Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K _M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol?L ^?1 and 164 ± 13.4 μmol?L ^?1 , respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for P_i-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

Effect of Mercury(II) Traces on Catalytic Activity of Peanut and Horseradish Peroxidases

Abstract

Mercury(II) in the range of 0.1–1 µg L^?1 concentrations was found to be a much more efficient inhibitor of native peanut peroxidase (PNP) than of horseradish peroxidase (HRP) in the reaction of o‐dianisidine oxidation with hydrogen peroxide. The possible reason for the different degree of mercury(II) effects on the catalytic activity of both enzymes was studied. It was shown that the different number of glycans in PNP and HRP molecules (three and eight, respectively), or their absence in the molecule of wild‐type recombinant horseradish peroxidase refolded from E. coli inclusion bodies (recHRP), does not play a significant role in the effects caused by mercury(II). The efficient inhibition of PNP by mercury(II) in the absence of any other additives (for example, thiourea) originates from a greater mobility of the distal calcium ion in the enzyme molecule. A model scheme for the interaction of the studied plant peroxidases with mercury(II) was proposed. The PNP‐based enzymatic method for mercury(II) determination with c _min =0.04 µg L^?1 (0.2 nmol L^?1) was developed and the possibility of PNP application for analysis of different samples was demonstrated.     

Synthesis and structures of platinum(II) mixed ligand complexes with purine or piyrimidine bases of DNA and imidazole derivatives. Part 2

Summary Platinum(II) mixed ligand complexes with either purine or pyrimidine and imidazole derivatives were prepared and characterized by i.r., Raman and electronic spectroscopy. The compounds had the general formula [PtL¹L²Cl₂], where L¹ = adenine, guanine, hypoxanthine, cytosine, 2-aminopyrimidine; L² =N-methylimidazole,N-ethyl-imidazole orN-propylimidazole. The platinum(II) complexes had a square planar structure withcis-halogens. Purine or pyrimidine and imidazole derivatived bases acted as monodentate ligands coordinated via the N(7) of purine and N(3) of pyrimidine and imidazole derivatives.     

Development,validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase

Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC 2.4.2.1) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The enzymatic assay (v = 0.5 mL, enzyme<0.2 μg/well, T = 37 °C) was performed with an overall time of about 15 min/plate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met the criteria of the CDER guidance of FDA. Kinetic parameters were in agreement with those reported in literature (HsPNP K_M = 0.150 ± 0.020 mM vs 0.133 ± 0.015 mM; MtbPNP K_M = 0.060 ± 0.009 mM vs 0.040 ± 0.003 mM for Ino), thus demonstrating the reliability of the newly developed enzymatic assay. Preliminary inhibition assays confirmed the effects reported for Acyclovir (Acv) and Formycin A (FA) against HsPNP and MtbPNP. The validated enzymatic assay was applied to the evaluation of a set of 8-halo-, 8-amino-, 8-O-alkyl-substituted purine ribonucleosides synthesized on purpose as potential inhibitors against MtbPNP. The assayed 8-substituted ribonucleosides did not exert a significant inhibitory effect against the tested enzymes up to 1 mM.     

Voltammetric determination of hypoxanthine based on the enhancement effect of mesoporous TiO2-modified electrode

A mesoporous TiO₂ was synthesized according to the reported method, and then used to modify the carbon paste electrode (CPE). The electrochemical behavior of hypoxanthine was investigated with great detail. Compared with the unmodified CPE, the mesoporous TiO₂-modified CPE greatly enhances the oxidation signal of hypoxanthine. Due to huge surface area, well-defined and special mesopores, the mesoporous TiO₂-modified CPE shows considerable enhancement effect toward hypoxanthine. Based on this, a sensitive, rapid and convenient electrochemical method was developed for the determination of hypoxanthine. The linear range is over the range from 2.0 × 10⁻⁷ to 5.0 × 10⁻⁵ mol L⁻¹, and the limit of detection is estimated to be 5.0 × 10⁻⁸ mol L⁻¹. The relative standard deviation (RSD) for 10 mesoporous TiO₂-modified CPEs is 5.7%. Finally, this sensing method was successfully used to determine hypoxanthine in human blood serum samples.     

