Deactivation studies of immobilized glucose oxidase

Studies have been performed in a tubular flow reactor to characterize the deactivation of immobilized glucose oxidase. The effects of oxygen concentration in the range of 0.09 to 0.467mM and hydrogen peroxide concentrations in the range of 0.1 to 10mM were studied. A simple mathematical model assuming first-order reaction and deactivation was found to describe the deactivation behavior adequately. The deactivation rate constant was found to increase with increasing levels of feed oxygen. Hydrogen peroxide was found to deactivate the enzyme severely and the deactivation rate constants were higher than those for oxygen deactivation. The influence of external and internal diffusion effects on the deactivation rate constant were examined. Although diffusional restrictions were negligible for oxygen transfer to the pellet, they were significant for transfer of hydrogen peroxide to the bulk stream. Increasing deactivation rates. Severe internal diffusion limitations were observed for the glucose oxidase system. However, for particle sizes in the range of 500 to 2000 μm, no effect on the rate of deactivation of the enzyme was observed.     

Fundamental studies of glucose oxidase immobilization on controlled pore glass

Immobilization experiments have been performed with glucose oxidase as enzyme and controlled-pore glass of different pore sizes as support for chemical coupling. The experimental results have been analyzed for comparison with the theoretical model predictions. Analysis of the initial stage of the process gives the fundamental characteristic of the immobilization reaction. These investigations allow us to study the influence of the degree of diffusional restriction on the evolution of the immobilization process and spatial distribution of immobilized enzyme. Nonuniformly distributed concentrations have been achieved within the porous matrix, and suggestions have been made in designing such profiles by choosing appropriate experimental parameters.     

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.     

Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles

Glutathione (L-gamma-glutamyl-L-cysteinyl-L-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC 1.1.3.4) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor.     

Determination of the inherent kinetics of immobilized glucose oxidase using a diffusion cell

The enzyme glucose oxidase (GO) was covalently immobilized onto a poly(vinyl alcohol) hydrogel, cross-linked with glutardialdehyde and a polyazonium salt. To compare the kinetic parameters of immobilized GO with the known kinetic parameters of soluble GO, the diffusion cell method was used.Between two compartments, containing solutions with different glucose concentrations, a GO-containing hydrogel membrane was placed. Simultaneous diffusion through and enzymatic reaction in the membrane occurred. In this way diffusional effects of the membrane could be eliminated from the effective kinetic parameters to yield the inherent kinetic parameters.It appeared that the enzymatic reaction is independent of the oxygen concentration at oxygen concentrations 0.22 mol m^–3 (Michaelis constant for oxygen < 0.22 mol m^–3). Further, the Michaelis constant for glucose does not change dramatically after immobilizing the enzyme. The maximal reaction rate is depending on the enzyme concentration. As the enzyme concentration in the membrane is not exactly known (mainly due to leakage of enzyme out of the membrane during membrane preparation), only an estimation of the turnover number can be made.The diffusion cell method is easy to carry out. Still, some recommendations can be made on the performance.List of Symbols _g , _0x partition coefficient of glucose and oxygen, respectively - thickness of the wetted membrane (m) - A _m surface area of membrane (m^–2) - C constant (mol² m^–3) - c _g , c _0x concentration of glucose and oxygen, respectively (mol m^–3) - c _g,0 c _g, glucose concentration at the filter-paper/membrane interface next to compartment A and B, respectively (mol m^–3) - c _{g, A} c _{g, B} glucose concentration in compartment A and B, respectively (mol m^–3) - c _GO glucose oxidase concentration (mol m^–3) - D _eff effective diffusion coefficient (m² s^–1) - D _m , D _sl diffusion coefficient in, respectively, the membrane and the solution layer (m² s^–1) - d _dl , d _df , d _sl thickness of, respectively, the diffusion layer, the filter-paper and the solution layer (m) - h _B initial slope of concentration versus time curve of compartment B (mol m^–3 s^–1) - J flux (mol m^–2 s^–1) - J ₀ flux in the membrane at membrane/filter-paper interface next to compartment A and B, respectively (mol m^–2 s^–1) - J _A , J _B flux leaving compartment A and entering compartment B, respectively (mol m^–2 s^–1) - J _m flux through the membrane (mol m^–2 s^–1) - k total mass transfer coefficient (m s^–1) - k ₁ , k ₂ rate constant of a particular reaction step (m³ mol^–1 s^–1) - k_–1, k_–2 rate constant of a particular reaction step (s^–1) - k _cat (intrinsic) catalytic constant of turnover number (s^–1) - k _cat ^* inherent catalytic constant, determined by inserting D _m (s^–1) - k _cat ^** inherent catalytic constant, determined by inserting D _eff (s^–1) - k _m (g) (intrinsic) Michaelis constant for glucose (mol m^–3) - k _m (o) (intrinsic) Michaelis constant for oxygen (mol m^–3) - k _m ^* (g) inherent Michaelis constant for glucose (mol m^–3) - k _m ^* (o) inherent Michaelis constant for oxygen (mol m^–3) - m _GO number of moles of GO present (mol) - P _m permeability of glucose in the mebrane (m s^–1) - P _eff effective permeability (m s^–1) - V volume (m³) - v ₀ initial reaction velocity (mol m^–3 s^–1) - V _max ^** inherent maximal reaction velocity, determined by inserting D_eff (mol m^–3 s^–1) - x distance (m)     

