Photomicrobial sensors for selective determination of phosphate

A photomicrobial sensor consisting of immobilized Chlorella vulgaris and an oxygen electrode has been developed for selective determination of phosphate. When 40 mM phosphate was added to the sensor system, the photocurrent increased to a maximum under light irradiation with a response time of 1 min. The current increased with increasing phosphate concentration in the range 8–70 mM. Selectivity of the sensor was satisfactory. Good agreement was obtained between the phosphate concentrations in lake water determined by the photomicrobial sensor and by conventional colorimetry (correlation coefficient 0.96).     

Enzyme sensor for hypoxanthine and inosine determination in edible fish

Summary An enzyme sensor for hypoxanthine (Hx) and inosine (HxR), consisting of an enzyme membrane and an oxygen electrode, was constructed, Xanthine oxidase (XO) and nucleoside phosphorylase (NP) were both immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. The enzyme sensor responded to Hx and HxR in the presence of phosphate, while it responded only to Hx in the absence of phosphate. A linear correlation was observed between current decrease and the concentrations of Hx and HxR in the range 0.5–2.0 mM respectively. Correlation coefficients between the present enzyme sensor and a conventional enzymatic method were 0.98 and 0.94 for Hx and HxR respectively. The standard deviation was +-1.5 M and 0.75 M for Hx and HxR respectively in 100 experiments. A simple and rapid determination of Hx and HxR in fish meat was possible within 3 min with the enzyme sensor.     

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant: Isolation and Characterization 1

Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisum sativum L.) seedlings and was purified 65-fold. The purified enzyme exhibits one predominant protein band by polyacrylamide gel electrophoresis, which corresponds to the dehydrogenase activity as measured by the nitro blue tetrazolium technique. The reaction is readily reversible, the pH optima for the forward (nicotinamide adenine dinucleotide phosphate reduction) and reverse reactions being 8.4 and 6.0, respectively. The enzyme has different cofactor and inhibitor characteristics in the two directions. Manganese ions can be used as a cofactor for the reaction in each direction but magnesium ions only act as a cofactor in the forward reaction. Zinc ions, and to a lesser extent calcium ions, inhibit the enzyme at low concentrations when magnesium but not manganese is the metal activator. It is suggested that there is a fundamental difference between magnesium and manganese in the activation of the enzyme. The enzyme shows normal kinetics and the Michaelis contant for each substrate was determined. The inhibition by nucleotides, nucleosides, reaction products, and related compounds was studied. The enzyme shows a linear response to the mole fraction of reduced nicotinamide adenine dinucleotide phosphate when total nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate plus reduced nicotinamide adenine dinucleotide phosphate) is kept constant. Isocitrate in the presence of divalent metal ions will protect the enzyme from inactivation by p-chloromercuribenzoate. Protection is also afforded by manganese ions alone but not by magnesium ions alone There is a concerted inhibition of the enzyme by oxalacetate and glyoxylate.     

A ratiometric fluorescent sensor for the detection of phosphate

Over the past few years, ratiometric fluorescent nanoprobes have garnered substantial interest because of their self-calibration characteristics. This research developed a ratiometric fluorescent sensor to detect phosphate. Through encapsulating luminescent materials, gold nanoclusters (AuNCs) and carbon dots (CDs) into a zeolitic imidazolate framework-8 (ZIF-8), the fluorescence signal of AuNCs was enhanced, while that of CDs was suppressed. After phosphate was added, it could decompose ZIF-8, and AuNCs and CDs were released, which weakened the fluorescence signal of the AuNCs while restoring that of the CDs. Thereby, this makes CDs/AuNCs@ZIF-8 a potential fluorescent sensor for phosphate determination. The ratiometric sensor had facile synthesis, good selectivity, and a low detection limit. Therefore, this sensor was an effective tool for the detection of phosphate.     

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degrees C. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degrees C. Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme. An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells.     

