Urease Molecules Research Articles

Interfacial interactions in urease/stearic acid and urease/DOPC binary mixed systems at the air-water and the air-solid interfaces

In the present study, the protein-lipid interactions between water-soluble metalloenzyme urease (Urs) and insoluble stearic acid (SA) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were investigated at the air–water interface using the Langmuir-Blodgett (LB) technique. Surface pressure (π) - molecular area (A) isotherms were measured for the pure SA and pure DOPC monolayers in the presence of urease in the water subphase. From the (π – A) isotherms of the binary system, it was found that urease strongly interacts with SA and DOPC. The compressibility of the SA monolayer was reduced with the incorporation of urease; a similar trend was also found with DOPC. Moreover, an association of urease with lipid monolayers was found to be surface pressure (π) dependent. The presence of a little hump in the mixed isotherms of Urs-SA at low surface pressure supports this observation. However, the miscibility studies and the calculations of excess Gibb’s free energy indicate an attractive interaction between urease and SA molecules. The considerable morphological variations observed by atomic force microscopy (AFM) in urease-SA deposited films onto hydrophilic and hydrophobic Si(100) surfaces suggest that urease was strongly bound with the surrounding lipid SA on the hydrophilic surfaces but the urease molecules were rigorously denatured on the hydrophobic surfaces. The AFM analysis of Urs-DOPC mixed LB films at room temperature (RT) and elevated temperature suggests substantial denaturation of urease in a hydrophobic environment leading us to speculate the exposure of tryptophan moieties of the urease molecules to the hydrophobic air phase.

Zerumbone Inhibits Helicobacter pylori Urease Activity.

Helicobacter pylori (H. pylori) produces urease in order to improve its settlement and growth in the human gastric epithelium. Urease inhibitors likely represent potentially powerful therapeutics for treating H. pylori; however, their instability and toxicity have proven problematic in human clinical trials. In this study, we investigate the ability of a natural compound extracted from Zingiber zerumbet Smith, zerumbone, to inhibit the urease activity of H. pylori by formation of urease dimers, trimers, or tetramers. As an oxygen atom possesses stronger electronegativity than the first carbon atom bonded to it, in the zerumbone structure, the neighboring second carbon atom shows a relatively negative charge (δ−) and the next carbon atom shows a positive charge (δ+), sequentially. Due to this electrical gradient, it is possible that H. pylori urease with its negative charges (such as thiol radicals) might bind to the β-position carbon of zerumbone. Our results show that zerumbone dimerized, trimerized, or tetramerized with both H. pylori urease A and urease B molecules, and that this formation of complex inhibited H. pylori urease activity. Although zerumbone did not affect either gene transcription or the protein expression of urease A and urease B, our study demonstrated that zerumbone could effectively dimerize with both urease molecules and caused significant functional inhibition of urease activity. In short, our findings suggest that zerumbone may be an effective H. pylori urease inhibitor that may be suitable for therapeutic use in humans.

High Sensitivity Au-based Kretschmann Surface Plasmon Resonance Sensor for Urea Detection

Kretschmann-based Surface Plasmon Resonance (SPR) optical sensor was applied to detect the presence of kidney wastes such as urea in solutions. To enhance the sensitivity of the SPR sensor, nanolaminated gold film (thickness of 50 nm) was used. In this work, the SPR response to urea in various concentrations were measured and investigated using optical wavelengths of 670 nm and 785 nm. The signals were compared between pure urea solution versus mixed solution in the presence of the 0.227 μmol urease enzyme. The proposed mixed solution is to eliminate doping and gel entrapment process for enzyme immobilization in conventional method. Angular interrogation technique was used to measure the sensor performance in urea detection using pure and mixed solutions with urea concentration of 0 - 800 mM. Upon exposure to mixed solution using 785 nm optical wavelength, the nanolaminated gold film exhibited higher SPR sensitivity as much as 7.8 M-1 than a pure urea solution (1.4 M-1). The coupling activity between urea and urease molecules in the mixed solution near the nanolaminated gold film surface lead to sensitivity enhancement. Angle shifting of mixed solution on 50 nm-thick nanolaminated gold film using 670 nm optical wavelength was greater by ~50% compared to 785 nm. Sensorgram data shows a steady and linear increment in SPR incident angle shifting when urea concentration increased. To the best of our knowledge, this is the first time that Kretschmann-based SPR has been used for urea sensing at 670 nm and 785 nm optical wavelengths.

