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We report an Enzymatic Fuel Cell (EFC) combining an enzyme that can cleave carbon-carbon bonds (oxalate oxidase (OxOx)) with an organic catalyst (Pyrene-TEMPO (TEMPO = 2,2,6,6-tetramethyl piperidinyl-N-oxyl)) immobilized on the surface of modified carboxylated multi-walled carbon nanotubes (MWCNT-COOH). This combination gave a hybrid bi-catalyst electrode for complete ethylene glycol (EG) oxidation. The hybrid electrode provided ninefold enhanced catalytic activity (0.17 ± 6 × 10−3 mA cm−2) in the presence of EG as compared to the electrode in the absence of EG (0.018 ± 3 × 10−5 mA cm−2), indicating that the enzyme combined with the organic catalyst improved energy generation through deep EG electrooxidation. Electrochemical impedance spectroscopy reveals that the addition of the enzyme in the electrode containing MWCNT–COOH–Pyrene-TEMPO increased the charge transfer resistance (R c t ) and the capacitance of the double layer. Long-term electrolysis for 15 h showed that the hybrid electrode presented outstanding current density and stability. The EG oxidation products were identified and quantified by high-performance liquid chromatography (HPLC-UV/RID). The results confirmed complete EG oxidation in the presence of CO 2 in the solution, allowing 10 electrons to be collected from the fuel. Overall, this study illustrates the development of a simple and improved hybrid bi-catalyst electrode for promising applications in small electronic devices. [ABSTRACT FROM AUTHOR]

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, K m , 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 K m value of OsOxO3 was higher than the other three which had similar K m . 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 1 h 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. [ABSTRACT FROM AUTHOR]

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe ions, two residue clusters of OxOx were assigned as potential Fe binding sites, the mechanism of the inhibition of Fe was delineated. Importantly, the residues that interact with Fe ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe highlights insight into substrate binding site in OxOx. [ABSTRACT FROM AUTHOR]

Germin and germin-like proteins (GLPs) are a broad and diverse family of developmentally regulated proteins widely distributed in plants. Oryza sativa L. harbours a large family of GLPs and serves as a good model for their study. In the present study, a germin-like protein gene ( OsRGLP1) of rice origin was characterized by its heterologous expression in tobacco. The real-time PCR established almost a uniform expression of OsRGLP1 in leaves, stem, and roots of T Nicotiana tabacum cv. Samsun. Although no morphological difference was apparent between T transgenic and wild-type plants, leaves of mature transgenic plants showed necrotic lesions associated with an elevated content of HO, which was evidenced by in situ 3,3′-diaminobenzidine staining. A significantly higher activity of heat resistant superoxide dismutase (SOD) was observed in the transgenic plants as compared to the wild-type. The SOD activity in the transgenic plants was insensitive to potassium cyanide and sensitive to HO. [ABSTRACT FROM AUTHOR]

Multi-year transgenic field trials were conducted to assess the extent of pollen-mediated transgenic flow in Virginia to support a petition requesting deregulated status for Blight Blocker peanuts from USDA Animal and Plant Health Inspection Service (APHIS) Biotechnology Regulatory Services (BRS). We measured transgene flow from transgenic lines to their non-transgenic parental cultivars. A colorimetric method based on quantification of hydrogen peroxide released from oxalic acid in the presence of the oxalate oxidase was used to screen seed embryos from non-transgenic rows at various distances from the transgenic source. The overall transgene flow rate in three cultivars was 0.2094% based on screening over 85,000 seeds. In general, the transgene flow rate greatly declined past 10 m from the transgene source. However, a transgene flow rate of less than 0.05% did occur sporadically at greater distances than 10 m. In conclusion, transgene flow in peanut can be spatially confined to provide negligible rates using relatively short separation distances. The extremely low rate of transgene flow at greater distance was dependent on ecological and environmental contexts, particularly on foraging patterns and flight distance of pollinators. [ABSTRACT FROM AUTHOR]

