Enzyme analysis by means of the air-gap electrode-determination of urease and arginase by monitoring of the initial reaction rate

The air-gap electrode has been used in kinetic chemical analysis, illustrated by the assay of the activities of the enzymes urease and arginase. The determinations are based on monitoring the initial reaction rates of the selective release of ammoniacal nitrogen, which in the arginine—arginase system was ensured by adding excess of urease. The reaction rates measured are in the range 2.5 · 10^-5—1.5 · 10^-3M min^-1; the relative standard deviation is ca. 2.8 %.     

Surface acoustic wave sensor system applied to the kinetic study of urease in plant seeds

A new type of the surface acoustic wave (SAW) sensor system was delivered. Ure-ase from several kinds of plant seeds was extracted with different extracting solvents. The urease activity, Michaelis constant and other kinetic parameters were estimated for the first time by means of the new device-SAW sensor system. Some factors such as pH, temperature, activators and inhibitors are also discussed. The method can be applied to the determination of urea content in human urine and the experimental results consist with those reported.     

Determination of urea in urine using a conductivity cell with surface acoustic wave resonator-based measurement circuit

A conductivity cell employing a 61 MHz surface acoustic wave resonator-based measurement circuit was applied to the detection of the urea/urease reaction. The kinetic enzymatic parameters of the urease were estimated from the frequency shifts. The effects of pH, temperature and inhibitor on the response of the enzyme conductivity measurement system were investigated. The system was applied to rapid determination of urea in small urine samples. The lowest detection limit of urea was 30 ng/ml.     

Coupled enzyme reaction microarrays based on pin-printing of sol–gel derived biomaterials

We report on the development of a new class of protein microarrays based on the co-immobilization of multiple components within a single pin-printed sol–gel array element. In the first case, the microarraying of a coupled two enzyme reaction involving glucose oxidase and horseradish peroxidase along with the fluorogenic reagent Amplex Red is demonstrated to allow “reagentless” fluorimetric detection of glucose. The second system involved the detection of urea using co-immobilized urease and fluorescein dextran, which works on the basis of a pH induced change in fluorescein emission intensity upon production of ammonium carbonate owing to hydrolysis of urea. In both the cases, it is demonstrated that the changes in intensity from the array are time-dependent, consistent with the enzyme catalyzed reaction, showing that such arrays can be used for kinetic studies. The rate of intensity change was also found to be dependent on the concentration of analyte added to the array, showing that such arrays could be useful for quantitative multianalyte biosensing. Inhibition of urease by the competitive inhibitor thiourea is also demonstrated on a microarray, demonstrating that sol–gel-based microarrays may find use in high-throughput drug-screening applications.     

The hydrolysis of urea and the proficiency of urease

We present the results of a computational study of the solution phase decomposition of urea, which provides insight into probable reaction pathways for the urease-catalyzed reaction. Calculations, which were used to derive thermodynamic parameters that were further used for a kinetic analysis, have been done at the solvent-corrected MP2/6-311++G** level. Both elimination and hydrolytic pathways have been considered. Elimination is favored for the solution phase reaction, which proceeds by H-bond coordination of a water molecule to the amine nitrogen atoms. The coordination of one water molecule greatly facilitates the reaction by allowing it to proceed through a cyclic six-member transition state. Aspects of the water-urea H-bond interactions have also provided insights into critical aspects of the hydrogen bond pattern in the urease active site. On the basis of a kinetic analysis, we have estimated the proficiency of urease and have predicted that it is the most proficient enzyme identified to date.     

