[Ultrastructure of Penicillium vitale Pidopl. et Bilai--producer of catalase and glucose oxidase].
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Catalase↗
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Determination of glucose oxidase oxidation-reduction potentials and the oxygen reactivity of fully reduced and semiquinoid forms.
The oxidation-reduction potential values for the two electron transfers to glucose oxidase were obtained at pH 5.3, where the neutral radical is the stable form, and at pH 9.3, where the anion radical is the stable form. The midpoint potentials at 25 degrees were: pH 5.3 EFl1ox + e- H+ equilibrium EFlH. Em1 = -0.063 +/- 0.011 V EFlH. + e- + H+ equilibrium EFlredH2 Em2 = -0.065 +/- 0.007 V pH 9.3 EFlox + e- EFi- Em1 = -0.200 +/- 0.010 V EFi- + e- + H+ equilibrium EFlredH- Em2 = -0.240 +/- 0.005 V All potentials were measured versus the standard hydrogen electrode (SHE). The potentials indicated that glucose oxidase radicals are stabilized by kinetic factors and not by thermodynamic energy barriers. The pK for the glucose oxidase radical was 7.28 from dead time stopped flow measurements and the extinction coefficient of the neutral semiquinone was 4140 M-1 cm-1 at 570 nm. Both radical forms reacted with oxygen in a second order fashion. The rate at 25 degrees for the neutral semiquinone was 1.4 X 10(4) M-1 s-1; that for the anion radical was 3.5 X 10(4) M-1 s-1. The rate of oxidation of the neutral radical changed by a factor of 9 for a temperature difference of 22 degrees. For the anion radical, the oxidation rate changed by a factor of 6 for a 22 degrees change in temperature. We studied the oxygen reactivity of the 2-electron reduced form of the enzyme over a wide wavelength range and failed to detect either oxygenated flavin derivatives or semiquinoid forms as intermediates. The rate of reoxidation of fully reduced glucose oxidase at pH 9.3 was dependent on ionic strength.
Aspergillus niger↗
Active site generation of a protonically unstable suicide substrate from a stable precursor: glucose oxidase and dibromonitromethane.
Bromonitromethane is an inefficient suicide substrate for glucose oxidase (in contrast to the case of CH(3)CCl=NO(2)(-) and D-amino acid oxidase) because, in the enzyme-substrate encounter step, the required ionization states of enzyme (EH(0)(+), pK(a) approximately 3.5) and substrate (CHBr=NO(2)(-), pK(a) approximately 8.3) cannot be highly populated simultaneously. Because reprotonation of CHBr=NO(2)(-) is rapid at the pH value used for the assay of glucose oxidase, presentation of the enzyme with the preformed anion could not be exploited in this case. We circumvent this difficulty by allowing the enzyme to reductively dehalogenate CHBr(2)NO(2), thereby generating the desired protonically unstable suicide substrate in situ (E(r) + CHBr(2)NO(2) --> E(o) + CHBr=NO(2)(-) + HBr + H(+)). Irreversible inactivation of the enzyme, because of the formation of a dead-end N-5 formylflavin adduct, is more than 100-fold faster when CHBr=NO(2)(-) is generated in situ than when it is externally applied. The remaining competitive fates of CHBr=NO(2)(-) at the active site are protonation and release or oxidation to HCOBr (or HCONO(2)). Strong support for these conclusions comes from (1) the brisk evolution of CH(3)CBr=NO(2)(-) (which is too bulky to act further as an efficient suicide substrate) from the enzyme-catalyzed reductive debromination of CH(3)CBr(2)NO(2), (2) the 1:1 stoichiometry of enzyme inactivation, and (3) the identification of the modified flavin as 5-formyl-1, 5-dihydro-FAD.
Anions↗
1,4-Benzoquinone-based electrophoretic assay for glucose oxidase.
The communication demonstrates feasibility of an enzyme microassay for glucose oxidase with 1,4-benzoquinone as an acceptor of electrons. The protocol uses the plug-plug mode of electrophoretically mediated microanalysis, with nanolitre injected volumes of enzyme and reactant solutions. The reactant and product, 1,4-benzoquinone and hydroquinone, are separated during the assay by differential binding to sulfated-beta-cyclodextrin used as additive to the phosphate buffer (pH 7) and monitored at selected wavelengths in their UV spectra. The assay covers glucose oxidase concentration from 0.01 to 0.1mgml(-1). Due to the strong UV absorbance of the both reactant and product, there is no need for use of a second enzyme (peroxidase) in the present assay.
Benzoquinones↗
Enhancement of glucose oxidase production in batch cultivation of recombinant Saccharomyces cerevisiae: optimization of oxygen transfer condition.
