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We describe an enzymatic fuel cell (EFC) that can electrooxidize glucose completely. The EFC contains the hybrid Ni@Pt-CNT/OxOx bioanode, composed of a bimetallic composite catalyst (Ni@Pt-CNT) and the enzyme oxalate oxidase (OxOx), which can cleave carbon-carbon bonds. Ni@Pt-CNT/OxOx displayed 3-fold higher catalytic activity in the presence than in the absence of glucose (1.3 and 0.4 mA cm−2, respectively), indicating that Ni@Pt-CNT and OxOx acted synergistically. Electrochemical impedance spectroscopy showed that Ni@Pt-CNT/OxOx had higher charge transfer resistance and double layer capacitance than Ni@Pt-CNT. Long-term bulk electrolysis (18 h) revealed that the EFC operating with Ni@Pt-CNT/OxOx presented better current density and stability than the electrochemical cell operating with Ni@Pt-CNT, so deep glucose electrooxidation generated energy. The glucose oxidation products detected by HPLC-UV/RID confirmed that glucose was fully electrooxidized, and that 24 electrons were harvested from it. The hybrid Ni@Pt-CNT/OxOx bioanode developed herein could be used in cascade reactions, to provide an EFC with promising application in self-powered electronic devices.

Isothermal titration calorimetry (ITC) may be used to determine the kinetic parameters of enzyme-catalyzed reactions when neither products nor reactants are spectrophotometrically visible and when the reaction products are unknown. We report here the use of the multiple injection method of ITC to characterize the catalytic properties of oxalate oxidase (OxOx) from Ceriporiopsis subvermispora (CsOxOx), a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The multiple injection ITC method of measuring OxOx activity involves continuous, real-time detection of the amount of heat generated (dQ) during catalysis, which is equal to the number of moles of product produced times the enthalpy of the reaction (ΔHapp). Steady-state kinetic constants using oxalate as the substrate determined by multiple injection ITC are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase-catalyzed oxidation of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) and by membrane inlet mass spectrometry. Additionally, we used multiple injection ITC to identify mesoxalate as a substrate for the CsOxOx-catalyzed reaction, with a kinetic parameters comparable to that of oxalate, and to identify a number of small molecule carboxylic acid compounds that also serve as substrates for the enzyme.

Oxalic acid (OA) is considered as an important pathogenetic factor of some destructive diseases caused by some fungal pathogens such as Sclerotinia sclerotiorum. Oxalate degradation is important for plant health, and plants that contain oxalate oxidase (OXO) enzymes could breakdown oxalate into CO2 and H2O2, which subsequently evokes defense responses. However, some species, such as Arabidopsis thaliana, have no oxalate oxidase activity identified to date. The present study aims to develop transgenic Arabidopsis expressing a wheat oxalate oxidase, to test for the response to OA exposure and fungal infection by S. sclerotiorum. The results showed that the transgenic Arabidopsis lines that expressed the wheat OXO exhibited enhanced resistance to OA exposure and S. sclerotiorum infection in the tolerance assays. In the same manner, it could convert OA to CO2 and H2O2 to a higher extent than the wild-type. Intensive osmotic adjustments were also detected in the transgenic Arabidopsis lines. The higher level of produced H2O2 subsequently induced an elevated activity of antioxidant enzymes including superoxide dismutase (SOD) and peroxidase (POD) in the transgenic Arabidopsis plants. The present study indicated that the expression of a gene encoding wheat OXO could induce intensive osmotic adjustments and hydrogen peroxide related defense response, and subsequently increased tolerance to S. sclerotiorum in transgenic A. thaliana.

