Glucose oxidase — An overview

Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase; EC 1.1.2.3.4) catalyzes the oxidation of β-d-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with simultaneous production of hydrogen peroxide. Microbial glucose oxidase is currently receiving much attention due to its wide applications in chemical, pharmaceutical, food, beverage, clinical chemistry, biotechnology and other industries. Novel applications of glucose oxidase in biosensors have increased the demand in recent years. Present review discusses the production, recovery, characterization, immobilization and applications of glucose oxidase. Production of glucose oxidase by fermentation is detailed, along with recombinant methods. Various purification techniques for higher recovery of glucose oxidase are described here. Issues of enzyme kinetics, stability studies and characterization are addressed. Immobilized preparations of glucose oxidase are also discussed. Applications of glucose oxidase in various industries and as analytical enzymes are having an increasing impact on bioprocessing.     

The immobilization of enzymes on nylon structures and their use in automated analysis

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.     

Gel entrapment of enzymes: kinetic studies of immobilized glucose oxidase

Glucose oxidase (EC 1.1.3.4, from Aspergillus niger) has been entrapped in a crosslinked 2-hydroxycthyl methaerylate gel containing 20% poly(vinyl pyrrolidone). The kinetic behavior and thermal stability of the entrapped enzyme were found to closely approximate that of the free enzyme. The entrapped glucose oxidase shows a broadened pH profile which is attributed to a buffering effect of the gel. Stability of gel entrapped glucose oxidase is extremely good at room temperature, suggesting a variety ofanalytical and control uses for this system.     

Immobilization of enzymes on activated carbon: Properties of immobilized glucoamylase,glucose oxidase,and gluconolactonase

Apparent kinetics and pH–activity relationships have been determined for glucoamylase and glucose oxidase immobilized on activated carbon using a diimide method. Reaction rate expressions of Michaelis–Menten form adequately approximate the observed kinetics for both enzyme preparations over the ranges of substrate concentrations considered. Influences of external mass transfer as well as substrate and product adsorption on interpretation of the experimental data have been examined. Immobilization of a glucose oxidase–gluconolactonase enzyme mixture has been found to increase substantially the ratio of gluconolactonase to glucose oxidase activities compared to the corresponding activity ratio for these enzymes in solution.     

Immobilization of glucose oxidase and peroxidase to a urea derivative of granulated microcrystallized cellulose

Glucose oxidase and peroxidase were immobilized individually to a urea derivative of granulated microcrystallized cellulose activated by formaldehyde. The catalytic properties of the immobilized enzymes were studied and compared to those of the soluble enzymes. The immobilized glucose oxidase and peroxidase were used for the manual determination of the concentration of glucose in sera. The developed method is characterized by high analytical reliability and comparatively low cost. The results correlated well with those obtained by using a BECKMAN glucoanalyzer, utilizing soluble glucose oxidase.     

Immobilized glucose oxidase and D-amino acid oxidase: a convenient method for the purification of flavin adenine dinucleotide and its analogs.

The preparation of flavin adenine dinucleotide-affinity columns employing glucose oxidase and d-amino acid oxidase covalently linked to Sepharose-4B is described. Both immobilized enzymes have very good long-term stabilities, retaining at least half of their original flavin adenine dinucleotide-binding capacity over 11 months. In both cases pure flavin adenine dinucleotide is obtained readily in good yields.     

Properties of enzymes. V. Catalase inhibition of glucose oxidase reaction.

The radical produced aerobically in the glucose oxidase reaction reduces the oxidated cytochrome c. The extent of reduction depends on the concentrations of substrate (glucose) and enzyme. Superoxide dismutase purified from various sources does not inhibit the cytochrome c reduction, but catalase does, in proportion to its concentration. This inhibition, which might be utilized for quantitative catalase determinations, provides further evidence for the formation of H2O2 in the glucose oxidase reaction.     

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.     

Immunoaffinity layering of enzymes

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae. Received: 16 November 1998 / Received revision: 22 March 1999 / Accepted: 26 March 1999     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

Insolubilized enzymes. Kinetic behaviour of glucose oxidase bound to porous glass particles

1. The spectrophotometric and steady-state kinetic properties of glucose oxidase (EC 1.1.3.4, from Aspergillus niger) that is covalently linked to porous glass beads have been examined. These properties have been compared with those of soluble glucose oxidase, for which the kinetic mechanism at pH5.5 and 25 degrees C has been established previously by a combination of conventional and rapid-reaction techniques to be the following: [Formula: see text] where E(o) and E(r) represent oxidized and reduced forms of the enzyme, respectively. 2. The ratio k(+4)/k(+2) is unchanged after insolubilization, and evidence is presented which suggests that the absolute magnitudes of k(+4) and k(+2) are unchanged. 3. The kinetic efficiency of the insolubilized enzyme is greatly enhanced because of a 14-fold increase in the apparent affinity of glucose for E(o). This effect is attributed either to the binding of glucose to the glass surface or to a change in enzyme structure imposed by the insolubilization process. 4. Only 6% of the insolubilized enzyme which can be reduced by glucose is catalytically active. It is shown by calculation and direct experimental evidence that this fraction of catalytically active enzyme is bound to the exterior bead surface. The remaining 94% of the enzyme is bound within the pore network and may be subject to severe substrate diffusion control.     

