Pyranose 2-oxidase Research Articles

An electrospun nanofiber matrix based on organo-clay for biosensors: PVA/PAMAM-Montmorillonite

Diagnostic techniques based on biomolecules have huge a potential to be applied in the application in various areas such as food/beverage industries, diseases diagnostics, monitoring of bio-processes and environmental pollutants. Immobilization of biomolecules on a transducer is the key parameter to being able to prepare a highly stable diagnostic tests. Electrospun nanofibers are a good alternative to immobilize biomolecules. Here, electrospun nanofibers based on an organoclay were used to design the first generation amperometric enzyme biosensor. PAMAM G2 dendrimers were used to intercalate montmorillonite clay (Mt) and then the modification of Mt by PAMAM was characterized using FTIR, XRD, TGA and zeta potential measurements. After that nanofibers were prepared by electrospinning Mt and PAMAM-Mt using poly(vinyl) alcohol (PVA) as an auxiliary polymer and the formed PVA/PAMAM-Mt electrospun nanofibers were proved by SEM, TEM and AFM techniques. Finally, pyranose oxidases (PyOx) were immobilized on a glassy carbon electrode surface, which was modified using the PVA/PAMAM-Mt electrospun nanofibers. Amperometric measurements were carried out using buffer solution at −0.7 V under stirring conditions. The linear response for glucose was from 0.005 mM to 0.25 mM using PVA/Mt/PyOx and PVA/PAMAM-Mt/PyOx biosensors. The limit of detection was 0.7 µM glucose with PVA/PAMAM-Mt/PyOx biosensor. To detect glucose in real sample, measurements were carried out using soft drink cola as a substrate instead of glucose.

Biochemical and mutational analyses of a Trametes pyranose oxidase and comparison of its mutants in breadmaking

Pyranose oxidase (POx) is a homotetrameric flavoprotein that catalyzes the oxidation of pyranose-configured sugars at position C-2 to corresponding 2-ketoaldoses. The wide substrate specificity makes POx potential for application in various biotechnological industries. In the present study we reported the gene cloning and heterologous expression of a POx from the basidiomycete Trametes sp. and functionally expressed the gene in Escherichia coli BL21(DE3). Based on sequence alignment, three residues were chosen for site-directed mutagenesis to obtain two single mutants (K312E and E539K) and two double mutants (T166A/E539K and K312E/E539K). In comparison to the wild-type, K312E shifted its optimal pH to 5.5 while the optimal temperature of E539K and K312E/E539K increased by 10 °C. The mutants retained more activities over broader pH ranges and higher temperatures and catalyzed d-glucose at higher efficiency (5800‒12,667 M−1 s−1 for the mutants versus 5083 M−1 s−1 for the wild-type). The recombinant POx and its mutants were all useful in gluten agglomeration and enlarging the loaf volume, which depends on the amounts of enzymes added. Interestingly, adding the same amount (0.5 nkat/g of flour) of wild-type and mutant enzymes differed in the change of loaf volumes, pinpointing that the catalytic activity is not the sole determinant in applying POx in breadmaking.

Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase.

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [α-2H2]- p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s-1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H+ transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H+ from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes.

Colorimetric detection of 1,5-anhydroglucitol based on graphene quantum dots and enzyme-catalyzed reaction

Early diagnosis of diabetes yields significant clinical benefits. The serum level of 1,5‑anhydroglucitol (1,5‑AG) has been a new biochemical marker for postprandial hyperglycemia. In this study, a simple colorimetric method for 1,5‑AG detection has been designed based on highly efficient peroxidase mimetic activity of GQDs and enzyme-catalyzed reaction. By the catalytic action of pyranose oxidase (PROD), the 1,5‑AG was oxidized to 1,5‑anhydrofuctose and H2O2. The GQDs in the presence of H2O2 exhibited highly efficient catalytic activity toward the oxidation of 3, 3′, 5, 5′‑tetramethylbenzidine (TMB) to a blue colored product. The influence of relevant experimental variables was optimized. A linear relationship of optical signal with the concentration of 1,5‑AG in the range of 20.0–100.0μg/mL with the regression correlation coefficient of 0.9985 was obtained which could be monitored by colorimetry detection. The limit of detection (LOD) for 1,5‑AG detection was approximately 0.144μg/mL. All in all, the proposed 1,5‑AG detection system based on GQDs and PROD-catalyzed reaction showed better performances with simple operation, low-cost, higher selectivity.