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 ( O 2 - ) and hydrogen peroxide (H 2 O 2 ), 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 13C2-oxalate was used to allow for accurate detection of 13CO2 (m/z 45) despite the presence of adventitious 12CO2. 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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Oxalate contents of foods reliably determined with new oxalate oxidase method. [•] New method uses oxalate oxidase prepared from wheat bran. [•] Some commonly available foods in Southern China have high oxalate levels. [•] Chinese wolfberry, dragon and abalone fruit, Chinese torreya fruit: high in oxalate. [•] Water spinach, black rice, rice bean, are also high in oxalate. [Copyright &y& Elsevier]

Abstract: Graphene oxide nanoparticles (G@NP) were synthesized from graphite powder of lead pencil and characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD). A mixture of G@NPs and polypyrrole (PPy) was electropolymerized onto gold (Au) electrode followed by electrodeposition of a mixture of polyaniline (PANI) and chitosan (CHIT) onto G@NPs/PPy/Au to construct CHIT/PANI/G@NPs/PPy/Au electrode. An oxalate oxidase (OXO) purified from strawberry fruits was immobilized on to this modified Au electrode through chitosan. The enzyme electrode (OXO/CHIT/PANI/G@NPs/PPy/Au electrode) was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectrophotometry and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The OXO/G@NPs/PPy/PANI/CHIT/Au electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxillary electrode were connected through a potentiostat to fabricate an amperometric oxalate biosensor. The biosensor exhibited optimum response within 3s at pH 5.5, 35°C with a linearity, between 1 and 400μM for oxalic acid and a detection limit of 1μM. Apparent Michaelis–Menten constant (K m) for oxalate was 12.5μM while I max was 0.008mA. The optimized biosensor measured oxalate level in urine and plasma collected from apparently healthy persons and urinary stone formers. [Copyright &y& Elsevier]

Previous work on the adaptation of maize (Zea mays L.) primary root growth to water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. Cell wall proteomic analysis suggested that levels of apoplastic reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), may be modified in a region-specific manner within the growth zone of water-stressed roots. Apoplastic ROS may have wall loosening or tightening effects and may also have other growth regulatory functions. To gain an understanding of how apoplastic ROS levels change under water stress, cerium chloride staining was used in conjunction with transmission electron microscopy to examine the spatial distribution of apoplastic H2O2. The results revealed that apoplastic H2O2 levels increased specifically in the apical region of the growth zone under water stress, correlating spatially with the maintenance of cell elongation. The basal regions of the growth zone of water-stressed roots and the entire growth zone of well-watered roots exhibited relatively low levels of apoplastic H2O2. The increase in apoplastic H2O2 in the apical region under water stress probably resulted, at least in part, from a pronounced increase in oxalate oxidase activity in this region. By contrast, well-watered roots showed negligible oxalate oxidase activity throughout the growth zone. The results show that changes in apoplastic ROS levels in the root growth zone under water-deficit conditions are regulated in a spatially-specific manner, suggesting that this response may play an important role in maize root adaptation to water stress. [ABSTRACT FROM AUTHOR]

Abstract: Oxalate oxidase has potential to act as an oxygen scavenger in active packaging to increase the shelf-life of food and beverages, while simultaneously producing the protective packaging gas carbon dioxide. This study shows that oxalate oxidase from barley can be immobilized with retained catalytic activity through entrapment in a latex polymer matrix. Conditions for formation of film containing oxalate oxidase have been evaluated as well as effects of storage and latex on enzyme activity, migration of enzyme in films, and the ability of the latex films to resist higher temperatures. Drying of enzyme-containing latex films at 75°C prior to conditioning at 30°C resulted in higher activity than drying solely at 30°C, or drying at 95°C or 105°C followed by conditioning at 30°C. Storage of films in air at 4°C for 14 days did not negatively affect the enzymatic activity. Inclusion of catalase in films with oxalate oxidase effectively prevented release of hydrogen peroxide. The results suggest that the immobilized enzyme can successfully be used both as an oxygen scavenger and as an oxalic-acid scavenger. [Copyright &y& Elsevier]

