Occurrence of an oxalate oxidase in Sorghum leaves

An oxalate oxidase found in the 15 000 g supernatant of 10-day-old sorghum leaves exhibited a pH optimum of 5 and a temperature optimum of 45° and was unaffected by Na⁺. The enzyme activity remained linear up to 10 min and the apparent K_m for oxalate was 2.4 × 10^?5 M. The enzyme activity was strongly inhibited by sodium dithionite and α,α′-dipyridyl. Inhibition by the latter was specifically reversed by Fe²⁺. The activity of the dialysed enzyme was restored by the addition of Fe²⁺ and FAD. Inhibition of the enzyme by iodoacetate, p-chloromercuribenzoate and N-methylmaleimide revealed that SH groups at the active site are essential.     

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U?mg^?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6?h. The enzymatic activity was not influenced by metal ions and chemical agents (K⁺, Na⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺, glucose, urea, lactate) commonly found in serum and urine, with Cu²⁺ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca²⁺ and Fe²⁺. Its K_m and K_cat for oxalate were found to be 0.45?mM and 85?s^?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50?mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.     

State of translocated Ca2+ by sarcoplasmic reticulum inferred from kinetic analysis of calcium oxalate precipitation

Uptake of Ca²⁺ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca²⁺ is depressed by increasing the added Ca²⁺ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca²⁺ ($\text{V}{}_0$). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration ($\text{V}{}^{\mathrm{\infty }}$). With the combined extrapolated values a maximal expected precipitation rate could be calculated ($\text{V}{}_0{}^{\mathrm{\infty }}$). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca²⁺ were established. Entering $\text{V}{}_0{}^{\mathrm{\infty }}$ in the equations, an internal free Ca²⁺ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not supress the Ca²⁺ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca²⁺/mg protein. This amount presumably equals the internal free Ca²⁺ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca²⁺ gradients built up during the uptake of Ca²⁺, values of free Ca²⁺ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Nuclear pore complex oxalate binding protein p62: its expression on oxalate exposure to VERO cells

Oxalate rich stones are the most common among the various stones. Oxalate binding protein plays a vital role in the transport of oxalate. Nuclear pore complex (NPC) contains a protein of molecular weight 62 kDa and it has maximum oxalate binding activity. The physiological significance of the presence of oxalate binding protein in the NPC is not well understood. In order to study its function, the expression of this protein during oxalate stress condition and the morphological changes on oxalate exposure to synchronized VERO cells have been determined. VERO cells were synchronized at different stages of cell cycle using cell cycle blockers and expression of the NPC p62 was assessed using enzyme linked immunosorbent assay (ELISA) technique with p62 antibody (MAb 414). Expression of NPC p62 was more pronounced in 1.0 mM oxalate concentration in mitotic phase than in S phase, suggesting cell cycle dependency. During oxalate exposure there is cell aggregation and complete degeneration of cell morphology occurs, which in turn lead to the expression of certain genes, including the NPC oxalate binding protein p62. Thus, oxalate induces degeneration of cells (may be due to the lipid peroxidation) and leads to the expression of NPC oxalate binding protein and the expression is of cell cycle dependent manner.     

A secretion signal is present in the Collybia velutipes oxalate decarboxylase gene.

The oxalate decarboxylase (OXDC) gene from Collybia velutipes is overexpressed as an active form in Schizosaccharomyces pombe. The recombinant enzyme shows similar pH optima and stability, while substrate kinetic analysis shows a ninefold decrease in K(m) value with respect to native OXDC. Most of the expressed protein was present in periplasm and remained firmly bound to cell-wall materials. However, 20% of enzyme expressed was secreted out into the medium suggesting the presence of a secretion signal (C. velutipes) in the oxalate decarboxylase gene. This secretion signal is associated with the N-terminal of OXDC as is evident by secretion of nonsecretory genes AmA1 and beta-galactosidase. An expression vector using this signal is constructed for expression and secretion of heterologous proteins in S. pombe.     