Synthesis and structure of palladium(II) mixed complexes with DNA purine or pyrimidine bases and imidazole derivatives. Part I

Summary Palladium(II) mixed ligand complexes with purine or pyrimidine and imidazole derivatives were prepared and characterized by i.r., Raman and electronic spectroscopy. The compounds have the general formula [Pd(L₁)(L₂)(X₂)]; where L₁ = adenine, guanine, hypoxanthine, cytosine, 2-aminopyrimidine, 4(6)-hydroxypyrimidine; L₂ = N-methylimidazole, N-ethylimidazole or N-propylimidazole; X = Cl or Br. The complexes are square planar with cis-halogens. The purine, pyrimidine and imidazole bases act as monodentate ligands coordinated via the N(7) of purine and N(3) of pyrimidine and imidazole.     

Electro-catalytic effect of Al2O3 supported onto activated carbon in oxidizing phenol at graphite electrode

In this work, the electro-catalytic oxidation of phenol was studied using the aluminum oxide supported onto activated carbon (Al₂O₃/AC). The later was successfully prepared by impregnating aluminum particles in the activated carbon (AC) using heat treatment. Al₂O₃/AC was characterized by X-ray diffraction (XRD) and infrared spectroscopy (IR). The electro-catalytic performance of the Al₂O₃/AC for phenol oxidation was studied using cyclic voltammetry (CV), chronoamperometry, linear sweep voltammetry polarization, electrochemical impedance spectroscopy and differential pulse voltammetry (DPV) in 0.1 mol L^?1 Na₂SO₄. It has been shown that the proposed catalyst exhibits remarkably an electro-catalytic performance toward phenol oxidation. Moreover, the oxidation peak currents are linearly dependent on the concentration of phenol in the wide ranges from 1.0 × 10^?3 mol L^?1 to 1.0 × 10^?4 mol L^?1 and 8.0 × 10^?5 mol L^?1 to 1.0 × 10^?6 mol L^?1 with a detection limit of 1.51 × 10^?7 mol L^?1 (signal (S) to noise (N) ratio, S/N = 3) and response time of 3 min. The possible interferences were evaluated in 1.0 × 10^?5 mol L^?1 of phenol. The proposed catalyst also indicated suitable repeatability and stability. Moreover, the proposed Al₂O₃/AC–CPE has been successfully applied for the phenol analysis in natural waters and olive oil samples with good recoveries.     

Direct Electron Transfer of Glucose Oxidase and Glucose Biosensor Based on Nano-structural Attapulgite Clay Matrix

The direct electrochemistry of glucose oxidase (GOD) was achieved based on the immobilization of GOD on a natural nano‐structural attapulgite (ATP) clay film modified glassy carbon (GC) electrode. The immobilized GOD displayed a pair of well‐defined quasi‐reversible redox peaks with a formal potential (E^0′) of ?457.5 mV (vs. SCE) in 0.1 mol·L^?1 pH 7.0 phosphate buffer solution. The peak current was linearly dependent on the scan rate, indicating that the direct electrochemistry of GOD in that case was a surface‐controlled process. The immobilized glucose oxidase could retain bioactivity and catalyze the oxidation of glucose in the presence of ferrocene monocarboxylic acid (FMCA) as a mediator with the apparent Michaelis‐Menten constant K^app_m of 1.16 mmol·L^?1. The electrocatalytic response showed a linear dependence on the glucose concentration ranging widely from 5.0×10^?6 to 6.0×10^?4 mol·L^?1 (with correlation coefficient of 0.9960). This work demonstrated that the nano‐structural attapulgite clay was a good candidate material for the direct electrochemistry of the redox‐active enzyme and the construction of the related enzyme biosensors. The proposed biosensors were applied to determine the glucose in blood and urine samples with satisfactory results.     

Direct Electrochemistry and Electrocatalysis of Hemoglobin Based on Nafion‐Room Temperature Ionic Liquids‐Multiwalled Carbon Nanotubes Composite Film