Barley oxalate oxidase (OxO), a manganese-containing protein, is largely employed for determination of oxalate in various biologic materials. The present report describes in vitro effects of nanoparticles (NPs) of three metal oxides, i.e., zinc oxide (ZnO), copper oxide (CuO), and manganese oxide (MnO), on the activity and stability of OxO purified from barley roots. The transmission electron microscopy and X-ray diffraction studies of these NPs revealed their very fine crystalline structure with the dimeter in the range 30-70, 50-60, and 20-60 nm for ZnO NPs, CuO NPs, and MnO NPs, respectively. The addition of suspension of these three NPs into assay mixture of enzyme individually, led to the adsorption of OxO over their surface, as confirmed by Fourier transform infrared spectra and UV-Vis spectroscopic studies. Compared to free enzyme, MnO NPs-bound enzyme showed improved activity (35 % stimulation at 2.5 mg/ml concentration), while ZnO NPs- and CuO NPs-bound enzyme had no substantial improvement. The kinetic properties of individually NPs-bound enzyme were studied and compared with those of free enzyme. The MnO NPs-bound enzyme also showed marked improvement in its storage and thermal stability compared to free enzyme. [ABSTRACT FROM AUTHOR]

Background & objectives: High level of urinary oxalate substantially increases the risk of hyperoxaluria, a significant risk factor for urolithiasis. The primary goal of this study was to reduce urinary oxalate excretion employing liposome encapsulated oxalate oxidase in animal model. Methods: A membrane bound oxalate oxidase was purified from Bougainvillea leaves. The enzyme in its native form was less effective at the physiological pH of the recipient animal. To increase its functional viability, the enzyme was immobilized on to ethylene maleic anhydride (EMA). Rats were injected with liposome encapsulated EMA- oxalate oxidase and the effect was observed on degradation of oxalic acid. Results: The enzyme was purified to apparent homogeneity with 60-fold purification and 31 per cent yield. The optimum pH of EMA-derivative enzyme was 6.0 and it showed 70 per cent of its optimal activity at pH 7.0. The EMA-bound enzyme encapsulated into liposome showed greater oxalate degradation in 15 per cent casein vitamin B6 deficient fed rats as compared with 30 per cent casein vitamin B6 deficient fed rats and control rats. Interpretation & conclusions: EMA-oxalate oxidase encapsulated liposome caused oxalate degradation in experimental hyperoxaluria indicating that the enzyme could be used as a therapeutic agent in hyperoxaluria leading to urinary stones. [ABSTRACT FROM AUTHOR]

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10 mM of oxalic acid whereas only 15 isolates were grow at 50 mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23 %) after 60 min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents. [ABSTRACT FROM AUTHOR]

This study describes the influence of copper nanoparticles (CNPs) on activity and kinetic properties of a Cu2+ requiring sorghum oxalate oxidase (OxO). CNPs were synthesised by citrate-induced reduction of CuCl2 and their size (range 13–58 nm) was determined by transmission electron microscopy. The CNPs were characterised by scanning electron microscopy and X-ray diffraction studies. These CNPs enhanced OxO activity by 30%. Thermal and storage stability were increased, while Km value for oxalate decreased in the presence of CNPs. CNPs protected OxO against chelation by diethyldithiocarbamate, a Cu2+ specific chelator. The analytic use of OxO in the presence of CNP for determination of oxalate in food stuff is demonstrated. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The use of Au nanoparticles (AuNPs)–CaCO3 porous microsphere hybrid as a matrix for enzyme immobilization to increase the performance of an amperometric biosensor. Design and methods: A method is described for construction of an amperometric oxalate biosensor by immobilizing a barley root oxalate oxidase (OxOx) onto AuNPs–CaCO3 porous microsphere hybrid encapsulated in silica sol and deposited on Au electrode. Results: The biosensor showed optimum response within 4s at pH 5.0 and 30°C, when polarized at +0.4V vs. Ag/agCl. The biosensor possesses high sensitivity and measures oxalate concentrations as low as 1.0μmol/L. The working linear range was from 1.0 to 1000μmol/L of oxalate. The biosensor measured urinary and plasma oxalate of normal persons and stone formers. Conclusion: The use of AuNPs–CaCO3 porous microsphere hybrid as a support for immobilization of OxOx has resulted into an improved amperometric oxalate biosensor. [Copyright &y& Elsevier]