Conductometric immunoassay of alpha-fetoprotein in sera of liver cancer patients using bienzyme-functionalized nanometer-sized silica beads.

A conductometric immunoassay protocol was designed for the sensitive detection of a liver cancer biomarker, alpha-fetoprotein (AFP), in biological fluids by using enzyme-conjugated nanometer-sized enzyme-doped silica beads. Initially, urease molecules were doped into nanosilica particles by using the reverse micelle method. Thereafter, arginase-labeled anti-AFP antibodies were covalently conjugated onto the surface of the synthesized nanoparticles. The immunoreaction was carried out in a monoclonal anti-AFP capture antibody-coated microplate with a sandwich-type assay format by using bienzyme-functionalized silica nanobeads as the recognition elements. Upon l-arginine introduction, the substrate was cleaved into urea and l-ornithine on the basis of the arginase enzymatic reaction, and the as-produced urea was then decomposed into ammonia (NH4+) and bicarbonate (HCO3-) ions by the doped urease, thus causing the variation in the local conductivity of the detection solution on an interdigitated conductometric transducer. Under optimal conditions, the developed immunosensing system exhibited good conductometric responses toward target AFP within a dynamic linear range of 0.01-100 ng mL-1 at a relatively low detection limit of 4.8 pg mL-1 based on the 3sB criterion. Importantly, good reproducibility, high specificity and acceptable method accuracy were acquired for the analysis of human serum specimens in liver cancer patients.

A pH-responsive bioassay for paper-based diagnosis of exosomes via mussel-inspired surface chemistry

The analysis of exosomes, which shows an increasing potential as prognostic biomarkers for non-invasive cancer diagnosis, can reveal their biological functions and disease associations. Nevertheless, the development of a convenient and quantitative method for determination of exosomes is still challenging. Herein, a novel approach for exosome quantification using pH test paper is developed via HRP-mediated promotion of mussel-inspired surface engineering and reagent-free functionalization of urease molecules. Uerase can hydrolyse urea into ammonia and carbon dioxide, and simultaneously raise the pH value of the solution. By establishing the relationship between exosome recognition and the change of pH value of the sensing solution, we can directly employ the low-cost, widely used and commercially available pH test paper to quantitatively analyse exosomes. The pH-responsive bioassay enables sensitive detection of exosomes with a detection of limit down to 4.46 × 103 particles/µL and can be successfully applied for determination of exosomes in clinical specimens. The versatility and reliability of our sensing platform may open up opportunities towards paper-based diagnosis of cancer.

Influence of Enzyme Quantity and Distribution on the Self-Propulsion of Non-Janus Urease-Powered Micromotors.

The use of enzyme catalysis to power micro- and nanomachines offers unique features such as biocompatibility, versatility, and fuel bioavailability. Yet, the key parameters underlying the motion behavior of enzyme-powered motors are not completely understood. Here, we investigate the role of enzyme distribution and quantity on the generation of active motion. Two different micromotor architectures based on either polystyrene (PS) or polystyrene coated with a rough silicon dioxide shell (PS@SiO2) were explored. A directional propulsion with higher speed was observed for PS@SiO2 motors when compared to their PS counterparts. We made use of stochastically optical reconstruction microscopy (STORM) to precisely detect single urease molecules conjugated to the micromotors surface with a high spatial resolution. An asymmetric distribution of enzymes around the micromotor surface was observed for both PS and PS@SiO2 architectures, indicating that the enzyme distribution was not the only parameter affecting the motion behavior. We quantified the number of enzymes present on the micromotor surface and observed a 10-fold increase in the number of urease molecules for PS@SiO2 motors compared to PS-based micromotors. To further investigate the number of enzymes required to generate a self-propulsion, PS@SiO2 particles were functionalized with varying amounts of urease molecules and the resulting speed and propulsive force were measured by optical tracking and optical tweezers, respectively. Surprisingly, both speed and force depended in a nonlinear fashion on the enzyme coverage. To break symmetry for active propulsion, we found that a certain threshold number of enzymes molecules per micromotor was necessary, indicating that activity may be due to a critical phenomenon. Taken together, these results provide new insights into the design features of micro/nanomotors to ensure an efficient development.