Novel optical solid-state glucose sensor using immobilized glucose oxidase

Meso-tetra(4-carboxyphenyl)porphine (CTPP(4)) binds reversibly to immobilized glucose oxidase (GOD), resulting in an absorbance peak for the CTPP(4)-GOD complex at 427nm. The absorbance intensity of the 427nm peak is reduced upon exposure to glucose, which causes the dissociation of CTPP(4) from GOD. The change in absorbance at 427nm shows linear dependence on glucose concentration from 20 to 200mg/dL (1.1-11.1mM).     

Time-dependent inactivation of immobilized glucose oxidase and catalase

Homogeneous membranes containing immobilized glucose oxidase and catalase were stored in buffered solutions at 37 degrees C to determine the mechanisms and rates of catalyst inactivation. The experiments were designed so that inactivation occurred homogeneously throughout the membrane, thereby simplifying the analysis. The mechanism of inactivation is consistent with the reaction of hydrogen peroxide and certain catalytic intermediates of both enzymes. Based on this information, numerical simulations were developed that incorporate spatially heterogeneous catalytic and inactivation processes.     

Characterization of glucose oxidase immobilized on collagen

Summary The enzyme glucose oxidase (E.C. 1.1.3.4) was immobilized on collagen — a proteinaceous material found in biological systems as a structural material for a wide variety of cells and membranes. The novel technique of electrocodeposition, which utilizes the principles of electrophoresis, was used to deposit the enzyme-collagen complex on stainless steel helical supports. This technique has been developed in our laboratory. The mechanism of complex formation between collagen and enzyme involves multiple salt linkages, hydrogen bonds and van der Waals interactions.As a first step toward examining its feasible technical use, the kinetic behavior of the collagen-supported glucose oxidase was studied in a batch recycle type reactor and was compared with that for the soluble form. A novel reactor configuration consisting of multiple concentric electrocodeposited helical coils was used. The reactor was found to attain a stable level of activity which was maintained for several months under cyclic testing. The optimum levels of pH and temperature for the immobilized form of the enzyme were the same as those of the soluble enzyme, but the immobilized enzyme was more active than the soluble form at higher temperatures and pH. The values of the Michaelis-Menten parameters indicate that the overall reaction rate of the immobilized enzyme may be partially restricted by bulk and matrix diffusion.     

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.     