Characterization of rheological properties of rye arabinoxylans in buckwheat model systems

The aim of this investigation was to study the rheological properties (gelation profile, mixing and pasting properties) of two rye arabinoxylans (AXs) (water-extracted (WEAXs), calcium hydroxide-extracted (CEAXs)) in buckwheat model systems using wholemeal and white flour. To promote gelation in these systems, pyranose 2-oxidase (POx) was added. AX characterization in solution showed a higher gelation profile for the CEAXs (G’: 0.48 Pa, G’’: 0.25 Pa) compared with the WEAXs (G’: 0.21 Pa, G’’: 0.14 Pa), probably due to differences in chemical and structural properties. In buckwheat batter systems, highest rheological properties were achieved when POx was added to the control flours (for wholemeal flour: G’: 40.1 kPa, G’’: 8.6 kPa; for white flour: G’: 18.7 kPa, G’’: 1.4 kPa), whereas most AX concentrations improved these properties to a lower degree. Nearly all wholemeal flour systems reached higher viscoelastic properties when containing CEAXs (G’: 20.0–35.1 kPa; G’’: 4.2–6.7 kPa), while WEAXs improved the majority of these properties in systems made with white flour (G’: 10.4–12.7 kPa; G’’: 2.2–2.3 kPa). No additional effect was seen in the batter viscoelasticity when POx was combined with these AXs. Pasting and mixing properties of the flour systems were mostly reduced by the addition of AXs, while the presence of POx displayed little or no further effect. These observations indicate that AXs could be applied as natural structure-forming agents in GF bread, when used in the right amount.

Enzymatic Cascade Catalysis for the Synthesis of Multiblock and Ultrahigh‐Molecular‐Weight Polymers with Oxygen Tolerance

Synthesis of well-defined multiblock and ultrahigh-molecular-weight (UHMW) polymers has been a perceived challenge for reversible-deactivation radical polymerization (RDRP). An even more formidable task is to synthesize these extreme polymers in the presence of oxygen. A novel methodology involving enzymatic cascade catalysis is developed for the unprecedented synthesis of multiblock polymers in open vessels with direct exposure to air and UHMW polymers in closed vessels without prior degassing. The success of this methodology relies on the extraordinary deoxygenation capability of pyranose oxidase (P2Ox) and the mild yet efficient radical generation by horseradish peroxidase (HRP). The facile and green synthesis of multiblock and UHMW polymers using biorenewable enzymes under environmentally benign and scalable conditions provides a new pathway for developing advanced polymer materials.

Enzymatic Cascade Catalysis for the Synthesis of Multiblock and Ultrahigh‐Molecular‐Weight Polymers with Oxygen Tolerance

AbstractSynthesis of well‐defined multiblock and ultrahigh‐molecular‐weight (UHMW) polymers has been a perceived challenge for reversible‐deactivation radical polymerization (RDRP). An even more formidable task is to synthesize these extreme polymers in the presence of oxygen. A novel methodology involving enzymatic cascade catalysis is developed for the unprecedented synthesis of multiblock polymers in open vessels with direct exposure to air and UHMW polymers in closed vessels without prior degassing. The success of this methodology relies on the extraordinary deoxygenation capability of pyranose oxidase (P2Ox) and the mild yet efficient radical generation by horseradish peroxidase (HRP). The facile and green synthesis of multiblock and UHMW polymers using biorenewable enzymes under environmentally benign and scalable conditions provides a new pathway for developing advanced polymer materials.

Dynamics of the protein structure of T169S pyranose 2-oxidase in solution: Molecular dynamics simulation.

Pyranose 2-oxidase (P2O) from Trametes multicolor contains FAD as cofactor, and forms a tetramer. The protein structure of a mutated P2O, T169S (Thr169 is replaced by Ser), in solution was studied by means of molecular dynamics simulation and analyses of photoinduced electron transfer (ET) from Trp168 to excited isoalloxazine (Iso*), and was compared with wild type (WT) P2O. Hydrogen bonding between Iso and nearby amino acids was very similar as between T169S and WT protein. Distances between Iso and Tyr456 were extremely heterogeneous among the subunits, 1.7 (1.5 in WT) in subunit A (Sub A), 0.97 (2.2 in WT) in Sub B, 1.3 (2.1 in WT) in Sub C, 1.3 nm (2.0 in WT) in Sub D. Mean values of root of mean square fluctuation over all residues were greater by four times than those in WT. This suggests that the protein structure of T169S is much more flexible than that of WT. Electrostatic (ES) energies between Iso anion in one subunit and ionic groups in the entire protein were evaluated. It was found that more than 50% of the total ES energy in each subunit is contributed from other subunits. Reported fluorescence decays were analyzed by a method as WT, previously reported. Electron affinities of Iso* in T169S were appreciably higher than those in WT. Static dielectric constants near Iso and Trp168 were also quite higher in T169S than those in WT.

