Enzymatic Oxidation and Separation of Various Saccharides with Immobilized Glucose Oxidase

Glucose oxidase from Aspergillus niger, the specific enzyme for β-d-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of d-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for d-glucose and other saccharides examined. As a result, k _cat/K _M ratio for d-glucose was determined to be 42 times higher than d-mannose, 61.6 times higher than d-galactose, 279 times higher than d-xylose, and 254 times higher than for d-fructose and d-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove d-glucose from d-cellobiose, d-glucose from d-xylose, and d-xylose from d-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of d-glucose and d-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease d-glucose or d-xylose content to 0–0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides d-xylose, d-cellobiose, and d-lyxose, respectively, was observed.     

Catalase-conjugated liposomes encapsulating glucose oxidase for controlled oxidation of glucose with decomposition of hydrogen peroxide produced

The catalase-conjugated liposome encapsulating glucose oxidase (CLG) was prepared for developing a novel liposomal system for glucose oxidation with controllable enzyme activities. The catalase molecules were conjugated to the surface of liposome with 100 nm in mean diameter through coupling with the membrane-incorporated 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (NGPE) at its mole fraction f_G of 0.05 or 0.15. The average number of enzyme molecules per CLG with f_G of 0.15 was 8.7 for glucose oxidase and 6.5 for catalase. The CLG-catalyzed oxidation of glucose was performed at 40 °C for prolonged period up to 99 h. The CLG with f_G of 0.15 gave larger oxidation rate than that with f_G of 0.05. In the fed-batch oxidation of glucose catalyzed by the former CLG, the stable oxidation rate was observed for 75 h with negligible accumulation of H₂O₂ produced because of the durable catalytic actions of the liposomal enzymes. The oxidation rate of the CLG reaction increased to 1.1 mM-glucose/(h mM-lipid) at the acidic pH in the internal phase of liposome and the neutral pH in the external one corresponding to the optimal pH conditions for the activities of glucose oxidase and catalase, respectively. The oxidation rate catalyzed by the CLG could be controlled by adding sublytic concentrations of cholate to increase permeability of the liposome membrane to glucose. The catalase-conjugated liposomal system is potentially utilized for controlling the rate of reactions catalyzed by a variety of oxidases.     

Enzymatic formation of ions and their detection at a three-phase electrode

An electrochemical method for the detection of enzymatically created anions is described that uses a thin-film electrode with decamethylferrocene as an electroactive redox probe. The enzymatic oxidation of glucose with enzyme glucose oxidase produces gluconic acid as a final product. The oxidation of decamethylferrocene dissolved in the thin-nitrobenzene film, that is spread on the working graphite electrode and submerged in the aqueous solution containing glucose and glucose oxidase, is followed by the up-take of gluconate anions from the aqueous phase to nitrobenzene. The peak currents of the square-wave voltammetric responses of that system are a linear function of the glucose concentration in the milimolar range from 0.1 mmol/L to 0.7 mmol/L (R²=0.994).     

A nano-functionalized real-time electrochemiluminescent biosensor for alanine transaminase assay

An electrochemiluminescent (ECL) biosensor was constructed for selective assay of alanine aminotransferase (ALT) based on the enzymatically catalyzed oxidation of pyruvate by pyruvate oxidase (PYOD). The composite of potassium ferricyanide and carbon nanotube was adopted to pre-functionalize the basal platinum electrode while the potassium ferricyanide acted as the activator of PYOD. The ALT catalyzed the reaction of L-alanine and-ketoglutarate to produce pyruvate which could be further enzymatically oxidiz...     