Abstract: Oxalate oxidase (OXO) utilizes oxalate to generate hydrogen peroxide, and thereby acts as a source of hydrogen peroxide. The present study was carried out to investigate whether apoplastic OXO modifies the metabolism of cell wall-bound ferulates in wheat seedlings. Histochemical staining of OXO showed that cell walls were strongly stained, indicating the presence of OXO activity in shoot walls. When native cell walls prepared from shoots were incubated with oxalate or hydrogen peroxide, the levels of ester-linked diferulic acid (DFA) isomers were significantly increased. On the other hand, the level of ester-linked ferulic acid (FA) was substantially decreased. The decrease in FA level was accounted neither by the increases in DFA levels nor by the release of FA from cell walls during the incubation. After the extraction of ester-linked ferulates, considerable ultraviolet absorption remained in the hemicellulosic and cellulose fractions, which was increased by the treatment with oxalate or hydrogen peroxide. Therefore, a part of FA esters may form tight linkages within cell wall architecture. These results suggest that cell wall OXO is capable of modifying the metabolism of ester-linked ferulates in cell walls of wheat shoots by promoting the peroxidase action via supply of hydrogen peroxide. [Copyright &y& Elsevier]

American chestnut [Castanea dentata (Marshall) Borkhausen], a canopy dominant species across wide swaths of eastern North America, was reduced to an understory shrub after introduction of the blight fungus [Cryphonectria parasitica (Murrill) Barr] in the early 1900s. Restoration of American chestnut by using bioteehnology is promising, but the imprecise nature of transgenesis may inadvertently alter tree phenotype, thus potentially impacting ecologically dependent organisms. We quantified effects of genetic engineering and fungal inoculation of trees on insect herbivores by using transgenic American chestnuts expressing an oxalate oxidase gene and wild-type American and Chinese (C. mollissima Blume) chestnuts. Of three generalist folivores bioassayed, only gypsy moth [Lymantvia dispar (L.)] was affected by genetic modification, exhibiting faster growth on transgenic than on wild-type chestnuts, whereas growth of polyphemus moth [Antheraea polyphemus (Cramer)] differed between wild-type species, and fall webworm [Hyphantria cunea (Drury)] performed equally on all trees. Inoculation of chestnuts with blight fungus had no effect on the growth of two herbivores assayed (polyphemus moth and fall webworm). Enhanced fitness of gypsy moth on genetically modified trees may hinder restoration efforts if this invasive herbivore's growth is improved because of transgene expression. [ABSTRACT FROM AUTHOR]

Abstract: An amperometric oxalate biosensor using nanohybrid film of multi-walled carbon nanotubes (MWCNTs) and gold colloidal nanoparticles (GNPs) via carbodiimide chemistry by forming amide linkages between carboxylic acid groups on the CNTs and amine residues of cysteamine self-assembled monolayer (SAM) has been prepared. The c-MWCNTs were immobilized on the gold (Au) electrode and characterized by FTIR. The morphologies of the c-MWCNT/Au and GNPs/MWCNT/Au electrodes were investigated by scanning electron microscopy (SEM) and the electrochemical performance of the Au, c-MWCNT/Au and GNPs/c-MWCNT/Au electrodes were also studied amperometrically. The Cl− and NO3 − insensitive oxalate oxidase from grain sorghum was finally immobilized on this electrode. The influence of pH, temperature and oxalate concentration on electrode activity was studied. The electrode showed optimum response within 7s. The electrocatalytic response showed a linear dependence on the oxalic acid concentration ranging from 1 to 800μM with a detection limit of 1μM. The K m value for the oxalic acid sensor was 444.44μM. The enzyme electrode retained 30% of its initial activity after 5 months, when stored at 4°C. The electrode was employed for measurement of oxalic acid in serum, urine and foodstuffs. [Copyright &y& Elsevier]