IMMOBILIZATION OF OXALATE DECARBOXYLASE TO EUPERGIT AND PROPERTIES OF THE IMMOBILIZED ENZYME

Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5 mol/L (NH₄)₂SO₄, room temperature, and shaking at 30 rpm for 24 hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44 U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.     

Active site geometry of oxalate decarboxylase from Flammulina velutipes: Role of histidine-coordinated manganese in substrate recognition

Oxalate decarboxylase (OXDC) from the wood-rotting fungus Flammulina velutipes, which catalyzes the conversion of oxalate to formic acid and CO(2) in a single-step reaction, is a duplicated double-domain germin family enzyme. It has agricultural as well as therapeutic importance. We reported earlier the purification and molecular cloning of OXDC. Knowledge-based modeling of the enzyme reveals a beta-barrel core in each of the two domains organized in the hexameric state. A cluster of three histidines suitably juxtaposed to coordinate a divalent metal ion exists in both the domains. Involvement of the two histidine clusters in the catalytic mechanism of the enzyme, possibly through coordination of a metal cofactor, has been hypothesized because all histidine knockout mutants showed total loss of decarboxylase activity. The atomic absorption spectroscopy analysis showed that OXDC contains Mn(2+) at up to 2.5 atoms per subunit. Docking of the oxalate in the active site indicates a similar electrostatic environment around the substrate-binding site in the two domains. We suggest that the histidine coordinated manganese is critical for substrate recognition and is directly involved in the catalysis of the enzyme.     

Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease

Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO₂. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capable of degrading oxalate protect cells from oxidative damage and thus serve as a therapeutic option for prevention of CaOx stone disease.

     

Determination of serum oxalate using peroxyoxalate chemiluminescence of free oxalic acid

We describe a new sensitive and specific method for determination of oxalate in human serum. By using the chemiluminescence decay of monoperoxyoxalic acid very low concentrations of oxalate (200 nmol/L) can be determined. The mean serum oxalate level in apparently healthy controls was 14.5 ± 8.5 m?mol/L. Supplementation of ascorbic acid leads to an increase in serum oxalate level. While serum oxalate concentrations of calcium oxalate stone formers (x = 16.4 ± 9.8 m?mol/L) are not significantly different from the control group, an extreme increase of serum oxalate is evident in haemodialysis patients. The serum oxalate concentration decreased during dialysis treatment from 141.4 ± 32.1 m?mol/L to 36.4 ± 12.7 m?mol/L.     

Calcium and oxalate content of the leaves of Phaseolus vulgaris at different calcium supply in relation to calcium oxalate crystal formation

Soluble and insoluble oxalate and insoluble calcium were measured in the leaves of Phaseolus vulgaris. The plants were grown in nutrient solutions with two different concentrations of calcium. Two developmental stages of the leaves were studied. Although the content of insoluble calcium differs widely according to leaf age and growth conditions, the percentage bound in crystals is nearly the same in all cases. In the growing leaves, concentrations of total oxalate are independent of calcium supply, thus, showing that the known rise in numbers of crystals, and of cells containing them, is not induced via oxalate biosynthesis. Fully expanded leaves contain more oxalate when grown in a nutrient solution with higher calcium concentration. Amounts of oxalate in percent of dry weight are similar to those given in the literature for other legume leaves.     