Spectrophotometric cell comprising parallel rotating and stationary bioreactors: Application to the determination of glucose in serum samples

A spectrophotometric cell comprising parallel bioreactors facing each other and containing immobilized enzyme preparations is described. The lower reactor rotates to minimize diffusional constraints, and the upper reactor is fixed to provide an integrated design for the realization of coupled enzyme-catalyzed reactions. The operating characteristics of the cell are illustrated with the determination of glucose using glucose oxidase [EC 1.1.3.4] and horseradish peroxidase [EC 1.11.1.7] as immobilized enzymes (horseradish peroxidase on the rotating reactor and glucose oxidase on the stationary one). The H₂O₂ produced in the dissolved-oxygen oxidation of β- -glucose enters into oxidative coupling in a reaction with N,N-dimethylaniline and 4-aminophenazone which is catalyzed by horseradish peroxidase; the absorbance of the colored complex formed provides the basis for monitoring. The cell was incorporated into a continuous-flow/stopped-flow/continuous-flow operation, and the determination was based on the rate of response under stopped-flow conditions. The overall approach was applied to the determination of glucose in standards of human serum and samples of bovine blood serum.     

An improved enzymatic assay for glucose determination in blood serum using a l,1′-dimethylferricinium dye

l,l′-dimethylferricinium (DMFe⁺),a stable and pH-insensitive blue dye, was prepared via enzymatic oxidation of a 1,1′-dimethylferrocene (DMFe):2-hydroxypropyl-β-cyclodextrin (HPCD) watersoluble inclusion complex, using bilirubin oxidase immobilized onto porous aminopropyl glass beads via glutaraldehyde activation. In the presence of glucose, DMFe⁺ was reduced to DMFe by reacting with the reduced glucose oxidase (FADH₂), and the absorbance decrease was followed at 650 nm. In acetate pH 5.2 buffer, the response to glucose in blood serum was nonlinear, especially in the low concentration range, because of a competition for the reduced glucose oxidase between the DMFe⁺ dye and oxygen. At this pH, endogenous ceruloplasmin was also observed to oxidize residual DMFe (16%) in the dye preparation, causing an increase in absorbance at 650 nm. An assay protocol was then developed using maleate buffer, pH 6.5, to overcome these interferences as well as mutarotation of α-D-glucose. The results obtained for glucose in the blood serum samples agreed well with those of the reference hexokinase/glucose-6-phosphate dehydrogenase method.     

On the catalytic mechanism of choline oxidase

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with betaine aldehyde as an intermediate. In this study, primary deuterium and solvent kinetic isotope effects have been used to elucidate the mechanism for substrate oxidation by choline oxidase using both steady-state kinetics and rapid kinetics techniques. The D(kcat/Km) value with 1,2-[2H4]-choline at saturating oxygen concentration was independent of pH in the range between 6.5 and 10, with a value of approximately 10.6, indicating that CH bond cleavage is not masked by other titratable kinetic steps belonging to the reductive half-reaction. In agreement with this conclusion, a Dkred value of approximately 8.9 was determined at pH 10 for the anaerobic reduction of the flavin by choline, irrespective of whether aqueous or deuterated solvent was used. At pH 10, both the D2(O)(kcat/Km) and the D2(O)kred values were not different from unity with choline or 1,2-[2H4]-choline, while the Dkcat and D2(O)kcat values were 7.3 and 1.1, respectively. The kcat and kred values were 133 s(-1) and 135 s(-1) with betaine aldehyde and 60 s(-1) and 93 s(-1) with choline. These data are consistent with a chemical mechanism in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage and with chemical steps of flavin reduction by choline and betaine aldehyde being rate limiting for the overall turnover of the enzyme.     

Partial oxidation of glucose by a Pt|WO3 electrode

Electrochemical oxidation of (+)-glucose by a Pt|WO₃ electrode in sulphuric acid medium was investigated by HPLC and in-situ FTIR spectroscopy. The oxidation on Pt|WO₃ electrode was found to be a multi-step and multi-electron process. The major oxidation products were identified to be glucono-δ-lactone, CO₂ and a probable five-member ring lactone. Based on these observations, the mechanism of glucose oxidation on Pt|WO₃ was discussed.     

