Review Properties and Applications of Urease

Urease is a highly efficient catalyst for the hydrolysis of urea with a rate approximately 10 14 times the rate of the noncatalyzed reaction. It has a long and distinguished history in the development of enzymology. In this work the properties of urease and its applications in biotechnology are reviewed, including urea content analysis in blood, urine, alcoholic beverages, natural water and environmental wastewaters; analysis of heavy metal content in natural waters, wastewaters and soil; determination of creatinine, arginine and IgG; urea removal from artificial kidney dialyzates, alcohol beverages and fertilizer wastewaters; wastewater reclamation for life support systems in space; pH control or shift for multi-enzyme reaction system; and urea hydrolysis as sources of ammonia or carbon dioxide in special cases. Future research trends are also outlined.     

Review Properties and Applications of Urease

Urease is a highly efficient catalyst for the hydrolysis of urea with a rate approximately 10 14 times the rate of the noncatalyzed reaction. It has a long and distinguished history in the development of enzymology. In this work the properties of urease and its applications in biotechnology are reviewed, including urea content analysis in blood, urine, alcoholic beverages, natural water and environmental wastewaters; analysis of heavy metal content in natural waters, wastewaters and soil; determination of creatinine, arginine and IgG; urea removal from artificial kidney dialyzates, alcohol beverages and fertilizer wastewaters; wastewater reclamation for life support systems in space; pH control or shift for multi-enzyme reaction system; and urea hydrolysis as sources of ammonia or carbon dioxide in special cases. Future research trends are also outlined.     

Microcapsule artificial kidney: treatment of patients with acute drug intoxication

The microcapsule artificial kidney was used in the treatment of three patients with acute drug intoxication. The apparatus contains 300 g. of microencapsulated activated charcoal with a total membrane area available for diffusion of more than 2m.² The membrane thickness is only 500 A. These properties make possible a compact artificial kidney whose efficiency for the removal of uremic metabolites and drugs is much higher than standard hemodialysis apparatus. The microcapsules are made blood-compatible by coating with human albumin. A roller pump was used to propel the blood through the microcapsule artificial kidney at a flow rate of 300 ml./min. for two to three hours. The clearance values for glutethimide, methyprylon and methaqualone were much higher than those achieved by standard hemodialysis. Hemoperfusion quickly lowered the drug level in the blood with resulting clinical improvement.     

Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

The Yersinia pseudotuberculosis Yut protein,a new type of urea transporter homologous to eukaryotic channels and functionally interchangeable in vitro with the Helicobacter pylori UreI protein

Urea uptake in eukaryotes and prokaryotes occurs via diffusion or active transport across the cell membrane. Facilitated diffusion of urea in both types of organisms requires a single-component channel. In bacteria, these transport systems allow rapid access of urease to its substrate, resulting in ammonia production, which is needed either for resistance to acidity or as a nitrogen source. In Yersinia pseudotuberculosis, a ureolytic enteropathogenic bacterium, a gene of unknown function (yut) located near the urease locus was found to encode a putative membrane protein with weak homology to single-component eukaryotic urea transporters. When expressed in Xenopus oocytes, Yut greatly increases cellular permeability to urea. Inactivation of yut in Y. pseudotuberculosis results in diminished apparent urease activity and reduced resistance to acidity in vitro when urea is present in the medium. In the mouse model, bacterial colonization of the intestine mucosa is delayed with the Yut-deficient mutant. Although structurally unrelated, Yut and the Helicobacter pylori UreI urea channel were shown to be functionally interchangeable in vitro and are sufficient to allow urea uptake in both bacteria, thereby confirming their function in the respective parent organisms. Homologues of Yut were found in other yersiniae, Actinobacillus pleuropneumoniae, Brucella melitensis, Pseudomonas aeruginosa and Staphylococcus aureus. The Y. pseudotuberculosis Yut protein is therefore the first member of a novel class of bacterial urea permeases related to eukaryotic transporters.     

Molecular mechanics evaluation of the proposed mechanisms for the degradation of urea by urease

A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the NHis272-Ni1-O-Curea, and Ni1-O-Curea-Nurea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional "His320 acts as a base" mechanism is unlikely. while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.     

Urease Immobilization on Arylamine Glass Beads and its Characterization

Jack bean urease has been immobilized on arylamine glass beads (200–400 mesh size, 75–100 Å pore size) and its properties compared with soluble enzyme. The binding of urease was 13.71 mg per gram beads. The K_m for soluble and immobilized urease for urea was 4.20 mM and 8.81 mM, respectively. V_max values of urease decreased from 200 to 43.48 μmol of ammonia formed per min per mg protein at 37°C on immobilization. Both pH and buffer ions influenced the activities of soluble as well as immobilized urease. Soluble urease exhibited pH optima at 5.5 and 8.0. However, immobilized urease showed one additional pH optimum at 6.5. In comparison to phosphate buffer, citrate buffer was inhibitory to urease activity. Immobilization of urease on arylamine glass beads resulted in improved thermal, storage and operational stability. Because of inertness of support and stability of immobilized urease, the preparation can find applications in ‘artificial kidney’ and urea estimation in biological fluids viz., blood, milk etc.     

Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.     

Molecular Mechanics Evaluation of the Proposed Mechanisms for the Degradation of Urea by Urease

Abstract

A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the N_His272-Ni1-O-C_urea and Ni1-O-Curea-N_urea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional “His320 acts as a base” mechanism is unlikely, while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.     

Recycling of urea associated with the host plant urease in the silkworm larvae,Bombyx mori

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.     

Kinetic studies on urea hydrolysis by immobilized urease in a batch squeezer and flow reactor

The immobilization of urease on the reticulated polyurethane foam, and the kinetic phenomenon of urea hydrolysis by the resulting immobilized urease in both batch squeezer and circulated flow reactors were studied. Urease was immobilized with bovine serum albumin and glutaraldehyde on polyurethane foam support of 7 to 15 mum thickness. The residual apparent activity of urease after immobilization was about 50%. The good hydrodynamic property and flexibility of polyurethane foam were retained in solution after immobilization. A modified biofilm reactor model was used to describe the kinetic phenomenon of urea hydrolysis in both batch squeezer and circulated flow reactors. The characteristic parameters of the reactor model for both bioreactors were obtained by combining the Rosenbrock optimization method, the Rungs-Kutta method, and the Newton-Raphson method. The best-fit results were in good agreement with the experimental data. This study suggests another application of polyurethane foam in enzyme immobilization and immobilized enzyme reactors, which offers potential for practical applications in various bioreactors. (c) 1992 John Wiley & Sons, Inc.     

A Multistage Well‐Mixed Model for Urea Removal from Industrial Wastewater

A multistage well‐mixed model for urea removal from industrial wastewater has been proposed. The model incorporates reaction rate of urea hydrolysis and takes into account the effects of backmixing on the reactor performance. The model provides temperature and concentration distribution of different components along the height of reactor. The predicted data of the model were consistent with the plant data indicating the validity of the model. The impact of different parameters on the performance of urea hydrolyzer has been examined. The result of this work showed that an increase in inlet temperature of wastewater and steam flow rate would improve the urea removal efficiency.     

Optimisation of solute transport in dialysers using a three-dimensional finite volume model

Dialyser manufacturers only provide limited information about mass removal under well-defined flow and solute conditions in commercially available dialysers for hemodialysis. This computational study aimed at assessing the solute transport efficiency in a dialyser for different geometries (fiber lengths and diameters).

A three-dimensional finite volume model of a single fiber in a high flux polysulphone dialyser (Fresenius F60) was developed. Different equations describe blood and dialysate flow (Navier–Stokes), radial filtration flow (Darcy) and solute transport (convection–diffusion). Fluid and membrane properties were derived from in vitro and in vivo tests as well as from literature data. Urea (MW60) was used as marker to simulate small molecule removal, while middle molecule transport was modelled using vitamin B12 (MW1355) and inulin (MW5200). Keeping the fluid velocity in a single fiber constant, fiber diameter and length were changed in a wide range for evaluation of solute removal efficiency. Clearances were found enhanced by 13% (urea), 50% (vitamin B12) and 89% (inulin) for a fiber twice as long as a standard one and by 5.5% (vitamin B12) and 21% (inulin) for a fiber diameter of 150 μm instead of 200 μm. The impact of fiber dimensions was more pronounced for the middle molecules compared to urea.     

Urease immobilized on an aminated polysulphone membrane – inhibition by boric acid

An enzymatic membrane for application in the processes of decomposition and removal of urea from aqueous solutions was prepared: jack bean urease was immobilized on an aminated polysulphone membrane by adsorption. The inhibition of the system by boric acid was studied using procedures based on the MICHAELIS-MENTEN integrated equation (non-linear regression, and the linear transformations of WALKER and SCHMIDT, JENNINGS and NIEMANN, and BOOMAN and NIEMANN). The reaction was carried out in a 100 mM phosphate buffer of pH 7.0, containing 2 mM EDTA, obtained by neutralization of orthophosphoric acid with NaOH, at an initial urea concentration of 10 mM, and a temperature of 25 °C. The reaction was initiated by the addition of the enzyme to the urea solution, and was monitored by removing samples of the reaction mixture for NH₃ determinations by the phenol-hypochlorite method until the urea was exhausted. The results were compared with those obtained earlier under the same reaction conditions for free urease and urease covalently immobilized on chitosan. The inhibition was found to be competitive, similar to that of the free enzyme and urease immobilized on chitosan, with inhibition constants K_i equal to 0.36, 0.19 and 0.60 mM. The results show that adsorption of the enzyme on a polysulphone membrane changed the enzyme to a lesser degree than covalent immobilization of the enzyme on a chitosan membrane.     