A novel potentiometric sensor based on urease/ bovine serum albumin-poly(3,4-ethylenedioxythiophene)/Pt for urea detection

AbstractWe have presented a potentiometric urea sensor using an urease/bovine serum albumin (BSA)-poly(3,4-ethylenedioxythiophene)(PEDOT)/Pt electrode. A urea detection sensitivity of 15.2 mV/decade (order of magnitude) has been achieved. BSA trapped in the PEDOT matrix was employed to bond urease molecules on the surface of a BSA-PEDOT/Pt electrode via amide bonds formed between the carboxyl functional groups on the enzyme and the amines on the BSA. The effects of PEDOT thickness, pH value of the urea solution, urease concentration, and temperature on the urea detection sensitivity were also studied. The lifetime of the sensor was studied for a period of 10 weeks, and the average sensing degradation rate was about 9 % per week. This sensor displayed a high selectivity to urea over glucose, KCl, and NaCl.

Carbon isotope fractionation during calcium carbonate precipitation induced by urease‐catalysed hydrolysis of urea

Stable carbon isotopic fractionation during calcium carbonate precipitation induced by urease‐catalysed hydrolysis of urea was experimentally investigated in artificial water at a constant temperature of 30°C. Carbon isotope fractionation during urea hydrolysis follows a Rayleigh distillation trend characterized by a 13C‐enrichment factor of −20 to −22‰. CaCO3 precipitate is up to 17.9‰ 13C‐depleted relative to the urea substrate (−48.9±0.07‰). Initial CaCO3 precipitate forms close to isotopic equilibrium with dissolved inorganic carbon. Subsequent precipitation occurs at −2 to −3‰ offset from isotopic equilibrium, suggesting that the initial δ13C value of CaCO3 is reset through dissolution followed by reprecipitation with urease molecules playing a role in offsetting the δ13C value of CaCO3 from isotopic equilibrium. Potentially, this isotopic systematics may provide a tool for the diagnosis of ureolytically‐formed carbonate cements used as sealing agent. Moreover, it may serve as a basis to develop a carbon isotope tool for the quantification of ureolytically‐induced CO2 sequestration. Finally, it suggests carbon isotope disequilibrium as a hallmark of past enzymatic activity in ancient microbial carbonate formation.

In situ entrapment of urease in cryogels of poly(N‐isopropylacrylamide): An effective strategy for noncovalent immobilization of enzymes

AbstractSupermacroporous poly(N‐isopropylacrylamide) (PNIPAAm) cryogels containing urease were prepared via UV irradiation technique and hydrogen peroxide as initiator. Specifically, due to the cryostructuration phenomenon urease molecules were embedded into the dense cryogel walls. Thus, although the enzyme is physically entrapped, the system exhibited remarkable resistance against leaking due to the dense polymer network formed in the cryogel walls. The immobilized urease can catalyze the hydrolysis of urea in a broad temperature range in both batch and flow regime. The interconnected macropores assist for unhindered diffusion of the substrate and reaction products through the gel, thus, paving the way for consecutive reuse at a constant activity, in contrast to the conventional PNIPAAm hydrogel. Due to the spongy‐like morphology PNIPAAm cryogels containing urease can be exploited as highly permeable membrane for direct removal of traces of urea from continuously flowing feed solutions. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Comparative study on Pulsed Laser Deposition and Matrix Assisted Pulsed Laser Evaporation of urease thin films

Urease thin films were produced by Matrix Assisted Pulsed Laser Evaporation (MAPLE) and Pulsed Laser Deposition from two types of targets: frozen water solutions of urease with different concentrations (1–10% m/v) and pure urease pellets. The fluence of the ablating KrF excimer laser was varied between 300 and 2200 mJ/cm 2. Fourier transform infrared spectra of the deposited films showed no difference as compared to the original urease. Morphologic studies proved that the films consist of a smooth “base” layer with embedded micrometer-sized droplets. Absorption-coefficient measurements contradicted the traditional “absorptive matrix” model for MAPLE deposition. The laser energy was absorbed by urease clusters leading to a local heating-up and evaporation of the frozen matrix from the uppermost layer accompanied by the release of dissolved urease molecules. Significant enzymatic activity of urease was preserved only during matrix assisted transfer.