镀金和碳纳米管修饰金电极上吸附态葡萄糖氧化酶比活性的EQCM研究

采用石英晶体微天平(EQCM)技术监测了裸金电极、镀金和碳纳米管修饰金电极上葡萄糖氧化酶(GOD)的吸附过程. 通过EQCM测量吸附固定的GOD质量, 并实时检测酶反应产物H2O2的氧化电量, 求算了各表面上吸附态GOD的比活性(ESAi). 结果表明, 各表面上均可吸附一定的GOD, 且吸附态GOD均有一定的酶活性; 修饰CNTs可增大酶吸附量和酶电极对葡萄糖的响应电流, 但ESAi随CNTs修饰量的增大而降低; Au电极上电镀金后, 酶吸附量和酶电极对葡萄糖的响应电流亦增大, 但ESAi与裸金电极上的基本一致.     

Kinetic study of the performance of third-generation biosensors

A kinetic study of the performance of third-generation biosensors for glucose based on glucose oxidase immobilized on a microporous matrix of the conducting polymer poly(pyrrole) is presented. The mechanism of the enzymatically catalyzed oxidation of glucose will be different in this type of biosensor as the natural electron acceptor oxygen is replaced by the conducting polymer. Different kinetic parameters are found for the immobilized glucose oxidase than for the enzyme in solution. Mediation by the conducting polymer is found to be very effective and no significant electron transfer to oxygen is observed. In addition to substrate transport limitation in the microporous matrix, the enzymatic reaction in the biosensors is limited by the applied potential.     

3D Multilayered paper‐ and thread/paper‐based microfluidic devices for bioassays

In this paper we describe the fabrication of novel 3D microfluidic paper‐based analytical devices (3D‐μPADs) and a 3D microfluidic thread/paper‐based analytical device (3D‐μTPAD) to detect glucose and BSA through colorimetric assays. The 3D‐μPAD and 3D‐μTPAD consisted of three (wax, heat pressed wax‐printed paper, single‐sided tape) and four (hole‐punched single‐sided tape, blank chromatography circles, heat‐pressed wax‐printed paper, hole‐punched single‐sided tape containing trifurcated thread) layers, respectively. The saturation curves for each assay were generated for all platforms. For the glucose assay, a solution of glucose oxidase (GOx), horseradish peroxidase, and potassium iodide was flowed through each platform and, upon contact with glucose, generated a yellow‐brown color indicative of the oxidation of iodide to iodine. For the protein assay, BSA was flowed through each device and, upon contact with citrate buffer and tetrabromophenol blue, resulted in a color change from yellow to blue. The devices were dried, scanned, and analyzed yielding a correlation between either yellow intensity and glucose concentration or cyan intensity and BSA concentration. A similar glucose assay, using unknown concentrations of glucose in artificial urine, was conducted and, when compared to the saturation curve, showed good correlation between the theoretical and actual concentrations (percent differences <10%). The development of 3D‐μPADs and 3D‐μTPADs can further facilitate the use of these platforms for colorimetric bioassays.     

Determination of erythromycin base and 2'-acetylerythromycin in human plasma using high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the determination of plasma concentrations of erythromycin base and 2'-acetylerythromycin, an ester prodrug of erythromycin, is described. tert.-Butyl methyl ether extracts of 1-ml plasma samples (pH 10) were chromatographed on a C18 reversed-phase column. A three-electrode coulometric detector (oxidation potentials +0.65 and +0.85 V) was used for quantitation. Oleandomycin was used as an internal standard. The method has good precision and accuracy, is linear in the range 0.25-7.5 mg/l and has proved to be suitable for pharmacokinetic studies in humans. Correlation with a microbiological assay was satisfactory (r greater than or equal to 0.95), but the chromatographic method gave ca. 30% higher values.     

Preparation of the stabilized glycoenzymes by cross-linking their carbohydrate chains

Each of the three high-mannose type glycoproteins studied, acid phosphatase, invertase, and glucose oxidase, could be specifically cross-linked through its carbohydrate chains. The procedure involves periodate oxidation of carbohydrate residues followed by reaction of the generated aldehyde groups with adipic acid dihydrazide as a cross-linker. The amount and size as well as solubility of the formed polymers could be efficiently controlled by varying the reaction conditions, i.e., the oxidation degree and the concentrations of glycoproteins, cross-linker, and hydrogen ions during the cross-linking reaction. It was found that the quantity and size of polymers increased with oxidation degree and protein concentration and by lowering the pH. When the protein concentration was above and pH below certain values, depending on the glycoenzyme, insoluble polymers formed. The soluble cross-linked polymers retained a high level of original activity, and the minor decrease in specific activity noticed was shown to occur during the periodate oxidation step. The cross-linked glycoenzymes are much more resistant to denaturation by high temperature and by changes in pH, demonstrating the usefulness of this method in preparation of the stabilized glycoprotein derivatives.     

