentrations and observed the sharp collapse of the hydrophobic cleft and, in contrast, the gradual change of glucose oxidase.

Carrier Proteins↗

PubMed1976

Relative efficiencies of a soluble and immobilized two-enzyme system of glucose oxidase and catalase.

Dual catalysts of varying glucose oxidase (EC 1.1.3.4) and catalase (EC 1.11.1.6) activities were constructed by immobilization of the enzymes to silanized nickel silica alumina with glutaraldehyde. The amount of product formed and the efficiency of the systems were determined using either of the enzymes to initiate the cyclic reaction. The former depends on the activity of both enzymes in the system while efficiency describes how well the second enzyme in the sequence utilizes the intermediate of the reaction. Efficiency was a function both of the ratio of the two activities and their absolute values. An increase in the activity of the second enzyme of the reaction sequence of a decrease in the activity of the first enzyme increased the efficiency. A higher efficiency was observed with higher absolute activities of the enzymes when the ratios of the enzyme increased the efficiency. A higher efficiency was observed with higher absolute activities of the enzymes when the ratio of the enzymes was constant. Dual catalysts were compared to mixed catalysts (each enzyme immobilized to separate particles) and to soluble, homogeneous systems at equal total activities. In general, the dual catalysts were superior to either of the others. Computer simulations of the reaction rates indicated that all experiments were performed in the lag period of the two-step reaction.

Aluminum Silicates↗

PubMed1989

In vivo administration of glucose oxidase conjugated with monoclonal antibodies to angiotensin-converting enzyme. The tissue distribution, blood clearance, and targeting into rat lungs.

A conjugate between glucose oxidase (GO) and monoclonal antibody to human angiotensin-converting enzyme (ACE) cross-reacting with rat ACE (MoAb9b9) has been prepared by oxidation of the cardohydrate moiety of the enzyme with sodium periodate. The conjugate (GO-MoAb9b9) thus obtained retained both antigen-binding capacity and enzymatic activity. The fate of the conjugate in vivo after intravenous injection was studied using conjugates containing radiolabeled enzyme. GO-MoAb9b9 was specifically accumulated in rat lungs upon in vivo administration, as compared with free enzyme and nonimmune IgG-conjugated glucose oxidase. The specificity of the conjugate accumulation expressed as the localization ratio (the ratio between radioactivity of gram tissue to that of blood) (Loc. Ratio) reached a value up to 50 on the second day after injection, in contrast to native enzyme and to IgG-conjugated enzyme (Loc. Ratio was less than 0.5 for both preparations). The Loc. Ratio of GO-MoAb9b9 was even higher than that of the original antibody MoAb9b9 and was equal to 20, which is probably explained by an extremely rapid blood clearance of the conjugate from the circulation. The administration of excess free MoAb9b9 dramatically inhibited the conjugate targeting in the lung without any effect on liver uptake. At doses ranging from 10 to 1,000 micrograms/rat, the conjugate was accumulated in the lung without saturation of the antigen determinants of the target. At minimal doses, the efficiency of targeting achieved 5 to 7% of the conjugate injected. With elevation of the dose, the efficiency of targeting decreased to 2.5% of the injected dose (1 mg of GO-MoAb9b9 per rat). A sixfold greater accumulation of unmodified radiolabeled MoAb9b9 compared with the GO-MoAb9b9 conjugate in rat lung has been observed, though the kinetics of desorption from the target organ was similar for both the antibody and the conjugate. In the bloodstream, the conjugate persisted for at least 5 days without binding to blood cells; all circulating radioactivity was associated with proteins. A considerable part of the conjugate (to 50%) circulated as a tight antibody-enzyme complex for several days. The conjugate retained its antigen-binding capacity for at least 24 h; during this period, its enzymatic activity decreased by less than 40%. The results obtained provide the experimental ground for further attempts to apply glucose oxidase conjugates for local modulation of inflammation and elimination of the target cells.

Animals↗

PubMed1989

Immunotoxins containing glucose oxidase and lactoperoxidase with tumoricidal properties: in vitro killing effectiveness in a mouse plasmacytoma cell model.

We have tested the tumoricidal potency of enzyme immunotoxins constructed of antibodies conjugated to glucose oxidase and to lactoperoxidase. Murine plasmacytoma cells were targeted in vitro with the use of affinity-purified rabbit anti-plasmacytoma membrane antibodies (conjugated to glucose oxidase or lactoperoxidase) or rabbit serum raised against plasmacytoma microsome membranes followed by goat anti-rabbit immunoglobulin conjugates (to glucose oxidase or lactoperoxidase). Cytotoxicity was generated subsequently by incubation of the washed cells in a medium supplemented with glucose and sodium iodide, which were the substrates of these enzymes. This resulted in the presumed metabolic release of highly toxic reduced oxygen species and iodinated derivatives. Targeting of tumor cells with both conjugates, as opposed to one of them alone, produced a synergistic killing effect. The gain of specific versus unspecific cytotoxicity was upwards of 10,000-fold. The killing rates were elevated (t10 values less than 30 min) and linear over time. The resultant reduction in tumor cell viability was in the order of 5 to 6 logs after only 20 to 90 min of incubation in the glucose/NaI medium. Cytotoxicity was enhanced by the gamma-glutamyl cysteine synthetase inhibitor buthionine-S,R-sulfoximine and by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, while catalase was inhibitory. The results suggest that these enzyme immunotoxins may be suitable for the ex vivo purging of autologous bone marrow grafts.