Integrated urea sensor module based on poly(3-methylthiophene)-modified p-type porous silicon substrate

Integrated three-electrode system module composed of a porous silicon (PS)-based sensing electrode, an Ag/AgCl thin film reference electrode (REtf), and a Pt thin film counter electrode (CE) was fabricated for monitoring urea level of artificially-prepared body fluid. After thermal evaporation of the 200 nm thick Ag on the Ti-underlayered planar p-type silicon (p-Si) substrate, the Ag Film was oxidized in a FeCl3 solution to obtain the Ag/AgCl REtf. Multi-layered REtf was clearly shown from results of the auger electron spectroscopy (AES) depth profile. The nernstian slope of the REtf also showed good reproducibility. The PS layer was formed by electrochemical anodization with applying constant current to the p-Si substrate in an ethanolic HF solution and the macro PS (2 μm diameter and 10 μm depth) was obtained. The electrochemical active area (Aea) of the PS-based Pt thin film electrode was determined from the cyclovoltammetric result of redox reactions of K3Fe(CN)6 on the electrode surface and compared with the Aea of the planar silicon (PLS)-based Pt film substrate. After electropolymerization of the conductive poly(3-methylthiophene) (P3MT) on the PS-based Pt thin film, urease (isoelectric point ≈ 4.1) molecules were electrostatically doped into the P3MT film by applying positive bias. Amperometric calibration curves for both PS- and PLS-based sensing electrodes were compared in the range of 0.1–125 mM urea concentrations and the cross-sectional scanning electron microscopy (SEM) image of the PS-based sensing electrode was also shown.

Using phospholipid Langmuir and Langmuir–Blodgett films as matrix for urease immobilization

The immobilization of enzymes in organized two-dimensional matrices is a key requirement for many biotechnological applications. In this paper, we used the Langmuir–Blodgett (LB) technique to obtain controlled architectures of urease immobilized in solid supports, whose physicochemical properties were investigated in detail. Urease molecules were adsorbed at the air–water interface and incorporated into Langmuir monolayers of the phospholipid dipalmitoyl phosphatidyl glycerol (DPPG). Incorporation of urease made DPPG monolayers more flexible and caused the reduction of the equilibrium and dynamic elasticity of the film. Urease and DPPG–urease mixed monolayers could be transferred onto solid substrates, forming LB films. A close packing arrangement of urease was obtained, especially in the mixed LB films, which was inferred with nanogravimetry and electrochemistry measurements. From the blocking effect of the LB films deposited onto indium tin oxide (ITO) substrates, the electrochemical properties of the LB films pointed to a charge transport controlled by the lipid architecture.

Retention of activity of urease immobilized on grafted polymer films

AbstractExpanded poly(tetrafluoroethylene) (ePTFE) films grafted with 2‐hydroxyethyl methacrylate (HEMA) and 2‐hydroxyethyl acrylate (HEA) were applied to a polymer support for urease immobilization. The HEMA‐ and HEA‐grafted ePTFE (ePTFE‐g‐PHEMA and ePTFE‐g‐PHEA) films prepared by the combined use of the plasma treatment and photografting possessed high water‐absorptivities. Imidazole groups were introduced to grafted PHEMA and PHEA chains with 1,1′‐carbonyldiimidazole (CDI) in acetonitrile. The activity of urease covalently immobilized to the ePTFE‐g‐PHEMA and ePTFE‐g‐PHEA films in a pH 7.0 buffer at 4°C had the maximum value at the optimum pH value of 7.5 for native urease. Urease immobilized on the ePTFE‐g‐PHEMA films with the extent of CDI bonding of about 20% had the maximum activity, and the repeatedly measured activity was kept almost constant. The relative activity of immobilized urease stayed almost constant in the range of the immobilized amounts between 10 and 30 mg/g for both grafted ePTFE films, and decreased at higher immobilized amounts because of the crowding of immobilized urease molecules in the grafted layers. The relative activity of immobilized urease had the maximum values at the grafted amounts of 1.2 and 1.7 mmol/g for the ePTFE‐g‐PHEMA and ePTFE‐g‐PHEA films, respectively, and the further increase in the grafted amount resulted in the decrease in the relative activity. The optimum temperature of the activity for immobilized urease was shifted from 30 to 50°C for native urease by the covalent immobilization on both grafted ePTFE films and immobilized urease was repeatedly usable without a considerable decrease in the activity in the regions of the pH 6.0–9.0 and 10–60°C. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 4886–4896, 2006