Kinetic studies of the urease-catalyzed hydrolysis of urea in a buffer-free system

The kinetics of urea hydrolysis catalyzed by urease, mainly in the absence of buffers by use of the self-buffer effect of the products, was investigated. The effect of pH, temperature, and concentration of enzyme, substrate, product, salt ions, and buffers on the kinetic behavior of urease was examined. A kinetic model of a modified Michaelis-Menten form, incorporating substrate and product inhibition, pH dependence, and temperature effect, was developed to describe the reaction rate. Experimental data indicated that urease in a buffer-free solution was less susceptible to the inhibition of substrate product. The Michaelis constant keeps almost constant with the variation of pH and temperature, and increases with the addition of buffers and salts. The data also suggested that the noncompetitive pattern of the product inhibition, which is not significantly affected by temperature, increases gently with increasing pH. A Monod form rate expression was proposed to analyze the pH effect on the maximum rate. The proposed kinetic model was also examined by the long-time experiments in which pH, substrate, and product concentration varied obviously during the reaction course.     

pH-Sensitive Oscillatory Motion of a Urease Motor on the Urea Aqueous Phase

A self-propelled object coupled with an enzyme reaction between urease and urea was investigated at the air/aqueous interface. A plastic object that was fixed to a urease-immobilized filter paper was used as a self-propelled object, termed a urease motor, placed on an aqueous urea solution. The driving force of the urease motor is the difference in the surface tension around the object. Oscillatory motion or no motion was triggered depending on the initial pH of the urea solution. Both the frequency and maximum speed of the oscillatory motion varied depending on the initial pH of the water phase. The mechanisms underlying the oscillatory motion and no motion were discussed in relation to the bell-shaped enzyme activity of urease in the enzyme reaction and the surface tension around the urease motor.     

Studies on urea biosensors based on immobilized corynebacterium glutamicum and their kinetic response processes

Two novel biosensors for urea based on immobilized corynebacterium glutamicum 617 and corynebacterium glutamicum ATCC13032 in calcium alginate gel coupled with an ammonia gas-sensing electrode, were designed and constructed. Calibration plots of measured potential difference (mV) vs. log of urea concentration were linear in the range of 5.6 x 10(-5)-1.4 x 10(-2) and 5.6 x 10(-5)-1.1 x 10(-2) mol l(-1), with slopes of 59.2 and 61.3 mV per decade respectively, in pH 8.0, 0.1 mol l(-1) phosphate buffer solution at 30 degrees C. The relationship between the initial response velocity and the substrate concentration was also discussed. The results indicate that the kinetic response process of the reaction catalyzed by bacteria is similar to that by isolated enzyme. Using an Eadie-Hofstee plot, the apparent Michaelis constant K(m) and the maximum initial response velocity V(m) for urease in the immobilized bacterial membrane were determined. The two urea biosensors were successfully applied for the actual measurement of urea in urine and were relatively stable for 20 and 40 days respectively.     

Tungsten electrode for urea

Tungsten electrodes for urea were prepared via covalent linking of urease on oxidized metal surfaces in different ways. The most stable electrodes were obtained when tungsten was silanized and activated by glutaraldehyde or hexamethylene diisocyanate. The electrode with urease coupled via glutaraldehyde was tested for optimum conditions of use. The nature of the buffer and its concentration and ionic strength are particularly important.     

New Urea Biosensor Based on Urease Enzyme Obtained from Helycobacter pylori

The urease enzyme of Helicobacter pylori was isolated from biopsy sample obtained from antrum big curvature cell extracts. A new urea biosensor was prepared by immobilizing urease enzyme isolated from Helicobacter pylori on poly(vinylchloride) (PVC) ammonium membrane electrode by using nonactine as an ammonium ionophore. The effect of pH, buffer concentration, and temperature for the biosensor prepared with urease from H. pylori were obtained as 6.0, 5 mM, and 25 °C, respectively. We also investigated urease concentration, stirring rate, and enzyme immobilization procedures in response to urea of the enzyme electrode. The linear working range of the biosensor extends from 1 × 10(-5) to 1 × 10(-2) M and they showed an apparent Nernstian response within this range. Urea enzyme electrodes prepared with urease enzymes obtained from H. pylori and Jack bean based on PVC membrane ammonium-selective electrode showed very good analytical parameters: high sensitivity, dynamic stability over 2 months with less decrease of sensitivity, response time 1-2 min. The analytical characteristics were investigated and were compared those of the urea biosensor prepared with urease enzyme isolated from Jack bean prepared at the same conditions. It was observed that rapid determinations of human serum urea amounts were also made possible with both biosensors.     