Performance of rotating packed disk reactor with immobilized glucose oxidase

Summary Reactor performance was studied to investigate whether a rotating packed disk reactor (RPDR) can be used for the enzymatic oxidation of biochemicals. The disks were packed with calcium alginate beads with immobilized glucose oxidase and catalase, which catalyze the reaction of glucose and oxygen. The production rate of gluconic acid increased with the speed of rotation and the bulk flow rate. An optimum submergence for maximum productivity existed.     

Simultaneously immobilized glucose oxidase and catalase in controlled-pore titania

The immobilization of glucose oxidase and catalase by adsorption within the pores of controlled-pore titania has yielded a remarkably stable enzyme system. Catalase apparently acts as both a stabilizer and an activator for glucose oxidase within the pores of this material. Hydrogen peroxide concentrations and flow rates have a marked effect upon the apparent activity of the immobilized enzyme system. The carrier parameters were varied to obtain optimum loading and stability information.     

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.     

Renaturation studies of free and immobilized D-amino-acid oxidase

The renaturation of free and Sepharose-immobilized D-amino-acid oxidase (D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3), after its denaturation with 6 M guanidine hydrochloride, was investigated. No reactivation, or extremely limited reactivation (less than or equal to 4+), was obtained with the free enzyme, is spite of various attempts including the use of dialysis or buffers containing cofactors, different types of anions, surfactants and low concentrations of denaturing agents. The main obstacle to renaturation appeared to be the interaction among denatured or partially renatured monomers giving rise to inactive aggregates. In contrast, using the immobilized enzyme approach, substantial renaturation (up to 50%) of D-amino-acid oxidase was achieved. The denaturation-renaturation process was followed by monitoring the catalytic activity as well as the intrinsic protein fluorescence. An inverse correlation was found to exist between the degree of matrix activation by CNBr and the yield of enzyme reactivation. The anions of the lyotropic series markedly influenced the reactivation, showing an effectiveness opposite to their salting-out potential (thiocyanate congruent to iodide greater than chloride greater than phosphate congruent to sulphate congruent to citrate). Instead, the anions considerably increased the activity and stability of free and immobilized enzyme, according to their salting-out potential. Immobilized monomers of D-amino-acid oxidase, which in solution undergoes self-association, showed poor capacity to interact with the free enzyme: thus they appear unsuitable for analytical and preparative purposes.     

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.     

Glucose oxidase immobilized electrode for potentiometric estimation of glucose

Glucose oxidase has been immobilized onto a thin platinum strip, by co-crosslinking with bovine serum albumin and glutaraldehyde. The retention of redox characteristics of glucose oxidase has been verified by cyclic voltammetry. The activity of the immobilized enzyme reduces to a quarter of its value when the enzyme is in solution but improves when coimmobilized with 1 urea. The potentiometric response builds up and remains stable after 100 s. It is sensitive to the thickness of the immobilizing matrix, pH and temperature. An improvement in the performance of the electrode has been achieved by coimmobilizing 2 urea and metal ions such as Mg²⁺ and Mn²⁺. The presence of Cu has been proved to be detrimental. The electrode has been calibrated in the 0.1–5.0 mM glucose concentration range. It gives a stable response for more than 50 independent assays and can be stored for 60 days without significant loss of function.     

Electrochemical evaluation of glucose oxidase immobilized by different methods

Glucose oxidase electrodes were constructed on a platinum screen using polyacrylamide gel, glutaraldehyde crosslinking, and glutaraldehyde crosslinking with +0.04 volts dc on the platinum screen as the methods of enzyme immobilization. The electrodes were evaluated in an electrochemical cell for the oxidation of glucose at the enzyme electrode and the reduction of oxygen at a platinum auxiliary electrode, using constant current voltametry or under external load operation. The method of immobilization affected the extrapolated opencircuit potential as well as the half-cell potential and the steady current under external load operation. The charged glutaraldehyde electrode gave the best current performance; however, the small output (microamps) indicated that major problems in electron transfer from an enzyme catalyst to an external circuit must be resolved before such electrodes can be used in practical application.     

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.