Amperometric biosensor for determining human salivary phosphate

An amperometric biosensor was constructed for analysis of human salivary phosphate without sample pretreatment. The biosensor was constructed by immobilizing pyruvate oxidase (PyOD) on a screen-printed electrode. The presence of phosphate in the sample causes the enzymatic generation of hydrogen peroxide (H(2)O(2)), which was monitored by a potentiostat and was in proportion to the concentration of human salivary phosphate. The sensor shows response within 2s after the addition of standard solution or sample and has a short recovery time (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 to 625 microM phosphate with a detection limit of 3.6 microM. A total of 50 salivary samples were collected for the determination of phosphate. A good level of agreement (R(2)=0.9646) was found between a commercial phosphate testing kit and the phosphate sensor. This sensor maintained a high working stability (>85%) after 12h operation and required only a simple operation procedure. The amperometric biosensor using PyOD is a simple and accurate tool for rapid determinations of human salivary phosphate, and it explores the application of biosensors in oral and dental research and diagnosis.     

Phosphate-dependent glutaminase in enterocyte mitochondria and its regulation by ammonium and other ions

Summary.  The effects of ammonium and other ions on phosphate dependent glutaminase (PDG) activity in intact rat enterocyte mitochondria were investigated. Sulphate and bicarbonate activated the enzyme in absence and presence of added phosphate. In presence of 10 mM phosphate, ammonium at concentrations <1 mM inhibited the enzyme. This inhibition was reversed by increased concentration of phosphate or sulphate. The inhibition of PDG by ammonium in presence of 10 mM phosphate was biphasic with respect to glutamine concentration, its effect being through a lowering of V_max at glutamine concentration of ≤5 mM, and increased K_m for substrate concentration above 5 mM. The activation of the enzyme by bicarbonate was through an increase in V_max. Ammonium and bicarbonate ions may therefore be important physiological regulators of PDG. It is suggested that phosphate and other polyvalent ions may function by preventing product inhibition of the enzyme through promotion of PDG dimer formation. The dimerized enzyme may have a high affinity for glutamine and reduced sensitivity to inhibition by ammonium ions. Received August 10, 2001 Accepted April 1, 2002 Published online August 30, 2002 Acknowledgement This work was supported by University of Zimbabwe research grant to Dr. B. Masola. Authors' address: Dr. Bubuya Masola, Department of Biochemistry, University of Zimbabwe, P O Box MP167, Mount Pleasant, Harare, Zimbabwe, E-mail: masolab@yahoo.co.uk     

Enzyme mechanism and catalytic property of beta propeller phytase

BACKGROUND: Phytases hydrolyze phytic acid (myo-inositol-hexakisphosphate) to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the beta propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. RESULTS: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. CONCLUSIONS: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo-inositol triphosphates from phytate and the hydrolysis of myo-inositol monophosphates.     

Extracellular Ca2(+)-dependent inducible alkaline phosphatase from extremely halophilic archaebacterium Haloarcula marismortui.

When starved of inorganic phosphate, the extremely halophilic archaebacterium Haloarcula marismortui produces the enzyme alkaline phosphatase and secretes it to the medium. This inducible extracellular enzyme is a glycoprotein whose subunit molecular mass is 160 kDa, as estimated by sodium dodecyl sulfate-gel electrophoresis. The native form of the enzyme is heterogeneous and composed of multiple oligomeric forms. The enzymatic activity of the halophilic alkaline phosphatase is maximal at pH 8.5, and the enzyme is inhibited by phosphate. Unlike most alkaline phosphatases, the halobacterial enzyme requires Ca2+ and not Zn2+ ions for its activity. Both calcium ions (in the millimolar range) and NaCl (in the molar range) are required for the stability of the enzyme.     

Determination of sulfate after chromatography and toluidine blue complex formation

A rapid and simple method for the determination of sulfate involving a complex formation between inorganic sulfate and the dye, toluidine blue O, after chromatography, is presented. The method can be used for the determination of sulfate in the presence of interfering ions such as phosphate and citrate. Most of the ions have a different chromatographic migration in the solvent system employed. An added advantage is the measurement of the labile sulfate of mucopolysaccharides with accuracy.     

Alkaline phosphatase from rat osseous plates: purification and biochemical characterization of a soluble form

A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.     