AIMS: To obtain an optimal combination of agitation speed and aeration rate for maximization of specific glucose oxidase (GOD) production in recombinant Saccharomyces cerevisiae, and to establish a correlation between kLa vis-à-vis oxygen transfer condition and specific glucose oxidase production. METHODS AND RESULTS: The oxygen transfer condition was manifested indirectly by manipulating the impeller speed and aeration rate in accordance with a Central Composite Rotatory Design (CCRD). The dissolved oxygen concentration and the volumetric oxygen transfer coefficient (kLa) were determined at corresponding combinations of impeller speed and aeration rate. The maximal specific extracellular glucose oxidase production (3.17 U mg-1 dry cell mass) was achieved when the initial dissolved oxygen concentration was 6.83 mg l-1 at the impeller speed of 420 rev min-1 and at the rate of aeration of 0.25 vvm. It was found out that while impeller speed had a direct effect on the production of enzyme, a correlation between kLa and specific GOD production could not be established. CONCLUSION: At the agitation speed of 420 rev min-1 and at 0.25 vvm aeration rate, the degree of turbulence and the dissolved oxygen concentration were thought to be optimal both for cellular growth and production of enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined effect of agitation and aeration on recombinant glucose oxidase production in batch cultivation has not yet been reported in the literature. Therefore, this study gives an insight into the effect of these two important physical parameters on recombinant protein production. It also suggests that since there is no correlation between kLa and specific production of GOD, kLa should not be used as one of the scale-up parameters.
Bioreactors↗
Co-immobilization of glucose oxidase and hexokinase on silicate hybrid sol-gel membrane for glucose and ATP detections.
The co-immobilization of glucose oxidase (GOD) and hexokinase/glucose-6-phosphate dehydrogenase (HEX) in the silica hybrid sol-gel film for development of amperometric biosensors was investigated. The silica hybrid film fabricated by hydrolysis of the mixture of tetraethyl orthosilicate and 3-(trimethoxysiyl)propyl methacrylate possessed a three-dimension vesicle structure and good uniformity and conformability, and was ready for enzyme immobilization. The electrochemical and spectroscopic measurements showed that the silica hybrid sol-gel provided excellent matrice for the enzyme immobilization and that the immobilized enzyme retained its bioactivity effectively. The immobilized GOD could catalyze the oxidation of glucose, which could be used to determine glucose at +1.0 V without help of any mediator. The competition between GOD and HEX for the substrate glucose involving ATP as a co-substrate led to a decrease of the glucose response, which allowed us to develop an ATP sensor with a good stability. The fabricated silica hybrid sol-gel matrice offered a stage for further study of immobilization and electrochemistry of proteins.
Adenosine Triphosphate↗
Application of electron-donor properties of glucose oxidase and xanthine oxidase for reduction of microsomal NAD(P)H-dependent electron-transport chains.
The reduction of cytochromes b5 and P-450 in mammalian hepatic microsomes by glucose oxidase and xanthine oxidase has been investigated. Under anaerobic conditions cytochrome b5 is reduced by glucose oxidase to the "dithionite" level, while cytochrome P-450 remains oxidized. Under the same conditions xanthine oxidase completely reduces both hemoproteins. Besides, neither glucose oxidase nor xanthine oxidase reduces isolated cytochromes. They can be reduced only after addition of microsomes to incubation media. Only in this case are the cytochromes, both isolated and included in microsomal membranes, reduced. The participation of microsomal flavoproteins in the reduction reaction is discussed. The method suggested makes it possible to substantially decrease the rates of reduction of microsomal hemoproteins, thus permitting the investigation of interactions between microsomal NADH- and NADPH-dependent electron-transport chains and electron carriers.
Animals↗
Effects of sucrose, citric buffer and glucose oxidase on the stability of captopril in liquid formulations.
OBJECTIVE: To study the effects of sucrose, citric buffer and glucose oxidase on the stability of captopril in liquid formulations. METHOD: Captopril liquid formulations 1 mg/ml were prepared in various concentrations of syrup with and without citric buffer. The liquid formulations were stored in amber glass bottles at 5 degrees C. Samples were removed from these formulations at 0, 1, 2, 3, 4, 7, 10, 15, 20 and 30 days for assay of captopril by a stability-indicating high performance liquid chromatographic method. RESULTS: Our findings indicate that low concentration of citric buffer (0.03 M) had a small stabilising effect on captopril in liquid formulations containing no or low concentration of sucrose (10% w/v). However, in formulations with higher concentrations of sucrose (30 and 85% w/v, respectively), the stabilising effect was not apparent. Captopril in 1 mg/ml formulations containing 0.03 M citric buffer and 10% w/v glucose degraded rapidly in the presence of glucose oxidase. The degradation was particularly evident at higher temperature. Glucose oxidase is an antioxidant, which can extensively reduce oxygen concentration in liquid formulations. CONCLUSION: The stability of captopril is very sensitive to the presence of excipients. Many excipients can function as catalysts in its degradation. Even in the deficiency of oxygen, its degradation can also be substantial with suitable catalysts present in the liquid formulation.