A robust and effective composite film combined the benefits of Nafion, room temperature ionic liquid (RTIL) and multi‐wall carbon nanotubes (MWNTs) was prepared. Hemoglobin (Hb) was successfully immobilized on glassy carbon electrode surface by entrapping in the composite film. Direct electrochemistry and electrocatalysis of immobilized Hb were investigated in detail. A pair of well‐defined and quasi‐reversible redox peaks of Hb was obtained in 0.10 mol·L^?1 pH 7.0 phosphate buffer solution (PBS), indicating that the Nafion‐RTIL‐MWNTs film showed an obvious promotion for the direct electron transfer between Hb and the underlying electrode. The immobilized Hb exhibited an excellent electrocatalytic activity towards the reduction of H₂O₂. The catalysis current was linear to H₂O₂ concentration in the range of 2.0×10^?6 to 2.5×10^?4 mol·L^?1, with a detection limit of 8.0×10^?7 mol·L^?1 (S/N=3). The apparent Michaelis‐Menten constant (K_m^app) was calculated to be 0.34 mmol·L^?1. Moreover, the modified electrode displayed a good stability and reproducibility. Based on the composite film, a third‐generation reagentless biosensor could be constructed for the determination of H₂O₂.     

Rapid Establishment of <Emphasis Type="Italic">p-</Emphasis>Nitrophenol Biodegradation in Acetate-Fed Aerobic Granular Sludge

The aim of the study was to investigate the acclimation of precultivated acetate-fed aerobic granular sludge to a toxic xenobiotic biodegradation. Establishment of p-nitrophenol (PNP) biodegradation in acetate-fed aerobic granular sludge and concomitant changes in the microstructure and bacterial community were determined. Rapid establishment of PNP utilization was observed in the granular sludge when fed with PNP as the sole carbon source. The specific PNP removal was 36-mg h^-1 g^-1 granular biomass at an initial PNP concentration of 50 mg L^-1. The presence of PNP resulted in significant membrane damage in a subpopulation of the bacterial consortium, as shown by BacLight viability staining. This was coincided with a significant decrease in the culturable bacterial diversity of the granular biomass. PCR-DGGE analysis revealed a shift and decrease in number of bands during the establishment of PNP biodegradation. Scanning electron microscopy showed the dominance of rod-shaped bacteria in the PNP-utilizing microbial granules. Our results suggest that acetate-fed granular sludge could be quickly adapted for PNP biodegradation.     

Nanostructured Alpha‐Nickel Hydroxide Electrodes for High Performance Hydrogen Peroxide Sensing

Nanostructured alpha‐nickel hydroxide (α‐Ni(OH)₂) immobilized on a Fluorine‐doped Tin Oxide (FTO) surface was explored for the construction of hydrogen peroxide amperometric Flow Injection Analysis (FIA) sensors. Their notable electrocatalytic activity and heterogeneous electron‐transfer rate were confirmed by the appearance of a broad and intense peak associated with the oxidation of hydrogen peroxide and the enhancement of sensibility in hydrodynamic conditions. The α‐Ni(OH)₂ electrodes exhibited a broad dynamic range (5×10^?6 to 1×10^?3 mol L^?1), low detection limit (2×10^?7 mol L^?1), good repeatability (RSD=1.29 % for 20 successive analyses), and a sensitivity greater than 500 µA mmol^?1 L^?1 cm^?2.     

Studies of Penicillin G Acylase Immobilization Using Highly Porous Cellulose-Based Polymeric Membrane

The different ionic molecules/compounds were used as a ligand for the immobilization of penicillin G acylase on the highly porous cellulose-based polymeric membrane having buffer flux 1,746 LMH (L m⁻² h⁻¹) at 0.5 bar pressure. The immobilized enzyme activity around 250 U_App was obtained with the ligand such as proline, tryptophan, casein acid hydrolysate, and brilliant green. Comparatively, proline showed less IMY% (percentage immobilization yield—58) but higher RTA% (percentage of activity retention—71) and specific activity (145 U_App g⁻¹). However, the crosslinked preparation of brilliant green obtained using glutaraldehyde showed 82 ± 2.7% immobilized enzyme activity after the completion of successive five cycles. In comparison with the free enzyme, the enzyme immobilized on the brilliant green coupled membrane showed around 2.4-fold increase in K _m value (47.4 mM) as well as similar optimum pH (7.2) and temperature (40 °C). The immobilized enzyme retained almost 50% activity after 107 days and 50 cycles of operation. Almost 50% decrease in buffer flux after enzyme immobilization was observed. At the end of the 30 cycles, flux pattern shows around 38% decrease in buffer flux however, after 16 cycles of operation flux moves closer towards the steady state.     