We have described for the first time the localisation of oxalate oxidase (OXO, EC 1.2.3.4) in Abortiporus biennis cells, using histochemical and immunochemical methods coupled with transmission electron microscopy. Rabbit anti-oxalate oxidase immunoglobulins with anti-rabbit secondary antibody conjugated with 10-nm gold particles were used. Moreover, the formation of electron dense precipitation of reaction of diaminobenzidine (DAB) with horseradish peroxidase (HRP) for histochemical localisation of the enzyme was found. OXO was localised close to the membranous structures of the cell membranes, in membranous vesicles located close to the outer cell membrane, and vacuolar membranes surrounding vacuoles. The positive immunoreaction to OXO was also intense in cell wall areas. Moreover, we proved that gene coding for OXO was expressed in the same cultures. Corresponding mRNA was isolated, full length cDNA was synthesized, cloned and sequenced. Two copies of cupin domains were found in the sequence of amino-acids conserved domain coding for the cupin enzyme. Comparison of the genomic DNA and cDNA sequences has revealed the presence of seventeen introns in the gene. The isoelectric point of the protein was estimated at pH 4.5 and several possible N-glycosylation sites were predicted., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Electrochemical ethanol oxidation was performed at an innovative hybrid architecture electrode containing TEMPO-modified linear poly(ethylenimine) (LPEI) and oxalate oxidase (OxOx) immobilized on carboxylated multi-walled carbon nanotubes (MWCNT-COOH). On the basis of chromatographic results, the catalytic hybrid electrode system completely oxidized ethanol to CO 2 after 12 h of electrolysis. The fact that the developed system can catalyze ethanol electrooxidation at a carbon electrode confirms that organic oxidation catalysts combined with enzymatic catalysts allow up to 12 electrons to be collected per fuel molecule. The Faradaic efficiency of the hybrid system investigated herein lies above 87%. The combination of OxOx with TEMPO-LPEI to obtain a novel hybrid anode to oxidize ethanol to carbon dioxide constitutes a simple methodology with useful application in the development of enzymatic biofuel cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make Peruvianin‑I an interesting model for further understanding of the action of laticifer fluids in plant defense., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Oxalic acid is a naturally occurring metabolite in plants and a common constituent of all plant-derived human diets. Oxalic acid has diverse unrelated roles in plant metabolism, including pH regulation in association with nitrogen metabolism, metal ion homeostasis and calcium storage. In plants, oxalic acid is also a pathogenesis factor and is secreted by various fungi during host infection. Unlike those of plants, fungi and bacteria, the human genome does not contain any oxalate-degrading genes, and therefore, the consumption of large amounts of plant-derived oxalate is considered detrimental to human health. In this review, we discuss recent biotechnological approaches that have been used to reduce the oxalate content of plant tissues., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The biochemical and enzymatic properties of four highly similar rice oxalate oxidase proteins (OsOxO1-4) were compared after their purification from the leaves of transgenic plants each overexpressing the respective OsOxO1-4 genes. Although alignment of their amino acid sequences has revealed divergence mainly in the signal peptides and they catalyze the same enzymic (oxalate oxidase) reaction, divergence in apparent molecular mass, Km, optimum pH, stability and responses to inhibitors and activators was uncovered by biochemical characterization of the purified OsOxO1-4 proteins. The apparent molecular mass of oligomer OsOxO1 was found to be similar to that of OsOxO3 but lower than the other two. The molecular mass of the subunit of OsOxO1 was lower than that of OsOxO3. The Km value of OsOxO3 was higher than the other three which had similar Km. OsOxO1 and OsOxO4 possessed peak activity at pH 8.5 which was close to that at the optimum pH 4.0. The activity of OsOxO2 at pH 8.5 was only 65% of that at its optimum pH 3.5, while the activity of OsOxO3 did not vary much at pH 6-9 and was also much lower than that at its optimum pH 3. OsOxO2 and OsOxO3 still maintained all their activities after being heated at 70°C for 1h while OsOxO1 and OsOxO4 lost about 30% of their activities. Pyruvate and oxaloacetic acid inhibited the activity of OsOxO3 more strongly than the other three. Interestingly, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-biphosphate related to photosynthetic assimilation of triose phosphate greatly increased the activities of OsOxO3 and OsOxO4. In addition to the differences in the biochemical properties of the four OsOxO proteins, an intriguing finding is that the purified OsOxO1-4 exhibited substrate inhibition, which is a typical of the classical Michaelis-Menten enzyme kinetics exhibited by a majority of other enzymes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Although reactive oxygen species (ROS) are highly toxic substances that are produced during aerobic respiration and photosynthesis, many studies have demonstrated that ROS, such as superoxide anion radical (O2(·-)) and hydrogen peroxide (H2O2), are produced in the plant cell wall in a highly regulated manner. These molecules are important signalling messengers playing key roles in controlling a broad range of physiological processes, such as cellular growth and development, as well as adaptation to environmental changes. Given the toxicity of ROS, especially of hydroxyl radical (·OH), the enzymatic ROS production needs to be tightly regulated both spatially and temporally. Respiratory burst oxidase homologues (Rboh) have been identified as ROS-producing NADPH oxidases, which act as key signalling nodes integrating multiple signal transduction pathways in plants. Also other enzyme systems, such as class III peroxidases, amine oxidases, quinone reductases and oxalate oxidases contribute to apoplastic ROS production, some especially in certain plant taxa. Here we discuss the interrelationship among different enzymes producing ROS in the plant cell wall, as well as the physiological roles of the ROS produced., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Germins and germin-like proteins (GLPs) comprise large families of extracellular plant glycoproteins that are structurally similar, yet they have been reported to have distinct biochemical activities: oxalate oxidase and superoxide dismutase activities, respectively. We expressed an azalea GLP (RmGLP2) in cultured cells of tobacco, and determined that the extracellular protein fraction and the recombinant RmGLP2 protein purified from these cells catalyzed the oxidation of oxalate. Notably, this activity is purportedly restricted to germin and has not been demonstrated for a GLP. Although the specific activity of the purified RmGLP2 protein was low compared with that of a previously characterized barley germin/oxalate oxidase, tobacco cells expressing RmGLP2 exhibited significantly reduced oxalate levels. Thus, RmGLP2 represents the first reported GLP with oxalate oxidase activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Membrane inlet mass spectrometry (MIMS) uses a semipermeable membrane as an inlet to a mass spectrometer for the measurement of the concentration of small uncharged molecules in solution. We report the use of MIMS to characterize the catalytic properties of oxalate oxidase (E.C. 1.2.3.4) from Ceriporiopsis subvermispora (CsOxOx). Oxalate oxidase is a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The MIMS method of measuring OxOx activity involves continuous, real-time direct detection of oxygen consumption and carbon dioxide production from the ion currents of their respective mass peaks. (13)C2-oxalate was used to allow for accurate detection of (13)CO2 (m/z 45) despite the presence of adventitious (12)CO2. Steady-state kinetic constants determined by MIMS are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). Furthermore, we used MIMS to determine that NO inhibits the activity of the CsOxOx with a KI of 0.58±0.06 μM., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)