Angiotensin-Converting Enzyme Inhibitors↗
Glucose biosensor using glucose oxidase immobilized in polyaniline.
A biosensor for glucose utilizing kinetics of glucose oxidase (EC 1.1.3.4.) was developed. The enzyme was immobilized on polyaniline by covalent bonding, using glutaraldehyde as a bifunctional agent. The system showed a linear response up to 2.2 mM of glucose with a response time of 2.5-4.0 min. In addition, the immobilized enzyme had a higher activity between pH 6.5 and 7.5. The system retained 50% of its activity after 30 d of daily use. The optical absorption spectra of the polyaniline/glucose oxidase electrode after glucose had been added to the buffer solution showed that the absorption band around 800 nm had changed considerably when glucose was allowed to react with the electrode. This optical variation makes polyaniline a very promising polymer for use as a support in optical sensor for clinical application.
Aniline Compounds↗
Enzyme binding to detect carbohydrate expression in tissue sections. 1. Native and cross-linked glucose oxidase.
Enzymes may be useful as highly specific histochemical probes to identify and localize macromolecular substrates in tissue sections. We have used glucose oxidase, a double-headed enzyme, to demonstrate beta-glucosyl groups in paraffin sections. Native glucose oxidase has two active sites per molecule. Soluble polymers formed by glutaraldehyde combine many active binding sites on to one molecule. Some of these bind to glucose in tissue sections, leaving others free to react with chromogenic substrate. The intensity of staining is directly related to the concentration of enzyme, duration of incubation with enzyme, temperature and pH. Polymeric forms of enzyme are about 100 times more effective than native. Glucose oxidase, particularly in a polymeric form, appears a simple reagent for the identification of glucose-containing structures. The use of native and polymerized enzymes as a histochemical probe has enormous potential in the analysis of normal tissues and in the detection of aberrant carbohydrate deposition in pathological tissues; this system serves as a useful model.
Animals↗
Active site similarities of glucose dehydrogenase, glucose oxidase, and glucoamylase probed by deoxygenated substrates.
The specificity constants, kcat/KM, were determined for glucose oxidase and glucose dehydrogenase using deoxy-D-glucose derivatives and for glucoamylase using deoxy-D-maltose derivatives as substrates. Transition-state interactions between the substrate intermediates and the enzymes were characterized by the observed kcat/Km values and found to be very similar. The binding energy contributions of individual sugar hydroxyl groups in the enzyme/substrate complexes were calculated using the relationship delta(delta G) = -RT ln [(kcat/KM)deoxy/(kcat/KM)hydroxyl] for the series of analogues. The activity of all three enzymes was found to depend heavily on the 4- and 6-OH groups (4'- and 6'-OH in maltose), where changes in binding energies from 10 to 18 kJ/mol suggested strong hydrogen bonds between the enzymes and these substrate OH groups. The 3-OH (3'-OH in maltose) was involved in weaker interactions, while the 2-OH (2'-OH in maltose) had a very small if any role in transition-state binding. The three enzyme-substrate transition-state interactions were compared using linear free energy relationships (Withers, S. G., & Rupitz, K. (1990) Biochemistry 29, 6405-6409) in which the set of kcat/KM values obtained with substrate analogues for one enzyme is plotted against the corresponding values for a second enzyme. The high linear correlation coefficients (rho) obtained, 0.916, 0.958, and 0.981, indicate significant similarity in transition-state interactions, although the three enzymes lack overall sequence homology.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age-dependent role change of the worker honeybee (Apis mellifera L.).
Worker honeybees change their behaviour from the role of nurse to that of forager with age. We have isolated cDNA clones for two honeybee (Apis mellifera L.) genes, encoding alpha-amylase and glucose oxidase homologues, that are expressed in the hypopharyngeal gland of forager bees. The predicted amino acid sequence of the putative Apis amylase showed 60.5% identity with Drosophila melanogaster alpha-amylase, whereas that of Apis glucose oxidase showed 23.8% identity with Aspergillus niger glucose oxidase. To determine whether the isolated cDNAs actually encode these enzymes, we purified amylase and glucose oxidase from homogenized forager-bee hypopharyngeal glands. We sequenced the N-terminal regions of the purified enzymes and found that they matched the corresponding cDNAs. mRNAs for both enzymes were detected by Northern blotting in the hypopharyngeal gland of the forager bee but not in the nurse-bee gland. These results clearly indicate that expression of the genes for these carbohydrate-metabolizing enzymes, which are needed to process nectar into honey, in the hypopharyngeal gland is associated with the age-dependent role change of the worker.