Immobilization of Enzymes on the Nano‐Au Film Modified Glassy Carbon Electrode for the Determination of Hydrogen Peroxide and Glucose

A new enzyme‐based amperometric biosensor for hydrogen peroxide was developed relying on the efficient immobilization of horseradish peroxidase (HRP) to a nano‐scaled particulate gold (nano‐Au) film modified glassy carbon electrode (GC). The nano‐Au film was obtained by a chitosan film which was first formed on the surface of GC. The high affinity of chitosan for nano‐Au associated with its amino groups resulted in the formation of nano‐Au film on the surface of GC. The film formed served as an intermediator to retain high efficient and stable immobilization of the enzyme. H₂O₂ was detected using hydroquinone as an electron mediator to transfer electrons between the electrode and HRP. The HRP immobilized on nano‐Au film maintained excellent electrocatalytical activity to the reduction of H₂O₂. The experimental parameters such as the operating potential of the working electrode, mediator concentration and pH of background electrolyte were optimized for best analytical performance of amperometry. The linear range of detection for H₂O₂ is from 6.1×10^?6 to 1.8×10^?3 mol L^?1 with a detection limit of 6.1 μmol L^?1 based on signal/noise=3. The proposed HRP enzyme sensor has the features of high sensitivity (0.25 Almol^?1cm^?2), fast response time (t_90%≤10 s) and a long‐term stability (>1 month). As an extension, glucose oxidase (GOD) was chemically bound to HRP‐modified electrode. A GOD/HRP bienzyme‐modified electrode formed in this way can be applied to the determination of glucose with satisfactory performance.     

Using flowerlike polymer–copper nanostructure composite and novel organic–inorganic hybrid material to construct an amperometric biosensor for hydrogen peroxide

A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping horseradish peroxidase (HRP) in the organic–inorganic hybrid material composed of zirconia–chitosan sol–gel and Au nanoparticles (ZrO₂–CS–AuNPs). The sensitivity of the biosensor was enhanced by a flowerlike polymer–copper nanostructure composite (pPA–FCu) which was prepared from co-electrodeposition of CuSO₄ solution and 2,6-pyridinediamine solution. Several techniques, including UV–vis absorption spectroscopy, scanning electron microscopy, cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy were employed to characterize the assembly process and performance of the biosensor. The results showed that this pPA–FCu nanostructure not only had excellent redox electrochemical activity, but also had good catalytic efficiency for hydrogen peroxide. Also the ZrO₂–CS–AuNPs had good film forming ability, high stability and good retention of bioactivity of the immobilized enzyme. The resulting biosensors showed a linear range from 7.80 × 10^?7 to 3.7 × 10^?3 mol L^?1, with a detection limit of 3.2 × 10^?7 mol L^?1 (S/N = 3) under optimized experimental conditions. The apparent Michaelis–Menten constant was determined to be 0.32 mM, showing good affinity. In addition, the biosensor which exhibits good analytical performance, acceptable stability and good selectivity, has potential for practical applications.     

Platinum(II) and palladium(II) chlorides complexes with purine,pyrimidine,N-ethylimidazole andN-propylimidazole

Summary Platinum(II) and palladium(II) chloride complexes with purine, pyrimidine (pyrimid),N-ethylimidazole(N-EtIm) andN-propylimidazole(N-PropIm) ligands have been prepared and characterized by analysis and spectroscopic methods. The compounds have general formula M(L₁)(L₂)Cl₂ where M=Pt^II, Pd^II; L₁=purine or pyrimid, L₂=N-EtIm orN-PropIm, except the complexes Pt(purine)(pyrimid)Cl₂ and [Pd(purine)(pyrimid)₂Cl]Cl and [Pt(purine)₂ (N-propIm)Cl]Cl·2H₂O.     

Amplified Electrochemical Response of Phenol by Oxygenation of Tyrosinase Coupling with Electrochemical‐chemical‐chemical Redox Cycle