Germin-like proteins (GLPs) play diversified roles in plant development and defense response. Here, we identified 36 expressed sequence tags (ESTs) encoding GLPs from peanut ( Arachis hypogaea L.). After assembly, these ESTs were integrated into eight unigenes named AhGLP1 to AhGLP8, of which, three ( AhGLP1-3) were comprised 14, ten, and seven EST clones, respectively, whereas the remaining ones were associated with one single clone. The length of the deduced amino acid (AA) residues ranged from 208 to 223 AAs except for AhGLP6 and AhGLP8, which were incomplete at the carboxyl terminus. All of the AhGLPs contained a possible N-terminal signal peptide that was 17 to 24 residues in length excluding AhGLP7, where there is likely a non-cleavable amino terminus. Phylogenetic analysis showed that these AhGLPs were classified into three subfamilies. Southern blot analysis indicated that AhGLP1 and AhGLP2 likely have multiple copies in the peanut genome. The recombinant mature AhGLP1 and AhGLP2 proteins were successfully expressed in Escherichia coli. The purified AhGLP2 has superoxide dismutase (SOD) activity in enzymatic assay, but not oxalate oxidase activity. The SOD activity of AhGLP2 was stable up to 70°C and resistant to hydrogen peroxide, suggesting that AhGLP2 might be a manganese-containing SOD. Furthermore, AhGLP2 could confer E. coli resistance to oxidative damage caused by paraquat, suggesting that the AhGLP2 likely protects peanut plants from reactive oxygen metabolites. Thus, information provided in this study indicates the diverse nature of the peanut GLP family and suggests that some of AhGLPs might be involved in plant defense response. [ABSTRACT FROM AUTHOR]

Abstract: Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. [Copyright &y& Elsevier]

Abstract: A highly sensitive, specific and rapid electrochemical oxalate biosensor was constructed by covalently immobilizing sorghum leaf oxalate oxidase on carboxylated multiwalled carbon nanotubes and conducting polymer, polyaniline nanocomposite film electrodeposited over the surface of platinum (Pt) wire using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry. The optimized oxalate biosensor showed linear response range of 8.4–272μM with correlation coefficient of 0.93 and rapid response within 5s at a potential of 0.4V vs Ag/AgCl. The sensitivity was approximately 0.0113μA/μM with a detection limit of 3.0μM. Proposed oxalate biosensor was successfully applied to human urine sample. [ABSTRACT FROM AUTHOR]

Background: Germin and germin-like proteins constitute a ubiquitous family of plant proteins. A role of some family members in defense against pathogen attack had been proposed based on gene regulation studies and transgenic approaches. Soybean (G. max L. Merr.) germin genes had not been characterized at the molecular and functional levels. Results: In the present study, twenty-one germin gene members in soybean cultivar 'Maple Arrow' (partial resistance to Sclerotinia stem rot of soybean) were identified by in silico identification and RACE method (GmGER 1 to GmGER 21). A genome-wide analyses of these germin-like protein genes using a bioinformatics approach showed that the genes located on chromosomes 8, 1, 15, 20, 16, 19, 7, 3 and 10, on which more disease-resistant genes were located on. Sequence comparison revealed that the genes encoded three germin-like domains. The phylogenetic relationships and functional diversity of the germin gene family of soybean were analyzed among diverse genera. The expression of the GmGER genes treated with exogenous IAA suggested that GmGER genes might be regulated by auxin. Transgenic tobacco that expressed the GmGER 9 gene exhibited high tolerance to the salt stress. In addition, the GmGER mRNA increased transiently at darkness and peaked at a time that corresponded approximately to the critical night length. The mRNA did not accumulate significantly under the constant light condition, and did not change greatly under the SD and LD treatments. Conclusions: This study provides a complex overview of the GmGER genes in soybean. Phylogenetic analysis suggested that the germin and germin-like genes of the plant species that had been founded might be evolved by independent gene duplication events. The experiment indicated that germin genes exhibited diverse expression patterns during soybean development. The different time courses of the mRNAs accumulation of GmGER genes in soybean leaves appeared to have a regular photoperiodic reaction in darkness. Also the GmGER genes were proved to response to abiotic stress (such as auxin and salt), suggesting that these paralogous genes were likely involved in complex biological processes in soybean. [ABSTRACT FROM AUTHOR]