Age Factors↗
Interaction of glucose oxidase with alkyl-substituted Sepharose 4B.
Glucose oxidase (GOD) is often used in immobilized forms for determination of glucose. To examine the possibility of its adsorption by hydrophobic interactions, palmityl-substituted Sepharose 4B (Sepharoselipid) was employed as an adsorptive matrix. Various conditions were used in tests to improve the limited immobilization of the enzyme observed under normal (native) conditions, including use of high concentrations of denaturing agents. Of the denaturants used, only the cationic detergent dodecyl trimethyl ammonium bromide was effective in denaturing the protein and exposing its hydrophobic sites for interaction with alkyl residues on the support. This, followed by the process of renaturation, provided catalytically active immobilized preparations. The apoenzyme, prepared by treatment of the holoenzyme with acidified (NH4)2SO4 or thermal denaturation, was totally immobilized on the support. Furthermore, it was shown that either flavin adenine dinucleotide (FAD) or the alkyl residues, not both, may interact with the nucleotide site at any given time. Results are discussed in terms of high rigidity of GOD molecule and limited exposure of hydrophobic sites in its native structure. The observations are in accord with suggestions in the literature that the FAD pocket is a very narrow channel of hydrophobic properties, adapted to accept its natural coenzyme.
Adsorption↗
Evidence for superoxide radical production by a simple flavoprotein: glucose oxidase.
Nitroblue tetrazolium was reduced to blue formazan during the oxidation of glucose by glucose oxidase. The rate of blue color formation was dependent on the concentrations of glucose, nitroblue tetrazolium and glucose oxidase. The rate of the reaction was negligible below pH 8.4, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, consistent with the participation of superoxide anion radical in the reaction.
Aspergillus niger↗
A glucose biosensor based on electrodeposited biocomposites of gold nanoparticles and glucose oxidase enzyme.
Electrodeposition was used for the codeposition of glucose oxidase enzyme and a gold nanoparticle-silicate network onto an indium tin oxide (ITO) glass electrode. This co-entrapment of glucose oxidase enzyme in a gold nanoparticle-silicate network imparts biocatalytic activity to the film. The gold nanoparticles in the network catalyse the oxidation and reduction of H2O2, the by-product of the enzymatic reaction. The low operating potential of the sensor eliminates the interference from common interferents, such as acetaminophen, ascorbic acid, dopamine, etc.
Journal Article↗
Preparation, characterization, and potential application of an immobilized glucose oxidase.
A simple, one-step process, using 0.25 M p-benzoquinone dissolved in 20% dioxane at 50 degrees C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated from Aspergillus niger. The coupling reaction was performed in 0.1 M potassium phosphate at pH 8.5 and 0-4 degrees C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50 degrees C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50 degrees C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.
Catalysis↗
Glucose-sensitive membranes containing glucose oxidase: activity, swelling, and permeability studies.
The development of membranes that swell in response to glucose is reported. The membranes may prove to be useful in glucose monitoring or glucose-dependent insulin delivery. The polymers were synthesized by the radiation-induced polymerization of frozen solutions containing hydroxyethylmethacrylate, N,N-dimethylaminoethyl methacrylate, tetraethylene glycol dimethacrylate, ethylene glycol, water, and glucose oxidase. The polymers were hydrogels, with water contents in the range of 60-90%, depending on the pH or glucose concentration. Changes in swelling and permeability of the hydrogel were caused by exposure to glucose solutions. The gluconic acid formed by the glucose oxidase catalyzed oxidation of glucose in the membrane lowered the pH of the system and thus caused the changes in the membrane. The retention of enzyme activity by the membranes in vitro and in vivo is also reported. The large differences in properties among membranes made with different chemical formulations suggest that glucose-sensitive membranes with performance characteristics needed for an artificial pancreas may be an achievable goal.
Acrylates↗
A new step to the mechanism of the enhancement effect of gold nanoparticles on glucose oxidase.
Quantum size effect was strongly supported by the experiment on correlation between size of gold nanoparticles (below 10 nm) and activity of glucose oxidase attached to them. The attractive enhancement effect of gold nanoparticles on the activity of glucose oxidase was then satisfactorily explained. In this experiment, 2 nm and 6 nm (average diameter) particles were obtained on monolayer matrix. The 2 nm particles proved to be much more able to increase the activity of glucose oxidase.
Adsorption↗