Bacteria can promote calcium oxalate crystal growth and aggregation

Our previous report showed that uropathogenic bacteria, e.g., Escherichia coli, are commonly found inside the nidus of calcium oxalate (CaOx) kidney stones and may play pivotal roles in stone genesis. The present study aimed to prove this new hypothesis by direct examining CaOx lithogenic activities of both Gram-negative and Gram-positive bacteria. CaOx was crystallized in the absence (blank control) or presence of 10⁵ CFU/ml E. coli, Klebsiella pneumoniae, Staphylococcus aureus, or Streptococcus pneumoniae. Fragmented red blood cell membranes and intact red blood cells were used as positive and negative controls, respectively. The crystal area and the number of aggregates were measured to initially screen for effects of bacteria on CaOx crystal growth and aggregation. The data revealed that all the bacteria tested dramatically increased the crystal area and number of crystal aggregates. Validation assays (spectrophotometric oxalate-depletion assay and an aggregation–sedimentation study) confirmed their promoting effects on both growth (20.17 ± 3.42, 17.55 ± 2.27, 16.37 ± 1.38, and 21.87 ± 0.85 % increase, respectively) and aggregation (57.45 ± 2.08, 51.06 ± 5.51, 55.32 ± 2.08, and 46.81 ± 3.61 % increase, respectively) of CaOx crystals. Also, these bacteria significantly enlarged CaOx aggregates, with the diameter greater than the luminal size of distal tubules, implying that tubular occlusion might occur. Moreover, these bacterial effects were dose-dependent and specific to intact viable bacteria, not intact dead or fragmented bacteria. In summary, intact viable E. coli, K. pneumoniae, S. aureus, and S. pneumoniae had significant promoting effects on CaOx crystal growth and aggregation. This functional evidence supported the hypothesis that various types of bacteria can induce or aggravate metabolic stone disease, particularly the CaOx type.     

Cytoplasmic streaming of calcium oxalate crystals in Callipsygma wilsonis (Bryopsidales, Chlorophyta)

Light microscopic study of the giant‐celled, marine green alga Callipsygma wilsonis J. Agardh (Udoteaceae, Bryopsidales) revealed numerous birefringent crystalline inclusions in the terminal segments of the assimilatory axes. The inclusions were thin plates with a triangular shape in face view, a base up to 75 μm in length, and a height that was one‐seventh the length of the base. Crystals of various sizes commonly were stacked face‐to‐face with one or more edges coinciding, but removal of organic material by treatment in sodium hypochlorite resulted in disaggregation. The crystals were soluble in dilute hydrochloric acid without effervescence but were insoluble in acetic acid. These diagnostic chemical solubility tests and a positive reaction to the Yasue staining reaction indicated that the crystals were composed of calcium oxalate. Scanning electron microscopy showed that most crystals had smoothly curving edges, but some had truncate or beveled margins. Calcium oxalate crystals have been reported to occur in the large central vacuoles of several bryopsidalean species, but the crystals in C. wilsonis were present in the parietal cytoplasm, which was evident from the presence of crystals in streaming cytoplasm. Calcium oxalate crystals, amyloplasts, chloroplasts, and other cytoplasmic constituents moved along cytoskeletal cables at rates of approximately 2.8 μm s⁻¹. These findings add to a growing body of evidence that calcium oxalate crystals in diverse algae may be present in cellular compartments other than the central vacuole.     

Dissolution and growth of calcium oxalate monohydrate I. Effect of magnesium and pH

The rate of dissolution of calcium oxalate monohydrate and of a calcium oxalate renal stone was measured in 0.9% NaCl solution at different levels of magnesium concentration and pH. The growth of calcium oxalate obtained by chemical reaction between Ca²⁺ and oxalate ions at a concentration similar to that existing in normal urine was also investigated as a function of pH and magnesium concentration. It was found that both magnesium and pH exert a fine kinetic control on the precipitation and growth of calcium oxalate monohydrate. Magnesium had no effect on the dissolution. The possible role of magnesium and pH in calcium oxalate urolithiasis has been discussed in the light of previous reports and of the data presented in this study.     

Recombinant Lactobacillus plantarum expressing and secreting heterologous oxalate decarboxylase prevents renal calcium oxalate stone deposition in experimental rats