Spectrophotometric quantification of lactose in solution with a peroxidase-based enzymatic cascade reaction system

A spectrophotometric assay was developed for the quantification of lactose in aqueous solution via a one-pot enzymatic cascade reaction at 25 °C and pH 7.2. Lactose (0.2-1.8 mM), E. coli β-galactosidase (β-Gal), Aspergillus niger glucose oxidase (GOD), horseradish peroxidase (HRP) and o-phenylenediamine (OPD) were incubated, and the increase in absorbance at 417 nm (A (417)) due to the formation of DAP (2,3-diaminophenazine), the dimeric oxidation product of OPD, was followed. The increase in A (417) was found to depend linearly on the initial lactose concentration via three consecutive but simultaneously occurring enzymatic reaction steps catalyzed by β-Gal, GOD, and HRP. No pre-incubation of lactose with β-Gal is needed with this simple lactose assay.     

Sensitive electrochemical immunoassay of carcinoembryonic antigen with signal dual-amplification using glucose oxidase and an artificial catalase

A new dual-amplification strategy of electrochemical signal based on the catalytic recycling of the product was developed for the antigen-antibody interaction by glucose oxidase (GOD)- conjugated gold-silver hollow microspheres (AuAgHSs) coupled with an artificial catalase, Prussian blue nanoparticles (PB), on a graphene-based immunosensing platform. The first signal amplification introduced in this study was based on the labeled GOD on the AuAgHSs toward the catalytic oxidation of glucose. The generated H(2)O(2) was catalytically reduced by the immobilized PB on the graphene nanosheets with the second amplification. With a sandwich-type immunoassay format, carcinoembryonic antigen (CEA) was monitored as a model analyte by using the synthesized AuAgHSs as labels in pH 6.0 phosphate buffer containing 10mM glucose. Under optimal conditions, the electrochemical immunosensor exhibited a wide dynamic range of 0.005-50 ng mL(-1) with a low detection limit (LOD) of 1.0 pg mL(-1) CEA (at 3σ). Both the intra- and inter-assay coefficients of variation (CVs) were lower than 10%. The specificity and stability of the immunosensor were acceptable. In addition, the assay was evaluated for clinical serum specimens, and received a good correlation with those obtained by the referenced electrochemiluminescent (ECL).     

Enzyme-conjugated hybridization chain reaction for magneto-controlled immunoassay of squamous cell carcinoma antigen with pH meter

A nanoparticle-based potentiometric immunoassay was designed for sensitive detection of squamous cell carcinoma antigen on a portable pH meter by coupling enzyme-labeled hybridization chain reaction with two alternating hairpin DNA probes for the signal amplification.     

A novel two-enzyme amperometric electrode for lactose determination.

Coimmobilization of beta-galactosidase and glucose oxidase in a redox polymer, polyvinylferrocenium perchlorate (PVF+ ClO4-), led to the development of an enzyme electrode for the determination of lactose. The amperometric response of the electrode was measured at +0.70 V vs. SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the substrate and buffer concentrations as well as the pH on the electrode response were elucidated.     

Integrated, electrically contacted NAD(P)+-dependent enzyme-carbon nanotube electrodes for biosensors and biofuel cell applications