Poly(3-methylthiophene)-based porous silicon substrates as a urea-sensitive electrode

Poly(3-methylthiophene) (P3MT)-based porous silicon (PS) substrates were fabricated and characterized by cyclic voltammetry, scanning electron microscopy, and auger electron spectroscopy. After doping urease (Urs) into the polymeric matrix, sensitivity and physicochemical properties of the P3MT-based PS substrate was investigated compared to planar silicon (PLS) and bulk Pt substrates. PS substrate was formed by electrochemical anodization in an etching solution composed of HF, H 2O, and ethanol. Subsequently, Ti and Pt thin-films were sputtered on the PS substrate. Effective working electrode area ( A eff) of the Pt-deposited PS substrate was determined from a redox reaction of Fe(CN) 6 3−/Fe(CN) 6 4− redox couple in which nearly reversible cyclic voltammograms were obtained. The i p versus v 1/2 plots showed that A eff of the PS-based Pt thin-film electrode was 1.62 times larger than that of the PLS-based electrode. Electropolymerization of P3MT on both types of electrodes were carried out by the anodic potential scanning under the given potential range. And then, urease molecules were doped to the P3MT film by the chronoamperometry. Direct electrochemistry of a Urs/P3MT/Pt/Ti/PS electrode in an acetonitrile solution containing 0.1 mol/L NaClO 4 was introduced compared to a P3MT/Pt/Ti/PS electrode at scan rates of 10 mV s −1, 50 mV s −1, and 100 mV s −1. Amperometric sensitivity of the Urs/P3MT/Pt/Ti/PS electrode was ca. 1.67 μA mM −1 per projected unit square centimeter, and that of the Urs/P3MT/Pt/Ti/PLS electrode was ca. 1.02 μA mM −1 per projected unit square centimeter in a linear range of 1–100 mM urea concentrations. 1.6 times of sensitivity increase was coincident with the results from cyclic voltammetrc analysis. Surface morphology from scanning electron microscopy (SEM) images of Pt-deposited PS electrodes before and after the coating of Urs-doped P3MT films showed that pore diameter and depth were 2 μm and 10 μm, respectively. Multilayered-film structures composed of metals and organics for both electrodes were also confirmed by auger electron spectroscopy (AES) depth profiles.

SURVIVING IN AN ACID BATH

THE ULCER-CAUSING BACTEria called Helicobacter pylori fascinate researchers. These bacteria live in human stomachs, which once were thought uninhabitable because of the extremely acidic conditions they harbor. H. pylori 's survival in the gut is known to be due to its high production of urease. When this enzyme hydrolyzes urea, the ammonia formed neutralizes the acidity around the bacterium, creating a livable microenvironment. Now, the crystal structure of H. pylori urease is providing more clues to the bacteria's survival tactics. The structure was obtained recently by Byung-Ha Oh and coworkers at Pohang University of Science & Technology and at Catholic University of Daegu, both in South Korea [ Nat. Struct. Biol. , 8,505(2001)]. Unlike other ureases, H.pylori urease molecules reside both inside the cytoplasm and on the cell surface. The surface molecules come from spontaneous lysis of other H.pylori , which releases ureases that then adsorb onto the surface of intact bacteria. Scientists have believed ...

Urease Encapsulation in Nanoorganized Microshells

Stable hollow polyelectrolyte capsules were produced by means of the layer-by-layer assembling of poly(allylamine), PAH, and poly(styrenesulfonate), PSS, on melamine formaldehyde microcores followed by the core decomposition at low pH. These capsules are nonpermeable for urease in water and become permeable in a water/ethanol mixture. The capsules were loaded with urease in water/ethanol mixture and then resuspended in water. The urease molecules are kept in the capsule, whereas the small urea molecules rapidly diffuse through the capsule wall providing a substrate for the biocatalytic reaction.