蔗糖-葡萄糖双功能酶传感器的研究

结合蔗糖转化酶(INV)酶管与葡萄糖氧化酶(GOD)-葡萄糖变旋酶(MUT)双酶电极构成一种新的蔗糖传感器。该传感器可以分别用于蔗糖及葡萄糖的测定。蔗糖经酶管作用产生α-D-葡萄糖,再用COD-MUT双酶电极定糖。若是样品中蔗糖和葡萄糖共存,比较样品流经不同路径(Ws和Wg)时传感器的响应值,可以排除葡萄糖对蔗糖测定的干扰。传感器的最适pH和温度范围分别为:5.0—6.5和30—40℃。在稳态法实验中,传感器的线性范围为:2.5×10~(-4)—5×10~(-3)mol/L。传感器的重复性很好,CV<1％。该传感器在用于测定发酵培养基(含葡萄糖)的蔗糖含量,平均回收率为97.9％。传感器与糖度计法测定的相关系数为0.997。传感器至少可以稳定使用8天以上。     

A screen-printed biosensor using pyruvate oxidase for rapid determination of phosphate in synthetic wastewater

A screen-printed phosphate biosensor based on immobilized pyruvate oxidase (PyOD, E.C. 1.2.3.3) has been developed for monitoring phosphate concentrations in a sequencing batch reactor (SBR) system. The enzyme was immobilized by a nafion matrix and covered a poly(carbamoyl) sulfonate (PCS) hydrogel on a screen-printed electrode. PyOD consumes phosphate in the presence of pyruvate and oxygen and generates hydrogen peroxide (H₂O₂), carbon dioxide and acetylphosphate. The electroactive H₂O₂, monitored at +420 mV vs Ag/AgCl, is generated in proportion to the concentration of phosphate. The sensor has a fast response time (2 s) and a short recovery period (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 M to 625 M phosphate with a detection limit of 3.6 M. There was good agreement (R²=0.9848) between the commercial phosphate testing kit and the phosphate sensor in measurements of synthetic wastewater in a SBR system. This sensor maintained a high working stability (>85%) after 12 h of operation and involved a simple operation procedure. It therefore serves as a useful tool for rapid and accurate phosphate measurements in the SBR system and probably for process control.     

UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase from Bacillus sphaericus: activation by potassium phosphate

During the course of purification of UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase, we observed a marked stimulation of the enzymatic activity in the presence of phosphate ions. This activation effect was studied with enzyme purified 979-fold from Bacillus sphaericus. Each salt tested stimulated the activity of the synthetase. The order of activation by different anions was HPO4(2-) greater than Cl- greater than SO4(2-). In every case, the potassium salt gave higher activity than the corresponding sodium salt. The activation in the presence of phosphate was quite pronounced (almost sevenfold with K2HPO4) and occurred at a relatively low concentration. The Ka for K2HPO4 was found to be 3.4 mM and the Hill coefficient was calculated to be 1.0. This would suggest that there is one phosphate-binding site per active centre. The presence of phosphate did not affect either the pH optimum of this enzyme or the optimum concentration of Mg2+ required. The presence of phosphate has little or no effect on the Km of any of the substrates. Thus, it appears that the presence of phosphate changes the enzyme conformation to a catalytically more active form. The activation of this enzyme in the presence of phosphate anion is all the more interesting because phosphate is a product of the reaction catalyzed by this enzyme.     

On the reliability of the lead salt precipitation method of acid phosphatase localization in plant cells

Summary Fixation of cells with glutaraldehyde (5.0%, pH 6.7) was found to facilitate both the penetration of substrate (p-nitrophenyl phosphate) into cells and the leaking out of intracellular phosphate ions. 64% of the original activity survived the fixation for at least 24 hours. Lead ions added to the incubation medium at 6 mM neither accelerated nonenzymatic hydrolysis of the substrate, nor completely inactivated the enzyme activity. Lead ions at concentrations above 6 mM formed an insoluble compound with p-nitrophenyl phosphate, resulting in a decrease in the concentration of free substrate and lead ions. Phosphate ions liberated from substrate could not be completely trapped by lead ions even at above 6 mM, suggesting the possibility of intracellular migration of phosphate ions.In the presence of 4 mM p-nitrophenyl phosphate, 6 mM lead nitrate, and 0.2 M sucrose at pH 6.5, lead salt precipitates were deposited on the outer surface of cell walls, within cell walls, at tonoplast membranes, in nuclei, and occasionally in proplastids. No deposition of lead salt was formed in the control test from which the substrate was omitted. When cells were treated at first with lead nitrate and then with potassium phosphate, lead salt deposits were formed in the same sites as those of cells incubated in a complete reaction medium.It is concluded that although the result of the lead salt precipitation procedure reflects the presence of enzyme activity, it cannot directly show the site of the enzyme.     