Background

Calcium oxalate (CaOx) is the major constituent of about 75% of all urinary stone and the secondary hyperoxaluria is a primary risk factor. Current treatment options for the patients with hyperoxaluria and CaOx stone diseases are limited. Oxalate degrading bacteria might have beneficial effects on urinary oxalate excretion resulting from decreased intestinal oxalate concentration and absorption. Thus, the aim of the present study is to examine the in vivo oxalate degrading ability of genetically engineered Lactobacillus plantarum (L. plantarum) that constitutively expressing and secreting heterologous oxalate decarboxylase (OxdC) for prevention of CaOx stone formation in rats. The recombinants strain of L. plantarum that constitutively secreting (WCFS1OxdC) and non-secreting (NC8OxdC) OxdC has been developed by using expression vector pSIP401. The in vivo oxalate degradation ability for this recombinants strain was carried out in a male wistar albino rats. The group I control; groups II, III, IV and V rats were fed with 5% potassium oxalate diet and 14^th day onwards group II, III, IV and V were received esophageal gavage of L. plantarum WCFS1, WCFS1OxdC and NC8OxdC respectively for 2-week period. The urinary and serum biochemistry and histopathology of the kidney were carried out. The experimental data were analyzed using one-way ANOVA followed by Duncan’s multiple-range test.

Results

Recombinants L. plantarum constitutively express and secretes the functional OxdC and could degrade the oxalate up to 70–77% under in vitro. The recombinant bacterial treated rats in groups IV and V showed significant reduction of urinary oxalate, calcium, uric acid, creatinine and serum uric acid, BUN/creatinine ratio compared to group II and III rats (P < 0.05). Oxalate levels in kidney homogenate of groups IV and V were showed significant reduction than group II and III rats (P < 0.05). Microscopic observations revealed a high score (4+) of CaOx crystal in kidneys of groups II and III, whereas no crystal in group IV and a lower score (1+) in group V.

Conclusion

The present results indicate that artificial colonization of recombinant strain, WCFS1OxdC and NC8OxdC, capable of reduce urinary oxalate excretion and CaOx crystal deposition by increased intestinal oxalate degradation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0086-y) contains supplementary material, which is available to authorized users.     

Gastrin-releasing peptide receptor gene silencing inhibits the development of the epithelial–mesenchymal transition and formation of a calcium oxalate crystal in renal tubular epithelial cells in mice with kidney stones via the PI3K/Akt signaling pathway

Between 1% and 15% of people are globally affected by kidney stones, and this disease has become more common since the 1970s. Therefore, this study aims to investigate the effects of gastrin-releasing peptide receptor (GRPR) gene silencing via the PI3K/Akt signaling pathway on the development of the epithelial–mesenchymal transition (EMT) and formation of a calcium oxalate crystal in renal tubular epithelial cells (TECs) of kidney stones. A total of 70 clean and healthy C57BL/6J mice were assigned into the normal ( n = 10) and kidney stones groups ( n = 60). The underlying regulatory mechanisms of GRPR were analyzed in concert with the treatment of shGRPR-1, LY294002, and shGRPR-1 + LY294002 in TECs isolated from mice with kidney stones. A series of experiments were conducted for the measurement of urinary oxalate and urinary calcium, the renal calcium salt deposition, the positive rate of GRPR, the expressions of renal TECs related genes and calcium oxalate regulation related genes, and the growth of calcium crystals induced by cells. After treatment of shGRPR-1 and shGRPR-1 + LY294002, levels of urinary oxalate and urinary calcium in the serum, as well as positive rate of GRPR, became relatively low, levels of E-cadherin enhanced, whereas levels of Akt, PI3K, GRPR, extents of PI3K and Akt phosphorylation, α-SMA, Vimentin and FSP-1, OPN, MCP-1, and CD44 decreased and a number of crystals reduced. Taken together, we conclude that GRPR gene silencing suppresses the development of the EMT and formation of the calcium oxalate crystal in renal TECs of kidney stones through the inactivation of the PI3K/Akt signaling pathway.     

