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.     

Physico-Chemical Characterization of Watermelon Urease upon Immobilization in Alginate Beads

Watermelon (Citrullus vulgaris) urease was immobilized in 3.5% alginate leading to 72% immobilization. There was no leaching of the enzyme over a period of 15 days at 4°C. It continued to hydrolyse urea at a faster rate upto 90 min of incubation. The immobilized urease exhibited a shift of apparent pH optimum by one unit towards acidic side (from pH 8.0 to 7.0). The K_m was found to be 13.3 mM; 1.17 times higher than the soluble enzyme (11.4 mM). The beads were fairly stable upto 50°C and exhibited activity even at ?10°C. The enzyme was significantly activated by ME and it exhibited two peaks of activation; one at lower concentration and another at higher concentration. Time-dependent ureolysis in presence of ME progressed at a much elevated rate. Unlike soluble enzyme, which was inhibited at 200 mM urea, the immobilized enzyme was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2000 mM urea. Moreover, the inhibition caused by high urea concentration was partially abolished by ME. The significance of the observations is discussed.     

Immobilization of watermelon (<Emphasis Type="Italic">Citrullus vulgaris</Emphasis>) urease in agarose gel for urea estimation

Urease from dehusked seeds of watermelon was immobilized in 1.5% agarose gel with 53.9% entrapment. There was negligible leaching (<10% at 4°C) and the same gel membrane could repeatedly be used for seven days. The immobilization exhibited no apparent change in the optimum pH but there was a significant decrease in the optimum temperature (50°C as compared to 65°C for soluble urease). The immobilized urease revealed an apparentK _m of 9.3±0.3 mM; 1.2 times lower than the soluble enzyme (11.4±0.2 mM). Unlike soluble enzyme which was inhibited at 200 mM urea, the immobilized urease was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2 M urea. The time-dependent thermal inactivation kinetics at 48 and 52°C was found to be biphasic, in which half of the initial activity was destroyed more rapidly than the remaining half. These gel membranes were also used for estimating the urea content of the blood samples from the University hospital. The results obtained matched well with those obtained by the usual method employed in the clinical pathology laboratory. The significance of these observations is discussed.     

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.     

以伴刀豆球蛋白为介质定向固定化脲酶的研究

将戊二醛将伴刀豆球蛋白(ConA)和壳聚糖载体交联,然后利用ConA与脲酶糖链的特异性结合作用,实现脲酶的定向固定化.定向固定化的最适条件为戊二醛浓度3.5%、ConA浓度1mg/mL、ConA溶液pH值7.0、脲酶浓度0.4mg/mL.定向固定化脲酶的最适pH 5.0～6.0、最适温度77℃,米氏常数Km11.76mmol/L,与游离酶及非定向固定化脲酶比较,定向固定化脲酶的最适pH向酸性范围发生了偏移并有更宽的pH适用范围,最适温度提高,与底物的亲和力较大,且有较好的操作稳定性.     

以伴刀豆球蛋白为介质定向固定化脲酶的研究

将戊二醛将伴刀豆球蛋白(ConA)和壳聚糖载体交联, 然后利用ConA与脲酶糖链的特异性结合作用, 实现脲酶的定向固定化。定向固定化的最适条件为戊二醛浓度3.5%、ConA浓度1 mg/mL、ConA溶液pH值7.0、脲酶浓度 0.4 mg/mL。定向固定化脲酶的最适pH 5.0~6.0、最适温度77°C、米氏常数Km11.76 mmol/L, 与游离酶及非定向固定化脲酶比较, 定向固定化脲酶的最适pH向酸性范围发生了偏移并有更宽的pH适用范围, 最适温度提高, 与底物的亲和力较大, 且有较好的操作稳定性。     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

Highly multilayered urease decomposes highly concentrated urea

Urease was immobilized at a density of 1.2 g of urease per gram of a matrix via ion-exchange binding of urease to an anion-exchange polymer chain grafted onto a pore surface of a porous hollow-fiber membrane and subsequent cross-linking of urease with transglutaminase. Urea was hydrolyzed during the permeation of a urea solution, the concentration of which ranged from 2 to 8 M, through the pores of the resultant membrane with a thickness of approximately 1 mm. Quantitative hydrolysis of 4 M urea was achieved at a permeation rate lower than 1 mL/h, i.e., a residence time longer than 5.1 min, at ambient temperature. This performance is ascribed to convective transport of urea through the pores rimmed by the urease-immobilized polymer chains at a high density. Urease was denatured in the presence of urea at concentrations higher than 6 M while hydrolyzing urea.     