In this work, an novel electrochemical‐chemical‐chemical (ECC) redox cycle was designed in an enzyme‐based sensor for acquiring additional signal amplification. The tyrosinase (Tyr) was entrapped in a sulfonated polyaniline?chitosan (SPAN?CS) composite which was used as a redox capacitor on a glass carbon electrode. Firstly, the substrate, phenol was catalyzed to catechol and further catalyzed to o‐benzoquinone by Tyr. Next, in the presence of Ru(NH₃)₆Cl₂, the reduced state of SPAN(SPAN_red) was reacted with o‐benzoquinone to form it's oxidized state (SPAN_ox) and catechol, then SPAN_ox was reduced back to SPAN_red by Ru(II) in the solution. Finally, the amplified anodic current of catechol was obtained on electrode through above ECC redox cycle system. In addition, the ECC redox cycling led to a high signal‐to‐background ratio. The voltammetric response showed excellent analytical performance to phenol over two linear range of 3.5 to 200.0 nmol L^?1 and 200.0 to 2000.0 nmol L^?1 with a high sensitivity of 2204 μA mM^?1. The detection limit was obtained to be 0.8 nmol L^?1 (S/N=3). Furthermore, the proposed approach exhibited good repeatability, stability and specificity, and could offer practicality in the detection of phenol in tap water.     

Electrochemiluminescence for probing interactions between a mimic enzyme and biological molecules

The present work proposed a novel ECL protocol to probe the interactions between mimic enzymes and small biological molecules. Iron(II) phthalocyanine (FePc) and two imidazoles (imidazole and histidine) were chosen as model molecules of mimic enzyme and small biological molecules, respectively. The interactions between FePc and the imidazoles were probed by a sensitive luminol–O₂ ECL system. Before complexing with the imidazoles, FePc can inhibit luminol–O₂ ECL due to its electrocatalysis towards O₂, however, after complexing with the imidazoles, FePc decreases the electrocatalysis, leading to the observation of an enhanced luminol–O₂ ECL. Additionally, the proposed protocol enables detection limits of 1.0 × 10^?8 mol L^?1 and 1.0 × 10^?7 mol L^?1 to be achieved, respectively, for imidazole and histidine under the physiological pH condition (pH 7.4).     

A New Amperometric Biosensor Based on HRP/Nano-Au/L-Cysteine/Poly（o-Aminobenzoic acid）-Membrane-Modified Platinum Electrode for the Determination of Hydrogen Peroxide

The third generation amperometric biosensor for the determination of hydrogen peroxide （H2O2） has been described. For the fabrication of biosensor, o-aminobenzoic acid （oABA） was first electropolymerized on the surface of platinum （Pt） electrode as an electrostatic repulsion layer to reject interferences. Horseradish peroxidase （HRP） absorbed by nano-scaled particulate gold （nano-Au） was immobilized on the electrode modified with polymerized o-aminobenzoic acid （poABA） with L-cysteine as a linker to prepare a biosensor for the detection of H2O2. Amperometric detection of H2O2 was realized at a potential of ＋20 mV versus SCE. The resulting biosensor exhibited fast response, excellent reproducibility and sensibility, expanded linear range and low interferences. Temperature and pH dependence and stability of the sensor were investigated. The optimal sensor gave a linear response in the range of 2.99×10^-6 to 3.55×10^-3 mol·L^-1 to H2O2 with a sensibility of 0.0177 A·L^-1·mol^-1 and a detection limit （S/N = 3） of 4.3×10^-7 mol·L^-1. The biosensor demonstrated a 95% response within less than 10 s.     

Nicke(II) halide complexes with hypoxanthine and xanthine

Summary Ni(LH)₃LX complexes (LH=hypoxanthine or xanthine; X=Cl, Br or I) are formed by boiling under reflux 2:1 molar mixtures of LH and hydrated NiX₂ in HC(OEt)₃–MeCO₂Et. The new complexes appear to be linear chain-like polymers, characterized by bidentate monoanionic L^– ligands singly bidging between adjacent Ni²⁺ ions. A coordination number six is attained by the presence of three terminal unidentate LH and one X ligand in the first coordination sphere of each Ni²⁺ ion. The neutral LH and monoanionic L^– ligands bind exclusivelyvia ring nitrogens to Ni^II. The probable binding sites of the uni- and bi-dentate hypoxanthine and ligands in the new complexes are discussed.Presented in part at the 3rd Chem. Congress of North America (LH=xanthine) and the XXVI ICCC (LH=hypoxanthine), see refs. 1 and 2, respectively.     

Production of Butyric Acid from Glucose and Xylose with Immobilized Cells of <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> in a Fibrous-bed Bioreactor

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g⁻¹, respectively, in batch fermentation when 30 g L⁻¹ glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L⁻¹ and yield 0.40 g g⁻¹ of butyric acid were obtained with 25 g L⁻¹ xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L⁻¹ with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).