Animals↗

PubMed1994

Glucose oxidase electrodes: effect of hydrogen peroxide on enzyme activity?

This paper presents a simple procedure to assess the amount of hydrogen peroxide arising in amperometric glucose oxidase sensors. At an external glucose concentration of 30 mmol/l, which represents the linear range of calibration, a hydrogen peroxide concentration of approximately 0.18 mmol/l is generated. Taking into consideration that in medical applications the main range of interest would be within euglycaemia which is between 4 and 9 mmol/l, it is concluded that in the type of glucose electrode investigated, hydrogen peroxide produced during enzymatic glucose measurement does not appreciably damage the enzyme glucose oxidase.

Biosensing Techniques↗

PubMedDate not supplied

[Effect of conditions of culturing Penicillium funiculosum G-15 on production of extracellular glucose oxidase].

Effects of conditions of Penicillium funiculosum G-15 cultivation on the production of extracellular glucose oxidase were studied. The data showed that surface and submerged methods of cultivation can be used for obtaining a glucose oxidizing enzyme. The optimum conditions for submerged cultivation (25 degrees C, initial pH 5.0, and aeration of 3 l/l per min) and surface cultivation (temperature 25 degrees C and initial pH 4.0) providing the maximum levels of glucose oxidase synthesis were determined.

Extracellular Space↗

PubMed1999

An immunosensor based on the glucose oxidase label and optical oxygen detection.

A novel optical immunosensor setup is described which uses glucose oxidase enzyme as a label in conjunction with a luminescence lifetime-based oxygen sensor and phase measurements. The oxygen sensor membranes prepared on microporous filters were used as a solid phase on which the immunoassay was carried out. These sensing materials in combination with a new measurement setup provided high sensitivity for the detection of oxidase enzymes, being at nanogram per milliliter level, i.e., 10(-11)-10(-12) M, with respect to glucose oxidase and its conjugates. Experimental data on the sensitivity were validated using theoretical equations and calculations. Using the new measurement setup and IgG-anti-IgG as a model, a number of different sensing materials were studied aimed to optimize the immunosensor and evaluate its performance. This approach was then applied to a practical system for the detection of human lactate dehydrogenase isoenzymes. It provided similar sensitivity of approximately 1 ng/mL, which is comparable to that of standard ELISA. The attributes of the new immunosensor approach are discussed with respect to performance and versitility.

Biosensing Techniques↗

PubMed1993

Comparison of blue dye visualization and glucose oxidase test strip methods for detecting pulmonary aspiration of enteral feedings in intubated adults.

STUDY OBJECTIVE: To compare the relative utility of blue dye visualization with a glucose oxidase test strip method for detecting aspiration of enteral feedings. DESIGN: Tracheally intubated adults were prospectively monitored for aspiration of enteral feedings. SETTING: Intensive care units of two community hospitals in Michigan. INTERVENTIONS: None. PATIENTS: The experimental group consisted of 15 patients receiving enteral feedings. The control group included 14 patients not enterally fed. MEASUREMENTS AND RESULTS: Blue food coloring was added to feeding formulas to obtain a visible blue color. At 8-h intervals, tracheal secretions were examined for blue discoloration, followed by measurement of glucose concentration using a calibrated glucose meter. Clinically significant aspiration was defined to require the following: (1) a bloodless positive glucose reading (> or = 20 mg/dl); (2) one or more signs of systemic inflammation; and (3) one or more signs of respiratory deterioration. Eight (53 percent) of 15 patients in the experimental group experienced at least one episode of presumptive aspiration as defined by either a bloodless positive glucose reading or visible blue discoloration of tracheal secretions. Clinically significant aspiration occurred in 5 (33 percent) of 15 patients in whom bloodless glucose readings were positive in 13 (19 percent) of 67 samples; among patients not developing this complication, glucose was found in only 3 (5 percent) of 60 samples; (p = 0.005). Inspecting tracheal secretions for blue dye usually failed to detect aspiration episodes identifiable by the glucose oxidase test strip method (relative sensitivity, 13 percent). Blue dye visualization performed no better among patients developing clinically significant aspiration (relative sensitivity, 15 percent). Patients who developed clinically significant aspiration received more of their enteral feedings in the supine position than patients without this complication (98 percent vs 21 percent; p < 0.001). CONCLUSIONS: Inspecting tracheal secretions for blue discoloration failed to detect most episodes of enteral feeding aspiration. Glucose oxidase test strip methods should replace blue dye visualization for detecting aspiration of enteral feedings in intubated adults.

Adult↗

PubMed1990

Purification of the glycoprotein glucose oxidase from Penicillium amagasakiense by high-performance liquid chromatography.

Fast protein liquid chromatography (FPLC) in combination with ion-exchange chromatography on a Mono Q column was used to purify glucose oxidase from Penicillium amagasakiense to homogeneity. Purification was performed with a mixed pH and salt gradient, with 20 mM phosphate buffer (pH 8.5) as starting buffer (A) and 50 mM acetate buffer (pH 3.6) with 0.1 M NaCl as elution buffer (B). Elution conditions were optimized to permit the simultaneous purification and separation of the glucose oxidase isoforms. Three peaks, each consisting of 1-2 isoforms and exhibiting a homogeneous titration curve profile, were resolved with a very flat linear gradient of 5.0-5.1% B in 40 ml. Three more peaks, each consisting of several isoforms, were eluted at 10%, 30% and 100% B. Optimization of the elution conditions and separation of the glucose oxidase isoforms was only possible because of the rapidity of each purification step and the high resolution provided by FPLC and Mono Q.

Buffers↗