Stable calcium isotope speciation and calcium oxalate production within beech tree (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) organs

In this study, we linked Ca speciation with isotope composition in plants. To do this, we performed leachate experiments to access the soluble Ca, structurally bound Ca and insoluble Ca (i.e., water and weak acid resistant) within beech tree organs (Fagus sylvatica L.). Ca isotopic measurements were combined with infrared spectroscopy and calcium oxalate biomineralization identification. The results from our study indicate that bark and leaves are the most enriched in monohydrated calcium oxalate crystals (whewellite), which are observable in parenchyma and sclerenchyma tissues, whereas roots and wood are enriched in structurally bound Ca. Our leaching experiments also show decreasing δ^44/40Ca isotopic signatures in the order of soluble Ca > structurally bound Ca > insoluble Ca. This finding implies that because leaves degrade faster than wooden organs and because Ca linked to pectate decomposes faster than Ca linked to oxalate crystals, differential Ca isotopic signatures are expected to be observed during litter degradation.     

Nucleation of calcium oxalate crystals on an imprinted polymer surface from pure aqueous solution and urine

Calcium oxalate (CaOx) is the most common component of human kidney stones. Heterogeneous nucleation is regarded as the key mechanism in this process. In this study, we have used an imprinted 6-methacrylamidohexanoic acid/divinylbenzene co-polymer as a biomimetic surface to nucleate CaOx crystal formation. The polymer was imprinted with either calcium oxalate monohydrate (COM) or dihydrate (COD) template crystals. These were washed out of the polymer, which was then immersed in various test solutions. The test solutions were an aqueous solution of calcium chloride and sodium oxalate, artificial urine and a sample of real urine. Crystals that formed on the polymer surface were characterized by X-ray powder diffraction, Fourier transform infrared spectroscopy, atomic absorption spectroscopy and scanning electron microscopy. Results showed that in the aqueous solution the COM-imprinted polymer induced the nucleation of COM. The COD-imprinted polymer induced only trace amounts of COD crystallization, together with larger quantities of COM. In artificial and real urines, COM also specifically precipitated on the COM-imprinted surface. The results show that, at least to some extent, the imprinted polymers direct formation of their morphologically matched crystals. In the case of COD, however, it appears that either rapid hydrate transformation of COD to COM occurs, or the more stable COM polymorph is directly co-precipitated by the polymer. Our results support the hypothesis that heterogeneous nucleation plays a key role in CaOx stone formation and that the imprinted polymer model could provide an additional and superior diagnostic tool for stone researchers to assess stone-risk in urine.Abbreviations COD calcium oxalate dihydrate - COM calcium oxalate monohydrate - COT calcium oxalate trihydrate - dvb divinylbenzene - 6-maaha 6-methylacrylamidohexanoic acid     

Determination of urinary oxalate with alkylamine glass-bound sorghum oxalate oxidase and horseradish peroxidase

Oxalate in urine was analyzed using sorghum oxalate oxidase and horseradish peroxidase immobilized on alkylamine glass through glutaraldehyde. The minimum detection limit was 0.46 g/0.1 ml urine. The recovery of added oxalate was 97.5%. Within and between assay coefficients of variation were <3.5% and <6.5% respectively. A good correlation (r=0.9234) was found between oxalate values obtained by a commercial kit method and the present method. The method is unaffected by Cl– and NO3– found in urine.     

草酸对土壤胶体与矿物表面酶的吸附及活性影响

采用平衡批处理法,研究了模拟根系分泌物--草酸溶液的浓度、pH对酸性磷酸酶在针铁矿、高岭石及黄棕壤和砖红壤胶体(＜2μm)上的吸附及比活的影响.结果表明,针铁矿对磷酸酶的吸附量受草酸浓度的影响较小,其它供试胶体对蛋白的吸附量随草酸浓度的升高,一般表现为先急剧降低(0～5mmol·L^-1),之后逐渐升高到与对照相当或略低.这与草酸在土壤胶体和矿物表面的配位形态及其对载体表面的电荷改变、溶解有关.草酸体系中,供试胶体对磷酸酶的吸附顺序为针铁矿>黄棕壤＞高岭石＞砖红壤.酶在草酸体系中的最大吸附点位一般出现在蛋白的等电点(IEP)和供试胶体的PZC之间,而酶在草酸体系中被固定到供试胶体上之后,其最适比活点随胶体类型的不同而没有变化或有所高移.