Integrated, electrically contacted beta-nicotinamide adenine dinucleotide- (NAD(+)) or beta-nicotinamide adenine dinucleotide phosphate- (NADP(+)) dependent enzyme electrodes were prepared on single-walled carbon nanotube (SWCNT) supports. The SWCNTs were functionalized with Nile Blue (1), and the cofactors NADP(+) and NAD(+) were linked to 1 through a phenyl boronic acid ligand. The affinity complexes of glucose dehydrogenase (GDH) with the NADP(+) cofactor or alcohol dehydrogenase (AlcDH) with the NAD(+) cofactor were crosslinked with glutaric dialdehyde and the biomolecule-functionalized SWCNT materials were deposited on glassy carbon electrodes. The integrated enzyme electrodes revealed bioelectrocatalytic activities, and they acted as amperometric electrodes for the analysis of glucose or ethanol. The bioelectrocatalytic response of the systems originated from the biocatalyzed oxidation of the respective substrates by the enzyme with the concomitant generation of NAD(P)H cofactors. The electrocatalytically mediated oxidation of NAD(P)H by 1 led to amperometric responses in the system. Similarly, an electrically contacted bilirubin oxidase (BOD)-SWCNT electrode was prepared by the deposition of BOD onto the SWCNTs and the subsequent crosslinking of the BOD units using glutaric dialdehyde. The BOD-SWCNT electrode revealed bioelectrocatalytic functions for the reduction of O(2) to H(2)O. The different electrically contacted SWCNT-based enzyme electrodes were used to construct biofuel cell elements. The electrically contacted GDH-SWCNT electrode was used as the anode for the oxidation of the glucose fuel in conjunction with the BOD-SWCNT electrode in the presence of O(2), which acted as an oxidizer in the system. The power output of the cell was 23 muW cm(-2). Similarly, the AlcDH-SWCNT electrode was used as the anode for the oxidation of ethanol, which was acting as the fuel, with the BOD-SWCNT electrode as the cathode for the reduction of O(2). The power output of the system was 48 microW cm(-2).     

Characterization of immobilized glucose oxidase—catalase and their deactivation in a fluid-bed reactor

2-Amino-4-chloro-s-triazine, a derivative of DEAE-cellulose, and acrolein/styrene copolymer were used as supports for the immobilization of glucose oxidase and catalase after being modified with diaminohexane followed by glutaraldehyde. Immobilization was carried out with optimum glucose oxidase-catalase ratios. The activity variations of the immobilized dual-enzyme systems were investigated in relation to pH and temperature. Time-dependent gluconic acid production resulting from the oxidation of glucose was monitored in a recycling fluid-bed reactor. The deactivation rates of glucose oxidase and catalase were investigated according to the first-order reaction kinetics depending on the presence of the intermediate product H₂O₂.     

Oxygen- and temperature-dependent kinetic isotope effects in choline oxidase: correlating reversible hydride transfer with environmentally enhanced tunneling

Choline oxidase catalyzes the flavin-linked oxidation of choline to glycine betaine, with betaine aldehyde as intermediate and oxygen as electron acceptor. Here, the effects of oxygen concentration and temperature on the kinetic isotope effects with deuterated choline have been investigated. The D(kcat/Km) and Dkcat values with 1,2-[(2)H4]-choline were pH-independent at saturating oxygen concentrations, whereas they decreased at high pH to limiting values that depended on oxygen concentration at < or = 0.97 mM oxygen. The kcat/Km and kcat pH profiles had similar patterns reaching plateaus at high pH. Both the limiting kcat/Km at high pH and the pKa values were perturbed to lower values with choline and < or = 0.25 mM oxygen. These data suggest that oxygen availability modulates whether the reduced enzyme-betaine aldehyde complex partitions forward to catalysis rather then reverting to the oxidized enzyme-choline alkoxide species. At saturating oxygen concentrations, the D(kcat/Km) was 10.6 +/- 0.6 and temperature independent, and the isotope effect on the preexponential factors (A(H)'/A(D)') was 14 +/- 3, ruling out a classical over-the-barrier behavior for hydride transfer. Similar enthalpies of activation (deltaH(double dagger)) with values of 18 +/- 2 and 18 +/- 5 kJ mol(-1) were determined with choline and 1,2-[(2)H4]-choline. These data suggest that the hydride transfer reaction in which choline is oxidized by choline oxidase occurs quantum mechanically within a preorganized active site, with the reactive configuration for hydride tunneling being minimally affected by environmental vibrations of the reaction coordinate other than those affecting the distance between the donor and acceptor of the hydride.