Adsorption of urease on PE-MCM-41 and its catalytic effect on hydrolysis of urea

Pore-expanded MCM-41 (PE-MCM-41) silica exhibits a unique combination of high specific surface area (ca. 1000 m(2)/g), pore size (up to 25 nm) and pore volume (up to 3.5 cm(3)/g). As such, this material is highly suitable for the adsorption of large biomolecules. The current study focused primarily on the application of PE-MCM-41 material as suitable host for urease (nickel-based large metalloenzyme) in controlled hydrolysis of urea. Urease adsorbed on PE-MCM-41, regular MCM-41 and silica gel (SGA) were used as catalysts for urea hydrolysis reaction. Adsorption studies of urease on these materials from aqueous solution at pH 7.2 revealed that the adsorption capacity of PE-MCM-41 (102 mg/g) is significantly higher than that of MCM-41 (56 mg/g) and SGA (21 mg/g). The equilibrium adsorption data were well fitted using the Langmuir-Freundlich model. Furthermore, the kinetic study revealed that the uptake of urease follow the pseudo-first order kinetics. The in vitro urea hydrolysis reaction on pristine urease and different urease-loaded catalysts showed that the rate of hydrolysis reaction is significantly slower on U/PE-MCM-41 compared to that of bulk urease and urease on MCM-41 and SGA. This technique could be an alternative means to the use of urease inhibitors to control the ammonia release from urea fertilizer.     

An amperometric urea biosensor based on covalent immobilization of urease on copolymer of glycidyl methacrylate and vinylferrocene

A unique urea biosensor construction based on the direct covalent attachment of urease onto a polymeric electron transfer mediator, poly(glycidyl methacrylate-co-vinylferrocene)-coated electrode is described. Amperometric response was measured as a function of urea concentration, at a fixed potential of +0.35 V vs. Ag/AgCl in phosphate-buffered saline (pH 7.0). Covalent immobilization of the urease directly to the functionalized ferrocene copolymer surface produced biosensors with a short response time (about 3 s) and provided low detection limits. The stability, reusability, pH, and temperature response of the biosensor, besides its kinetic parameter, were also studied.     

The burden borne by urease

At the active site of urease, urea undergoes nucleophilic attack by water, whereas urea decomposes in solution by elimination of ammonia so that its rate of spontaneous hydrolysis is unknown. Quantum mechanical simulations have been interpreted as indicating that urea hydrolysis is extremely slow, compared with other biological reactions proceeding spontaneously, and that urease surpasses all other enzymes in its power to enhance the rate of a reaction. We tested that possibility experimentally by examining the hydrolysis of 1,1,3,3-tetramethylurea, from which elimination cannot occur. In neutral solution at 25 degrees C, the rate constant for the uncatalyzed hydrolysis of tetramethylurea is 4.2 x 10-12 s-1, which does not differ greatly from the rate constants observed for the uncatalyzed hydrolysis of acetamide (5.1 x 10-11 s-1) or N,N-dimethylacetamide (1.8 x 10-11 s-1) under the same conditions. We estimate that the proficiency of urease as a catalyst, (kcat/Km)/knon, is 8 x 1017 M-1, slightly higher than the values for other metalloenzymes (carboxypeptidase b and cytidine deaminase) that catalyze the hydrolysis of similar bonds.     

Effect of thermosensitive matrix-phase transition on urease-catalyzed urea hydrolysis

Temperature dependencies of kinetic and equilibrium parameters of urea hydrolysis catalyzed by native urease and the urease immobilized in a thermosensitive poly-N-isopropylacrylamide gel have been studied. The swelling ratio of the collapsed urease-containing gel is shown to increase in the presence of urea. Below a lower critical solution temperature (LCST) of the polymer, the immobilized u reaseactually has thesame catalytic properties as the native enzyme. At temperatures above LCST, the observed catalytic activity of the immobilized enzyme depends chiefly not only on the thermoreversible matrix state, but also on gel water content.     