Mathematical model for internal pH control in immobilized enzyme particles

A mathematical model has been developed for the internal pH control in immobilized enzyme particles. This model describes the kinetics of a coupled system of two enzymes, immobilized in particles of either planar, cylindrical, or spherical shape. The enzyme kinetics are assumed to be of a mixed type, including Michaelis-Menten kinetics, uncompetitive substrate inhibition, and competitive and noncompetitive product inhibition. In a case study we have considered the enzyme combination urease and penicillin acylase, whose kinetics are coupled through the pH dependence of the kinetic parameters. The hydrolysis of urea by urease yields ammonia and carbon dioxide, whereas benzylpenicillin (Pen-G) is converted to 6-amino penicillanic acid and phenyl acetic acid by penicillin acylase. The production of acids by the latter enzyme will cause a decrease in pH. Because of the presence of the ammonia-carbon dioxide system, however, the pH may be kept under control. In order to obtain information about the optimum performance of this enzymatic pH controller, we have computed the effectiveness factor and the conversion in a CSTR at different enzyme loadings. The results of the computer simulations indicate that a high conversion of Pen-G may be achieved (80-90%) at bulk pH values of about 7.5-8.     

Immobilization imparts stability to watermelon urease to work in water miscible organic media

The behaviour of alginate immobilized and soluble watermelon (Citrullus vulgaris) urease in water miscible organic solvents like, acetonitrile, dimethylformamide (DMF), ethanol, methanol, and propanol is described. The organic solvents exhibited a concentration dependent inhibitory effect on both the immobilized and the soluble urease in the presence of urea. Pretreatment of soluble enzyme preparations with organic solvents in the absence of substrate for 10 min at 30°C led to rapid loss in the activity, while similar pretreatment of immobilized urease with 50% (v/v) of ethanol, propanol, and acetonitrile was ineffective. Time-dependent inactivation of immobilized urease, both in the presence and in the absence of urea, revealed stability for longer duration of time even at very high concentration of organic solvents. The soluble enzyme, on the other hand, was rapidly inactivated even at fairly lower concentrations. The results suggest that the immobilization of watermelon urease in calcium alginate make it suitable for its application in organic media. the observations are discussed.     

Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

Hydrolysis of urea by gelatin-immobilized urease: separation of kinetic and diffusion phenomena in a model immobilized-enzyme reactor system.

Experiments and appropriate mathematical models are presented in an attempt to elucidate and separate the effects of mass transfer and immobilization on the apparent kinetics of hydrolysis of urea by urease immobilized within a crosslinked gelatin film. Diffusion of urea through the gelatin matrix appears to exert the major influence on the observed kinetics. Diffusion coefficients are measured, and a model for the "effectiveness factor" is presented, accounting for this aspect of mass transfer control. A secondary, but significant, influence on apparent kinetics arises because the reaction products lead to an increased pH level which, because of diffusion resistance, remains high within the gelatin matrix. For pH levels in the 6.7 to 9.0 range the activity of urease is a strongly decreasing function of pH. An approximate model accounting for ionic equilibrium allows this pH-diffusion effect to be introduced in such a way as to lead to predictions of the apparent kinetics that are compared with experimental observations. Examination of these results indicates that the immobilization procedure leads to some loss of activity due to an interaction of the gelatin crosslinking reaction with the enzyme itself.     

Urea-hydrolyzing activity of a T-strain mycoplasma: Ureaplasma urealyticum.