An amperometric oxalate biosensor was developed and optimized by immobilizing a Cl- and [image omitted] insensitive oxalate oxidase purified from sorghum leaves, on carbon paste electrode. Enzymatically produced H2O2 from oxalate was split into 2H+ + O2 + 2e- at 60 mV and flow of electron (current) was measured. The biosensor showed optimum response at pH 4.5, 35°C and within 45 sec. The current measured (mA) by biosensor was linear for oxalate up to 20 µg/ml. The biosensor was employed for determination of oxalate in urine (from both apparently healthy individuals and urinary stone formers) and different brands of beer. The percent recoveries of added oxalate in urine of healthy and stone formers were 96.6% and 98.2%, respectively. Within and between batch coefficients of variation (CV) for oxalate determination were <7.5% and <8.5% in urine of healthy persons and <5.0% and <6.5% in the urine of stone formers, respectively. There was a good correlation (r = 0.91) between urinary oxalate values by present method and the most commonly used enzymatic colorimetric method. The enzyme electrode was used for 150-times during the span of 35 days, when stored in a reaction buffer at 4°C. [ABSTRACT FROM AUTHOR]

The effect of cadmium (Cd) on the expression and activity of NADPH oxidase, peroxidase and oxalate oxidase as well as on the expression of aquaporins and dehydrins was studied in barley root tip. The root tip represented intact apical part of the barley root containing the root cap, meristems and elongation zone. Except stress induced by Cd, barley root tips were analysed after their exposure to phytotoxic concentration of mercury (Hg)-, hydrogen peroxide (H2O2)- or polyethylene glycol (PEG)-induced water stress in order to compare the Cd-induced changes with changes induced by these other stress factors. Cd, Hg, H2O2 and with some exceptions also PEG treatments caused similar alterations in the gene expression of reactive oxygen species (ROS)-generating and water deficiency-related genes, and in the activity of ROS-generating enzymes. These evidences support our opinion that ROS accumulation and water imbalance are the common symptoms of these stress factors and that the elevated production of H2O2 plays, probably as a signal molecule, a key role in the induction of plant responses to abiotic stresses in barley root tip. On the other hand, H2O2 at permanent high concentration is probably the main toxic factor during stress conditions. [ABSTRACT FROM AUTHOR]

Abstract: The germin protein family is comprised of two main subgroups in plants, oxalate oxidases (OXOs) and germin-like proteins (GLPs). These proteins are implicated in a variety of plant processes including germination, development, pollen formation, and response to abiotic and biotic stress. Here, we examine the phylogenetic relationships and functional diversity of the germin gene family across diverse genera, and then focus on rice (Oryza sativa) as a model. In general, germin genes are expressed in all tissue types and are induced by biotic and/or abiotic stresses. In rice, many of the stress-induced germin genes physically co-localize with quantitative trait loci (QTL) for disease resistance, and emerging evidence suggests that small RNAs may regulate their transcript abundance. We analyze the extensive sequence, gene expression and functional data for germin family proteins and relate them to QTL map locations to predict additional candidate germin genes for future crop improvement. [Copyright &y& Elsevier]

Abstract: The effect of Cd on oxalate oxidase (OxO) activity and its localisation were analysed in barley root. In Cd-treated roots OxO activity was strongly induced in the region 2–4mm behind the root tip and in the area toward the root base. In situ analyses showed that Cd-induced OxO activity was localised to the cell wall (CW) of early metaxylem vascular bundles and surrounding parenchyma cells and was accompanied by lignification of metaxylem vessels. OxO activation was also observed during treatment with other heavy metals (HMs), salt treatment and at elevated non-optimal temperature. In contrast to HM activation of OxO and lignification, high temperature and NaCl indeed activated OxO but did not induce lignification of metaxylem vessels. These results suggest that oxalate oxidase as an H2O2-generating enzyme is activated in response to several stresses, however the ectopic lignification of metaxylem vessels is activated specifically by HMs. This HM-induced premature root xylogenesis due to ectopic lignification of metaxylem vessels probably causes shortening of the root elongation zone and therefore a reduction in root growth. [Copyright &y& Elsevier]