Determination of inorganic phosphorus using immobilized pyruvate oxidase

A new method for the automated analysis of inorganic phosphorus using immobilized enzyme was established. The method was based on the determination of hydrogen peroxide formed by the action of pyruvate oxidase on inorganic phosphate and pyruvate. Since pyruvate oxidase required inorganic phosphate for its stability and therefore had to be kept in a buffer containing inorganic phosphate, it could hardly be used as a reagent in the form of aqueous solution for the determination of inorganic phosphorus. This difficulty was overcome by using immobilized pyruvate oxidase in column form. When the present method was applied to the determination of inorganic phosphorus in serum, it gave perfect linearity of the data up to 0.20 g inorganic phosphorus/L with satisfactory precision, reproducibility, high sensitivity, and accurate recoveries. The immobilized enzyme reactor unit showed enhanced heat stability and good operational stability for a one-month period, during which time it was used over 900 times for analyses. The enzyme column was not affected by organic phosphorus compounds. The results correlated satisfactorily with those obtained by another well-established method.     

The Selective Detection of Cyantraniliprole Insecticides Using Molecularly Imprinted Polymers Coupled with an Acetylcholinesterase Inhibition-Based Biosensor

Cyantraniliprole is one of the anthranilic diamide insecticides widely used in the agriculture sector. Due to its low toxicity and relatively fast degradation, there is need for a sensitive determination method for its residues. Nowadays, there is growing interest in the development of enzyme-based biosensors. The major drawback is the non-specific binding of many insecticides to the enzyme. This work employs Molecularly imprinted polymers (MIPs) to increase enzyme specificity and eliminate the organic solvent effect on the enzyme activity. The synthesized Cyan-Molecularly imprinted polymers (Cyan-MIP) possesses high affinity and selectivity toward cyantraniliprole. Acetylcholinesterase assay characteristics including enzyme concentration, substrate concentration, DTNB concentration, and acetonitrile concentration were optimized. Under optimal experimental conditions, the developed MIP-Acetylcholinesterase (MIP-AchE) inhibition-based sensor provides better precision than the AchE inhibition-based sensor with a wide linear range (15–50 ppm), limit of detection (LOD) 4.1 ppm, and limit of quantitation (LOQ) 12.6 ppm. The sensor was successfully applied for cyantraniliprole determination in spiked melon, giving satisfactory recoveries.     

Phosphate-stimulated breakdown of 5′-methylthioadenosine by rat ventral prostate

A soluble enzyme preparation catalysing the release of adenine from 5'-methylthioadenosine was purified some 30-fold from extracts of the rat ventral prostate. This reaction was completely dependent on addition of inorganic phosphate ions to the assay medium. This absolute requirement for phosphate ions suggests a phosphorolytic cleavage mechanism. After acid treatment, the other product of the reaction appeared to be 5-methylthioribose. The actions of some other well-characterized enzymes of nucleoside metabolism of 5'-methylthioadenosine were also investigated; purified purine nucleoside phosphorylases from calf spleen and human erythrocytes did not attack 5'-methylthioadenosine. The role of 5'-methylthioadenosine in mammalian tissues is discussed.     

Electrochemical sensor based on photopolymeric membranes for determining urea

A new electrochemical enzymatic sensor based on the ion selective field effect transistors (ISFETs) and photocurable membrane was developed for the determination of urea. For the immobilization of urease on the gate surface of the ISFET a simple method, involving the use of liquid photocurable compositions on the basis of vinylpirollidone, oligouretanemetacrylate and oligocarbonatemetacrylate, was applied. The linearange of the response of the developed electrochemical sensor lies in the range 0.05-20 mM. The latter corresponds to the claims of the medical practice. The overall time of the analysis is 5-10 min. The effects of the buffer concentration and its pH as well as temperature and presence of ammonia ions in the measuring medium on the amplitude of the sensor response were estimated. The duration of sensor work is as shortest 40 days. The proposed sensor on the basis of the ISFET is promising for the express analysis of the level urea in blood, while the developed method of membrane preparation with entrapped enzyme can be combined with the integral technology of producing of the biosensors semiconductor transducers.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.