Pareto-Optimal Operating Policies of a Three-Phase- Fluidized-Bed Reactor Used for the Oxidation of D-Glucose on Co-Immobilized Pyranose Oxidase and Catalase

Pareto-Optimal Operating Policies of a Three-Phase- Fluidized-Bed Reactor Used for the Oxidation of D-Glucose on Co-Immobilized Pyranose Oxidase and Catalase

Carbon Nanotube Modified Screen Printed Electrodes: Pyranose Oxidase Immobilization Platform for Amperometric Enzyme Sensors

Here, a novel enzymatic biosensor was developed using multiwalled carbon nanotube including screen printed electrodes (MWCNT-SPE). Pyranose oxidase (PyOx) was immobilized on the electrode surface by way of gelatin membrane and then cross-linked using glutaraldehyde. Glucose was detected at -0.7 V (vs. Ag/AgCl) by watching consumed oxygen in enzymatic reaction after addition substrate. After optimization of pH and enzyme loading, the linearity was found in the range of 0.1–1.0 mM of glucose. After that, the effect of MCNT on the current was tested. Also the enzymatic biosensor including glucose oxidase instead of pyranose oxidase was prepared and the biosensor response followed for glucose. Furthermore, this system was tested for glucose analysis in soft drinks.

Light-addressable potentiometric sensor with gold nanoparticles enhancing enzymatic silver deposition for 1,5-anhydroglucitol determination

In this study, a light-addressable potentiometric sensor (LAPS) based on gold nanoparticles (AuNPs) enhancing enzymatic silver (Ag) deposition was developed for 1,5-anhydroglucitol (1,5-AG) determination. The pyranose oxidase enzyme (PROD), immobilized on the surface of AuNPs modified LAPS chip, could catalyze the hydrolysis of 1,5-AG and produce a reductive product H2O2 that enhanced the deposition of Ag. When a bias voltage was provided to the LAPS, a photocurrent which depended on the concentration of 1, 5-AG can be generated. Because the concentration of Ag ions in the electrolyte solution decreased with the deposition of metallic Ag on the surface of AuNPs modified LAPS chip, the potential shift (ΔV) has originated from local surface potential changes and reflected the changes of 1,5-AG concentration. The linear relationship between ΔV and 1,5-AG concentration were very well over the range from 60μg/mL to 225μg/mL with a correlation coefficient of 0.9977. The limit of detection was 40μg/mL at signal/noise [S/N]=3. In addition, LAPS detection system also showed higher specificity, acceptable stability and excellent recovery for 1, 5-AG detection.

Operation of a mechanically agitated semi-continuous multi-enzymatic reactor by using the Pareto-optimal multiple front method

One essential engineering problem when developing an industrial enzymatic process concerns the choice of the reactor operating alternative based on à-priori knowledge of the process kinetics and enzyme inactivation characteristics. For a multi-enzymatic system, involving complex interactions among enzymes that exhibit optimal activity on different parametric domains, and a high-order deactivation, this problem requires an extended analysis. The optimization and control engineering problems become very challenging due to the process high nonlinearity and the presence of a large number of complex constraints. All these will translate into a multi-objective optimization problem to be solved for each case. An elegant option developed in this paper is to obtain sets of Pareto optimal solutions, also called Pareto-optimal fronts, successively generated for pairs of adverse objectives. Then, the final choice of the enzymatic reactor operating policy results from the comparative analysis of these Pareto-fronts. Exemplification is made for the complex case of the oxidation of D-glucose (DG) to 2-keto-d-glucose (kDG) in the presence of P2Ox (Pyranose oxidase, EC 1.1.3.10) and catalase, continuously operated in a three-phase mechanically agitated semibatch reactor (MASCR) with co-immobilized enzymes on alginate beads. Optimal operating policy choice is based on the minimum amount of required P2Ox that ensures an imposed reaction conversion and maximum reactor productivity under various technological constraints, and for catalase/P2Ox ratios of 200–300U/U.