Specificity and kinetic parameters of recombinant Microdochium nivale carbohydrate oxidase

A new carbohydrate oxidase from Microdochium nivale heterologously expressed in Aspergillus oryzae (rMnO) has been characterized. The carbohydrate oxidase is a flavoenzyme which oxidizes glucose and other mono- or oligosaccharides. It shows a broad substrate specificity towards carbohydrates reacting with aldoses in the 1-position. The rMnO oxidizes the β-form of -glucose, and the product of -glucose oxidation is -gluconic acid.

The mechanism of carbohydrate oxidation by oxygen and artificial electron acceptors has been described by a ping-pong scheme. Compared to Aspergillus niger glucose oxidase (GOx) the reactivity of rMnO at pH 7.0 is significantly lower; k_cat is 20, k_ox 11 and k_red 22 times less, using oxygen as electron acceptor. Also with other two electron acceptors, like DPIP, the activity is low. However, compared to oxygen the rMnO shows 2–10 times higher activity towards some artificial single electron acceptors (AAs). The enzyme activity increases at higher ionic strength of the solution, if positively-charged AAs are used.

The high activity towards AAs and low rate for oxygen as well as broad specificity to carbohydrates indicates that rMnO may have some advantages compared to the most used GOx in connection with enzyme use for analytical devices and for biotechnological purposes.     

Glucose oxidase biosynthesis using immobilized mycelium of Aspergillus niger

A simple method is described for the immobilization of Aspergillus niger GIV-10 which produces an extracellular glucose oxidase. A. niger conidia were immobilized on sintered glass Raschig rings, pumice stones or polyurethane foam. Mycella growing out from the spores produced extracellular glucose oxidase: the highest production was with the pumice stone carrlers. This technique facilitates the growth of the filamentous cultures in the spongy structure of a support with continuous accumulation of biomass. After 24 to 36 h, a culture liquid with 2.7 to 3.1 U of glucose oxidase/ml was obtained. This procedure also made possible repeated batch enzyme production and as many as 25 subsequent 24-h batches could be fermented by using the same carrier with only a small loss of glucose oxidase activity.The authors are with the Institute of Microbiology, M. Curie-Sklodowska University, Akademicka 19, 20-003 Lublin, Poland.     

Glucose oxidase synthesis by Aspergillus niger GIV-10 on starch.

The effect of various kinds of starch, as the sole source of organic carbon, on the biosynthesis of glucose oxidase by A. niger GIV-10 was examined. A. niger grown on 6% wheat starch medium provided extracellular and intracellular glucose oxidase with the highest enzymatic activities. A new method of intracellular glucose oxidase extraction (without disruption of mycelium), developed and discussed in this paper, increased 2 to 3.8-times glucose oxidase yield, as compared to that described earlier.     

Macrokinetics and operational stability of immobilized glucose oxidase and catalase

Glucose oxidation by immobilized glucose oxidase (GlO) and catalase (Cat) has been investigated in batch and continuous reactions for operational studies. The macrokinetics of the process depend on coupled reaction steps and diffusion rates. The problem may be approximated by a simple pseudohomogeneous model taking into account both substrates of glucose oxidase and the intermediate reaction product H₂O₂. The effectiveness of both enzymes is enhanced in the coupled reaction path, the overall effectiveness nevertheless is very low. H₂O₂ causes the inactivation of both GlO and Cat. The rates of deactivation depend on the oxidation rates of glucose that give different quasistationary levels of H₂O₂ concentration. As a first approximation, the deactivation rates may be described by first-order reactions with respect to H₂O₂.     

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.     

Concerted action of lactate oxidase and pyruvate oxidase in aerobic growth of Streptococcus pneumoniae: role of lactate as an energy source

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H(2)O(2)-forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.     

Glucose oxidase immobilized polyethylene-g-acrylic acid membrane for glucose oxidase sensor

A polyethylene-g-acrylic acid (PE-g-AA) graft copolymer was prepared via gamma-ray-irradiation-induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water-soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE-g-AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 degrees C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme-immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0-1000 ppm. The obtained glucose oxidase-immobilized PE-g-AA membranes were kept in pH 5. 6 acetate buffer solution at 4 degrees C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.     

Activity staining of blotted enzymes by reaction coupling with transfer membrane-immobilized auxiliary enzymes

A blotting method is described to detect enzymes that do not normally yield a colored product. The method can be used for dot blotting as well as blotting after gel electrophoresis of many enzymes if the reactions they catalyze can be coupled to an oxidase or a dehydrogenase. The latter, designated "auxiliary enzymes," are preimmobilized on membranes of nitrocellulose or positively charged nylon and the reaction they catalyze is coupled with reduction of tetrazolium salt to yield colored formazan on areas of the transfer membrane occupied by the blotted enzymes. In the examples reported here, preimmobilized glucose oxidase, L-amino acid oxidase, xanthine oxidase, malate dehydrogenase, and a mixture of hexokinase and glucose-6-phosphate dehydrogenase were used as auxiliary enzymes to detect blotted invertase, leucine aminopeptidase, purine nucleoside phosphorylase, fumarase, and adenylate kinase, respectively. Detection limits varied, but never exceeded 100 ng for these enzymes. After blotting from polyacrylamide gels, the fumarase assay was the most sensitive of those investigated, detecting 10 ng of enzyme used for electrophoresis. Invertase, a glycoprotein, was detected with higher sensitivity on nitrocellulose membranes when concanavalin A was present on the membrane in addition to the auxiliary enzyme, glucose oxidase. On blots from isoelectric focusing gels, the assay detected two isozymes of purine nucleoside phosphorylase in a sample from calf spleen and at least five isozymes of this enzyme in lysates from human red cells.