Abstract: Egg shell membrane is a novel, robust, microporous, cost effective, easily available organic support material. In recent studies egg shell membranes were utilized as organic support for enzyme immobilization. But low conjugation yield limits its application as good support for biotechnological industries. In present study egg shell membrane was chemically treated to introduce free functional groups for covalent linkage of proteins to increase its conjugation yield and stability of conjugate complex. Many enzymes were tested for immobilization on modified egg shell membrane like oxalate oxidase, glucose oxidase, peroxidase and lipase. A fifteen to sixteen fold increase in conjugation yield was observed when immobilization was performed after chemical treatment in comparison to immobilization on native membrane with slight change in specific activity of immobilized enzyme which ranges from 5% to 15%. Egg shell membrane bound enzymes showed slight changes in their kinetic properties after immobilization. Egg shell membrane bound oxalate oxidase shows detection limit of 1.5μM when used for urinary oxalate determination. Egg shell membrane support shows no interference to enzyme activity and a good correlation of 0.99 was observed with the values estimated using commercially available Sigma kit. The immobilized oxalate oxidase, glucose oxidase, peroxidase and lipase were stable up to duration of 180 days and there is respective loss of 10%, 13%, 24%, and 33% of initial activity. Overall result strengthens our view of using chemically modified egg shell membrane as solid support for better immobilization of enzymes and can be used in various biotechnological applications. [Copyright &y& Elsevier]

Oxalate oxidase (OxO, EC 1.2.3.4.) was purified to homogeneity from wheat ( Triticum aestivum) seedlings by sequential thermal treatment, ultrafiltration, Sephadex G-100 gel filtration and affinity chromatography with concanavalin A. The enzyme was purified 66.11-fold with a recovery of 21.97%. It showed a subunit molecular mass of 32.6 kDa on SDS-PAGE and a native molecular mass of 170 kDa on Sephadex G-150 filtration, suggesting that it is a pentamer. The wheat OxO had a maximum activity at pH 3.5. Its K m for oxalate was 0.21 mM. Chemical modification revealed that cysteine, lysine and carboxylate residues were essential for OxO activity, whereas arginine, serine, threonine and tryptophane residues were not essential. [ABSTRACT FROM AUTHOR]

Rice oxalate oxidase genes (OXO) may play a role in resistance to Magnaporthe oryzae. Genome analyses showed four tandemly duplicated OXO genes, OsOXO1– OsOXO4, which mapped to a blast resistance QTL in chromosome 3. These genes have >90% nucleotide and amino acid identity, but they have unique gene structures, conserved motifs, and phylogeny compared to the 70 other members of the cupin superfamily in the Nipponbare genome, which were divided into several classes. In resistant and susceptible Vandana/Moroberekan advanced backcross lines, only OsOXO4 was expressed during rice– M. oryzae interactions, and its expression increased earlier in resistant than susceptible lines. The earlier expression of OsOXO4 in resistant lines correlated with a 26-bp promoter insertion containing an additional copy of the bacterial responsive nodulation cis-element. Our results showed that OsOXO1– 4 are in a separate class of rice cupin genes and supports a role for the promoter variant of OsOXO4 in resistance to M. oryzae. [ABSTRACT FROM AUTHOR]

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide-generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily. [ABSTRACT FROM AUTHOR]

Abstract: Enzymatically catalyzed decomposition of oxalic acid in bleaching filtrates from the pulp and paper industry offers a possibility to enduringly prevent oxalate scaling problems by specific removal of the oxalic acid in the system rather than by attempting to avoid calcium oxalate precipitation by countermeasures aiming at improved solubility. To achieve a broad evaluation of various oxalate-degrading enzymes and to cover conditions encountered in various types of processes, 16 different bleaching filtrates were collected from pulp mills engaged in mechanical pulping of softwood, mechanical pulping of aspen, and kraft pulping of softwood. A novel oxalate-degrading enzyme provided by Novozymes was compared with commercially available oxalate oxidase from barley and oxalate decarboxylase from Aspergillus niger. The activity of the enzymes in the filtrates was investigated using kinetic analysis and multivariate data analysis. Kinetic analysis indicated that the degradation rates were governed more by inhibitors in the filtrates than by the concentration of oxalic acid. Multivariate data analysis suggested links between high concentrations of certain compounds in the filtrates and high or low enzyme activity, as exemplified by the link between high concentrations of chelators in filtrates from mechanical pulping and low activity of oxalate oxidase from barley. All three enzymes could degrade oxalic acid in all filtrates, despite the fact that very high concentrations of residual hydrogen peroxide were found in several of the filtrates. [Copyright &y& Elsevier]

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape ( Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO2 and H2O2. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat ( Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H2O2 levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. [ABSTRACT FROM AUTHOR]