Kinetics of donor–acceptor complex polymerization. III. Assessment of some approximations in the kinetics

The mathematical solution of the equations derived from a kinetic scheme previously developed for donor–acceptor complex polymerization was based on steady-state conditions and the applicability of initial concentration conditions over a range of conversion. These assumptions are scrutinized and tested by computer simulation and by the exact differential equations utilizing Runge-Kutta method. The analysis shows that for case I conditions of low concentration of complexing-agent, the degree of conversion is not critical and that the previous approximate solutions are valid. The steady-state and non-steady-state conditions are compared, and the range of validity of the assumptions is established. The approximate solutions are found inapplicable in the instances of non-steady-state conditions coupled with a low concentration of either monomer.     

Amperometric Urea Biosensor Using Aminated Glassy Carbon Electrode Covered with Urease Immobilized Carbon Sheet,Based on the Electrode Oxidation of Carbamic Acid

A large oxidation current can be observed when ammonium carbamate aqueous solution is electrolyzed using a glassy carbon electrode (GCE) at a potential exceeding 1.0 V vs. Ag/AgCl and amino groups are introduced at the surface of the GCE. Aminated GCE exhibits the electrocatalytic activity of the oxidation of ammonium carbamate that is produced from urea as an intermediate product of urease reaction, and a distinct oxidation current is observed when the aminated GCE is used to oxidize the urea in the urease solution. A novel amperometric determination method to detect urea has been developed. This method is based on the electrooxidation of carbamic acid produced during urease reactions. Urease is immobilized to polymaleimidostyrene (PMS) coated on the insulated amorphous carbon sheet set on the aminated GCE surface. A good linear relationship is observed between urea concentration and the electrolytic current of the urease‐immobilized electrode in the concentration range from 0.5 mM to 21.0 mM. The proposed urea biosensor has an effective merit in that the interference resulting from ammonia and pH change caused by the urease reaction can be eliminated, differing from conventional urea biosensors.     

催化反应热诱导激光光偏转分析法测定尿素

报道用催化反应热诱导激光光偏转法测定痕量尿素。发现将脲酶制成一定浓度的乙醇浓液能显著地增强激光光偏转信号强度。探讨了增强作用机理以及光偏转号强度与尿素浓度的关系，并应用于尿样中尿素含量的测定，结果满意。     

酶法测定水中的尿素含量

利用脲酶、谷氨酸脱氢酶偶联催化尿素水解的原理,通过测定还原型烟酰胺腺嘌呤二核苷酸吸光度变化率得出其酶促反应速度,对应不同的尿素浓度制得标准曲线,讨论了ｐＨ值和抑制剂对测定的影响,实测了水样中尿素的含量。     

The application of the concept of reaction layer to the study of electrode processes coupled with the second-order chemical reactions at microelectrodes under steady-state conditions

Under steady-state conditions, the current equations of the second-order EC, ECE and DISPI reactions at microdisk, microspherical and microring electrodes are derived with the aid of the concept of the reaction layer. The conditions under which these equations would be valid are also discussed. Using these equations, methods to determine the kinetic parameters for the second-order EC, ECE and DISPI reactions are presented. The reduction of 2,6-diphenyl-pyrylium cation and oxidation of triphenylamine were investigated as examples of the second-order EC and ECE reactions.     

Periodic Nucleation of Calcium Phosphate in a Stirred Biocatalytic Reaction

Highly ordered superstructures composed of inorganic nanoparticles appear in natural and synthetic systems, however the mechanisms of non‐equilibrium self‐organization that may be involved are still poorly understood. Herein, we performed a kinetic investigation of the precipitation of calcium phosphate using a process widely found in microorganisms: the hydrolysis of urea by enzyme urease. With high initial ratio of calcium ion to phosphate, periodic precipitation was obtained accompanied by pH oscillations in a well‐stirred, closed reactor. We propose that an internal pH‐regulated change in the concentration of phosphate ion is the driving force for periodicity. A simple model involving the biocatalytic reaction network coupled with burst nucleation of nanoparticles above a critical supersaturation reproduced key features of the experiments. These findings may provide insight to the self‐organization of nanoparticles in biomineralization and improve design strategies of biomaterials for medical applications.