Flow Injection Analysis for the Determination of Urea in Cow’s Milk

An inexpensive and easily automated flow injection method for determination of urea in cow’s milk was evaluated. Urea is hydrolysed by urease and in a gas diffusion cell the ammonia formed passes a membrane into an indicator solution. The resulting colour change of the indicator is measured at 590 nm. The repeatability of the analysis, expressed as the coefficient of variation (C.V.), was between 0.5 and 1.2%. Measured (y) and expected (x) milk urea concentrations after addition of known amounts of urea were related according to the equation y = 1.00× – 0.12 with a C.V. for the regression of 1.8%. Recommended amounts (0.02 %) of the preservative bronopol (2-bromo-2-nitropropane-1,3-diol) added to the milk did not affect the results (P > 0.05).     

Mathematical Model of a Countercurrent Flow Multi-fibre Dialyser

Summary The closed form solution to a distributed parameter mathematical model of a countercurrent flow dialyser is presented. The model consisting of a bundle of hollow fibres in a shell accounts for axial convection and radial diffusion. The proposed model relates the fractional removal of a solute to mass transfer parameters such as Sherwood number, length Peclet number and system geometry. Excellent agreement with experimental beer dialysis data is demonstrated for the removal of alcohol using 8-lm thick 200-lm diameter cuprophane hollow-fibre membrane fibres. Potentially, this model could be very helpful in designing new processes involving dialysis. For example, removal efficiency better than 90% is achievable in systems operating with a Sherwood number of 2.0, length Peclet number of 5 · 105, unit tube-side/shell-side volumetric flow and length-to-diameter ratio of 5000. Results were obtained in this work from only the first eigenvalue and the case of no solute in the incoming dialysate stream.     

A poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) composite hydrogel membrane for urease immobilization to enhance urea hydrolysis rate by temperature swing*

A composite membrane made of cross-linked poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) (p(NIPAAm-NAS-HEMA)) hydrogel on polyester nonwoven support has been synthesized. The composite membrane shows temperature-responsive properties similar to conventional PNIPAAm hydrogels beads, which reversibly swells below and de-swells above the lower critical solution temperature of PNIPAAm (around 32 to 33 degrees C). Diffusion of urea through the membrane was temperature-dependent with the effective diffusion coefficient at 20 degrees C being 18 times that at 60 degrees C. Urease was immobilized directly to the membrane by forming covalent bonds between its amino groups and the succinimide ester groups of the membrane. Membrane prepared with NIPAAm to NAS molar ratio of 9, and then reacted in pH 7 buffer with 6 mg of urease gave the best immobilized enzyme, where 0.102 mg protein and 5.71 U activity per cm(2) membrane, and 55% relative specific activity could be obtained. There was negligible internal mass transfer resistance for this preparation judging from the calculated effectiveness factor. Urease shows enhanced thermal stability after immobilization with the first-order inactivation rate constant at 70 degrees C decreased to 1/8 of that of free urease. Membrane-immobilized urease could be utilized in a two-compartment membrane reactor with temperature swing to substantially enhance urea hydrolysis rate. The best operating condition of the membrane reactor was with temperature cycling between 60 to 20 degrees C and with temperature change every 10 min, where concentration of product ammonia after 3 h reaction increased 3.8-folds when compared with isothermal operation at 60 degrees C.     

Occurrence of urease in T strains of Mycoplasma

A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.     

Properties of urease immobilized on the functional organic silica surface

The paper deals with kinetics of the urea hydrolysis by microbial-origin urease dissolved and immobilized on the organic silica surface. It is shown that hydrolysis kinetics for soluble urease is described by the Michaelis-Menten equation until the concentration of urea reaches 1 M. Two fractions differing in the Michaelis constant are revealed for silochrome immobilized urease. The rate of urea hydrolysis by native and immobilized urease was studied depending on the pH value in presence of the substrate in the 1 M and 5 mM concentration. The hydrolysis rate of 1 M urea in the buffer-free solution by silochrome-immobilized urease is practically independent of pH within 4.5-6.5. Application of a 2.5 mM phosphate-citrate buffer as a solvent causes an increase in the hydrolysis rate within this pH range. For a soluble urease the 1 M urea hydrolysis rate dependence on pH is ordinary at pH 5.8-6.0. If the substrate concentration is 5 mM, the pH-dependences for the rate of the urea hydrolysis by silochrome- and aerosil-immobilized urease are close and at pH above 6.0 coincide with those for a soluble enzyme. The found differences in the properties of soluble and immobilized ureases are explained by the substrate and reaction products diffusion.