Synthesis of 3-Keto-levoglucosan Using Pyranose Oxidase and Its Spontaneous Decomposition via β-Elimination.

3-Keto-levoglucosan (3ketoLG) has been postulated to be the product of a reaction catalyzed by levoglucosan dehydrogenase (LGDH), a bacterial enzyme involved in the metabolism of levoglucosan (LG). To investigate the LG metabolic pathway catalyzed by LGDH, 3ketoLG is needed. However, 3ketoLG has not been successfully isolated from the LGDH reaction. This study investigated the ability of pyranose oxidase to convert LG into 3ketoLG by oxidizing the C3 hydroxyl group. During the oxidation of LG, 3ketoLG was spontaneously crystallized in the reaction mixture. Starting with 500 mM LG, the isolation yield of 3ketoLG was 80 %. Nuclear magnetic resonance analyses revealed that a part of 3ketoLG dimerized in aqueous solution, explaining its poor solubility. Even under normal conditions, 3ketoLG was unstable in aqueous solution, with a half-life of 16 h at pH 7.0 and 30 °C. The decomposition proceeded through β-elimination of the C–O bonds at both C1 and C5, as evidenced by decomposition products. This instability explains the difficulty in obtaining 3ketoLG via the LGDH reaction.

改良型減圧機構を用いたグルコース駆動式薬物放出システムに関する研究

In this work, an enhanced performance of an enzymatic chemo-mechanical actuator that can convert the chemical energy of glucose into mechanical energy for autonomous drug release without an electrical power is reported. The novel biochemical approach is based on increasing the decompression rate in a “vacuum unit” by fabricating enzyme co-immobilized membrane by multiple enzymes. Among the enzymes (glucose oxidase (GOD), pyranose oxidase (POD), alcohol oxidase (AOD)), which can oxidize glucose and/or glucono-1.5-lactone evaluated in co-immobilization designs within the vacuum unit, the highest decompression was obtained with POD+GOD, which was 3 times higher than that of the conventional organic engine with only GOD. Furthermore, the decompression rate of -7.4 Pa･cm3/sec in the vacuum unit necessary to drive the drug release system was obtained at 10 mmol/L glucose, which is close to the human blood sugar level. In conclusion, the vacuum unit is a promising device for development of a chemo- mechanical system driven by human blood sugar for the diabetes treatment.

Characterization of a bacterial pyranose 2-oxidase from Arthrobacter siccitolerans

In this study we provide the first biochemical characterization of a bacterial pyranose 2-oxidase (AsP2Ox) from Arthrobacter siccitolerans. The enzyme catalyzes the oxidation of several aldopyranoses at the C-2 position, coupling it to the reduction of dioxygen to hydrogen peroxide. Pyranose 2-oxidases belong to the glucose-methanol-choline oxidoreductase family. A structural model based on the known X-ray structure of P2Ox from Phanerochaete chrysosporium supports that AsP2Ox shares structural features with well-characterized fungal P2Oxs. The gene coding for AsP2Ox was cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme is a 64-kDa monomer containing a non-covalently bound flavin adenine dinucleotide (FAD) cofactor, distinct features as compared with fungal counterparts that are ∼ 270kDa homotetramers with covalent-linked FAD. AsP2Ox exhibits a redox potential of −50mV, an optimum temperature of 37°C and an optimum pH at 6.5. AsP2Ox oxidizes d-glucose at the highest efficiency, using additionally d-galactose, d-xylose, l-arabinose and d-ribose as electron donors, coupling their oxidation to the reduction of both dioxygen and 1,4-benzoquinone. AsP2Ox shows a relatively low thermal stability with a melting temperature (Tm) of 43°C and a half-life (t1/2) at 40°C of 25min. This work expands the repertoire of bacterial oxidoreductases with importance in biotechnological and diagnostic applications.