Ammonia as an accelerator of tumor necrosis factor alpha-induced apoptosis of gastric epithelial cells in Helicobacter pylori infection.

The mechanism by which Helicobacter pylori induces apoptosis remains unclear. In a previous study using biopsy samples, we found a significant correlation between the urease activity of an H. pylori strain and the apoptosis level induced by this strain. Therefore, in this study, we investigated whether urease and/or the ammonia generated by urease can induce apoptosis. Human gastric epithelial cell lines were cocultured with H. pylori, and the levels of apoptosis and ammonia production were measured. The medium was supplemented (or not supplemented) with urea and cytokines. While a large amount of ammonia (>30 mM) accumulated in the coculture containing urease-positive H. pylori and urea, no significant degree of apoptosis occurred. In the presence of tumor necrosis factor alpha (TNF-alpha), however, a marked acceleration of apoptosis was found in this coculture. Such enhancement of apoptosis was also induced by the addition of 4 to 8 mM ammonia to the cell culture without either H. pylori or urea but containing TNF-alpha. These results suggested that ammonia accelerates cytokine-induced apoptosis in gastric epithelial cells, while ammonia or urease molecules alone are unable to induce a significant degree of apoptosis.

Preparation of Grafted Expanded Poly(tetrafluoroethylene) Films for Immobilization of Urease.

Expanded poly(tetrafluoroethylene) (ePTFE) films grafted with 2-hydroxyethyl methacrylate (HEMA) were applied to a polymer support for urease immobilization. The amount of absorbed water for HEMA-grafted ePTFE (ePTFE-g-PHEMA) films prepared by the combined use of the plasma treatment and photografting increased with an increase in the grafted amount and the ePTFE-g-PHEMA films possessed high water-absorptivities. Imidazole groups were introduced to grafted PHEMA chains with 1, 1′-carbonyldiimidazole in acetonitrile, and then urease was covalently immobilized to the ePTFE-g-PHEMA films in a pH 7.0 buffer at 4°C. The activity of urease immobilized at pH 7.0 had the maximum value at the optimum pH level of 7.5 for native urease. The relative activity of immobilized urease was kept almost constant in the range below the immobilized amount of 40mg/g-ePTFE and decreased probably due to crowding of urease molecules in the grafted layers at further increased immobilized amounts. An increase in the grafted amount resulted in a decrease in the relative activity of immobilized urease because the diffusivity of the substrate urea into the grafted layer decreased. The optimum temperature of the activity for urease was shifted to 60°C from 30°C for native urease by the covalent immobilization onto the ePTFE-g-PHEMA films and immobilized urease was repeatedly usable without a considerable decrease in the activity in the regions of the pH 6.0 to 9.0 and 10 to 60°C.

A laponite clay-poly(pyrrole–pyridinium) matrix for the fabrication of conductimetric microbiosensors

The functionalization of an interdigitated microelectrode array by a composite matrix containing urease molecules and its application to the conductimetric detection of urea is described. Urease was entrapped in laponite clay gel and cross-linked by glutaraldehyde. The latter coating was adsorbed onto an electrochemically generated poly(pyrrole–pyridinium) film. The comparison of different organic and inorganic host matrices in terms of storage and operational stabilities clearly demonstrated the advantages of the composite matrix. Finally, in order to illustrate the potentialities of the composite matrix for the fabrication of conductimetric microbiosensors, the immobilization of glucose oxidase on microelectrode arrays and the conductimetric detection of glucose were successfully performed.

Urea determination using pH–enzyme electrode

A pH-membrane electrode with n-tridodecylamine (TDDA) as the hydrogen-ion-selective ionophore was used for the construction of a potentiometric biosensor for urea determination. The electrode was enzymatically modified by covalent binding of urease molecules directly to the surface of the potentiometric membrane. Incorporation of the urea biosensor into simple double-channel flow injection analysis (FIA) system allows reproducible urea determination in a millimolar range of concentration. The utility and limitations of the presented biosensor–FIA system for analysis of various real samples has been investigated. The system can be useful for some biomedical and pharmaceutical applications such as analyses of urine, posthaemodialysis fluid and extracts from pharmaceutical ointments containing urea.