The urea-hydrolyzing activity of a T-strain mycoplasma was studied in experiments using whole cells and cell-free enzyme preparations by measuring the release of 14CO2 from [14C]urea. Under the conditions used, the urea concentration optimum is approximately 5.6 X 10(-3) M urea. The activity is soluble and not membrane bound. It is stable at -70 C for several weeks but is more labile at higher temperatures. The pH optimum is between 5.0 and 6.0. The effect of several inhibitors on the activity was tested and revealed similarities, as well as differences, between T-strain mycoplasma urease activity and the urease activity of other organisms and plants.     

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.     

High throughput covalent immobilization process for improvement of shelf-life,operational cycles,relative activity in organic media and enzymatic kinetics of urease and its application for urea removal from water samples

High throughput covalent urease immobilization was performed through the amide bond formation between the urease and the amino-functional MNPs. The enzyme’s performances, including shelf-life, reusability, enzymatic kinetics, and the enzyme relative activity in organic media was improved. At optimal conditions, the immobilization efficiency was calculated about 95.0% with keeping 94.7% of the urease initial specific activity. The optimal pH for maximum activity of the free and immobilized urease was calculated as 7.0 at 37.0 °C and 8.0 at 60.0 °C, respectively. The kinetics studies showed the K_m of 26.0 mM and 8.0 mM and the V_max of 5.31 μmol mg⁻¹ min⁻¹ and 3.93 μmol mg⁻¹ min⁻¹ for the free and immobilized urease, respectively. The ratio K_cat/K_m as a measure of catalytic efficiency and enzyme specificity was calculated as 0.09 mg mL⁻¹ min⁻¹ and 0.22 mg mL⁻¹ min⁻¹ for the free and immobilized urease, respectively, indicating an improvement in the enzymatic kinetics. The shelf-life and operational studies of immobilized urease indicated that approximately 97.7% and 88.5% of its initial activity was retained after 40 days and 17 operational cycles, respectively. The immobilized urease was utilized to urea removal from water samples with an efficiency between 91.5–95.0%.     

Immobilization of glucose oxidase on silica-based supports

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.     

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

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.     

Assay for soil urease activity

Summary A procedure is described that allows assay of soil urease activity. The method uses a phosphate buffer (pH 8.8) and a urea substrate concentration of 0.007 M. Incubation for 4 h at 37°C is recommended and urease activity is estimated by determining the amount of ammonium produced by urea hydrolysis in soil. The method is precise, and compares favourably with other procedures. re]19750710     

A Determination and Comparison of Urease Activity in Feces and Fresh Manure from Pig and Cattle in Relation to Ammonia Production and pH Changes

Ammonia emission from animal production is a major environmental problem and has impacts on the animal health and working environment inside production houses. Ammonia is formed in manure by the enzymatic degradation of urinary urea and catalyzed by urease that is present in feces. We have determined and compared the urease activity in feces and manure (a urine and feces mixture) from pigs and cattle at 25°C by using Michaelis-Menten kinetics. To obtain accurate estimates of kinetic parameters V_max and K''_m, we used a 5 min reaction time to determine the initial reaction velocities based on total ammoniacal nitrogen (TAN) concentrations. The resulting V_max value (mmol urea hydrolyzed per kg wet feces per min) was 2.06±0.08 mmol urea/kg/min and 0.80±0.04 mmol urea/kg/min for pig feces and cattle feces, respectively. The K''_m values were 32.59±5.65 mmol urea/l and 15.43±2.94 mmol urea/l for pig feces and cattle feces, respectively. Thus, our results reveal that both the V_max and K''_m values of the urease activity for pig feces are more than 2-fold higher than those for cattle feces. The difference in urea hydrolysis rates between animal species is even more significant in fresh manure. The initial velocities of TAN formation are 1.53 mM/min and 0.33 mM/min for pig and cattle manure, respectively. Furthermore, our investigation shows that the maximum urease activity for pig feces occurs at approximately pH 7, and in cattle feces it is closer to pH 8, indicating that the predominant fecal ureolytic bacteria species differ between animal species. We believe that our study contributes to a better understanding of the urea hydrolysis process in manure and provides a basis for more accurate and animal-specific prediction models for urea hydrolysis rates and ammonia concentration in manures and thus can be used to predict ammonia volatilization rates from animal production.