Enzyme precipitate coating of pyranose oxidase on carbon nanotubes and their electrochemical applications

Pyranose oxidase (POx), which doesn't have electrically non-conductive glycosylation moiety, was immobilized on carbon nanotubes (CNTs) via three different preparation methods: covalent attachment (CA), enzyme coating (EC) and enzyme precipitate coating (EPC). CA, EC and EPC of POx on CNTs were used to fabricate enzymatic electrodes for enzyme-based biosensors and biofuel cells. Improved enzyme loading of EPC resulted in 6.5 and 4.5 times higher activity per weight of CNTs than those of CA and EC, respectively. After 34 days at room temperature, EPC retained 65% of initial activity, while CA and EC maintained 9.2% and 26% of their initial activities, respectively. These results indicate that precipitation and crosslinking steps of EPC have an important role in maintaining enzyme activity. To demonstrate the feasibility of POx-based biosensors and biofuel cells, the enzyme electrodes were prepared using CA, EC, and EPC samples. In the case of biosensor, the sensitivities of the CA, EC, and EPC electrodes without BQ were measured to be 0.27, 0.76 and 3.7mA/M/cm2, while CA, EC and EPC electrode with BQ showed 25, 25, and 60mA/M/cm2 of sensitivities, respectively. The maximum power densities of biofuel cells using CA, EC and EPC electrodes without BQ were 41, 47 and 53µW/cm2, while CA, EC and EPC electrodes with BQ showed 260, 330 and 500µW/cm2, respectively. The POx immobilization and stabilization via the EPC approach can lead us to develop continuous glucose monitoring biosensors and high performing biofuel cells.

Quick identification of a simple enzyme deactivation model for an extended-Michaelis-Menten reaction type. Exemplification for the D-glucose oxidation with a complex enzyme deactivation kinetics

Abstract One essential engineering problem when developing an industrial enzymatic process concerns the model-based design and optimal operation of the enzymatic reactor based on the process and enzyme inactivation kinetics. For a complex enzymatic system, the “default” used first-order enzyme deactivation model has been proved to lead to inadequate process design or sub-optimal operating policies. The present study investigates if a complex enzyme deactivation can be approximated with simple 1st, 2nd, or a novel proposed model with variable deactivation constant. The approached complex enzymatic system is those of the oxidation of D-glucose to 2-keto-D-glucose in the presence of pyranose 2-oxidase. The necessary “simulated experimental data” have been generated by means of an extended kinetic model from literature used to simulate a batch reactor under well-defined nominal conditions. The proposed enzyme deactivation model has been found to be the best lumping alternative, presenting several advantages: simplicity, flexibility, and a very good adequacy.

CTAB modified dellite: A novel support for enzyme immobilization in bio-based electrochemical detection and its in vitro antimicrobial activity

A novel support material for enzyme immobilization based on cetyltrimethylammonium bromide modified Dellite (CTAB-Del) was successfully synthesized and used to manufacture pyranose oxidase (PyOx) biosensors (CTAB-Del/PyOx). The intercalation of CTAB into Dellite was confirmed by FTIR, XRD and TGA techniques. PyOx was immobilized onto the glassy carbon electrode, via glutaraldehyde crosslinking, by using CTAB-Del as a support. In order to test the analytical performance of CTAB-Del/PyOx biosensors, chronoamperometric measurements were carried out using three electrodes configurations, at a constant potential of −0.7V in working buffer, under stirring, with successive addition of glucose. The linear response for CTAB-Del/PyOx biosensor ranged from 0.01 to 0.50mM with an equation of y=4.42x+0.004 (R2=0.998), and the limit of detection for glucose was calculated to be 0.081μM (S/N=3). In order to confirm its practical use, the CTAB-Del/PyOx biosensor was also applied for glucose measurement in various beverages. In addition, the antimicrobial activities of Del and CTAB-Del were screened in vitro by means of the disc diffusion susceptibility test, selecting a yeast, three different Gram-positive, and five different Gram-negative bacteria. The obtained results showed a moderate antibacterial activity of CTAB-Del against Gram-positive bacteria.

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.

Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity.

This study presents the first electrochemical characterization of the pyranose oxidase (POx) variant N593C (herein called POx-C), which is considered a promising candidate for future glucose-sensing applications. The resulting cyclic voltammograms obtained in the presence of various concentrations of glucose and mediator (1,4-benzoquinone, BQ), as well as the control experiments by addition of catalase, support the conclusion of a complete suppression of the oxidase function and oxygen reactivity at POx-C. Additionally, these electrochemical experiments demonstrate, contrary to previous biochemical studies, that POx-C has a fully retained enzymatic activity towards glucose. POx-C was immobilized on a special screen-printed electrode (SPE) based on carbon ink and grafted with gold-nanoparticles (GNP). Suppression of the oxygen reactivity at N593C-POx variant is a prerequisite for utilizing POx in electrochemical applications for glucose sensing. To our knowledge, this is the first report presented in the literature showing an absolute conversion of an oxidase into a fully active equivalent dehydrogenase via a single residue exchange.