Kinetics Of Urease Research Articles

In vitro anti-Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou.

Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism. Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein. Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 μg/mL, and minimum bactericidal concentration (MBC) values were 160-320 μg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity. Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.

Bioremediation potential and primary mechanism of Sporosarcina pasteurii for cadmium (Cd) and lead (Pb) in contaminated tailings

ABSTRACT Efficient bioremediation technology has drawn extensive attention from scholars due to the serious damage caused by heavy metals (HMs) to the environment. This study explored the remediation potential and mechanisms of the S. pasteurii strain in alleviating toxicity of Cd and Pb in tailings. The results showed that the decrease in DTPA-Cd and -Pb was associated with the transfer of exchangeable HMs to carbonate-bound HMs in bacteria-treated tailings. Biomineralization, extracellular adsorption, and intracellular accumulation were observed to result in removal rates above 95% for HMs in tailings solution. Mineralisation products driven-urease were identified as carbonate minerals, including otavite CdCO3, cerussite PbCO3 and hydrocerussite Pb3(CO3)2(OH)2. However, the increased Km in the urease kinetics study indicated that HMs reduced urease activity and biomineralization by minimum inhibitory concentrations (Cd 0.5 mM and Pb 40 mM). The functional groups (-OH, -NH2, -COOH and -PO4 3-) on the bacterial surface were involved in immobilising HMs, while the biosorption capacity (qm) was inhibited by metal ions (Pb2+ > Cd2+) according to Langmuir sorption isotherm. In conclusion, the biomineralization-adsorption of S. pasteurii was the mainstay for stabilising Cd and Pb in tailings, and the contribution of biomineralization was found to be much higher than that of biosorption.

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 Km of 26.0 mM and 8.0 mM and the Vmax 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 Kcat/Km 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%.

The Impact of pH on Catalytically Critical Protein Conformational Changes: The Case of the Urease, a Nickel Enzyme.

Urease uses a cluster of two NiII ions to activate a water molecule for urea hydrolysis. The key to this unsurpassed enzyme is a change in the conformation of a flexible structural motif, the mobile flap, which must be able to move from an open to a closed conformation to stabilize the chelating interaction of urea with the NiII cluster. This conformational change brings the imidazole side chain functionality of a critical histidine residue, αHis323, in close proximity to the site that holds the transition state structure of the reaction, facilitating its evolution to the products. Herein, we describe the influence of the solution pH in modulating the conformation of the mobile flap. High-resolution crystal structures of urease inhibited in the presence of N-(n-butyl)phosphoric triamide (NBPTO) at pH 6.5 and pH 7.5 are described and compared to the analogous structure obtained at pH 7.0. The kinetics of urease in the absence and presence of NBPTO are investigated by a calorimetric assay in the pH 6.0-8.0 range. The results indicate that pH modulates the protonation state of αHis323, which was revealed to have pKa =6.6, and consequently the conformation of the mobile flap. Two additional residues (αAsp224 and αArg339) are shown to be key factors for the conformational change. The role of pH in modulating the catalysis of urea hydrolysis is clarified through the molecular and structural details of the interplay between protein conformation and solution acidity in the paradigmatic case of a metalloenzyme.

Disposable Fluidic Devices of Bionanochannels for Enzymatic Monitoring and Energy Harvesting.

Nature produces a plethora of nanochannels to carry out highly complex biological tasks in a sophisticated manner. There have been several studies to understand the characteristics of these channels; however, efforts to apply them for technological advancements are still scarce. Here, we have demonstrated that the fluidic channels of biomaterials can be harvested as nanofluidic devices to produce energy from enzymatic chemical reactions. The bionanochannel-based nanofluidic devices exhibit various nanofluidic phenomena like surface-charged-governed ionic conductivity and development of the transmembrane potential. The mobility of ions in the hydrated bionanochannels are found to be higher than that of bulk water. The cation-selective nature of the biochannels was also exploited to harvest a continuous supply of power up to 74 mW m-2 for 3 h from the enzymatic decomposition of urea. The transmembrane potential across the biochannels was also explored for label-free electrical monitoring of the enzymatic reaction inside the biological medium. Electrical monitoring on the kinetics of urease at different reaction temperatures suggested that inside biological medium the reaction goes through a pathway of lower activation energy (31.1 kJ) than that in the bulk environment (34.1 kJ). Enzyme urease was found to be more sustainable in bionanochannels than in glass vials.

A combined temperature-pH study of urease kinetics. Assigning pKa values to ionizable groups of the active site involved in the catalytic reaction

A combined temperature-pH study of urease kinetics was performed with the primary objective to assign the observed pKa values to the ionizable groups of the enzyme active site involved in the reaction. This was done in view of the fact that the identity of the groups detected by pH studies reported in the literature has never been conclusively resolved. Accordingly, herein the urease kinetic parameters KM and vmax were measured at eight pHs between 5.0 and 8.3 in noninteracting biological buffers (MES and HEPES), at each pH at five temperatures between 15 and 35°C. The determined values of pKas and of the enthalpies of ionizationΔHiono allowed the ionizable groups to have the pKas assigned. Unlike previously assumed to have pKa≈6.5, the determined acidic pK2≈5.0 was assigned to a histidine residue (most likely His320 by Klebsiella aerogenes numbering). By contrast, the basic pK1≈8.8 was assigned to the Ni–Ni bridging hydroxyl ion, a feature that supports the mechanism operative in the urease activity in which this hydroxyl is a general acid. Further, the pH-dependent analysis of the obtained KM and vmax values revealed that even though both the thermodynamic and activation parameters of the urease reaction were little pH-dependent, their values indicated that the most favorable conditions for the substrate binding are at pH 5.5–6.0, and those for the catalytic step, at pH 6.5–7.0. Taken together, the work brings in new mechanistic information of significance to the understanding of the activity of urease.

Whole cell kinetics of ureolysis by Sporosarcina pasteurii.

Ureolysis drives microbially induced calcium carbonate precipitation (MICP). MICP models typically employ simplified urea hydrolysis kinetics that do not account for cell density, pH effect or product inhibition. Here, ureolysis rate studies with whole cells of Sporosarcina pasteurii aimed to determine the relationship between ureolysis rate and concentrations of (i) urea, (ii) cells, (iii) NH4+ and (iv) pH (H(+) activity). Batch ureolysis rate experiments were performed with suspended cells of S.pasteurii and one parameter was varied in each set of experiments. A Michaelis-Menten model for urea dependence was fitted to the rate data (R(2) =0·95) using a nonlinear mixed effects statistical model. The resulting half-saturation coefficient, Km , was 305mmoll(-1) and maximum rate constant, Vmax , was 200mmoll(-1) h(-1) . However, a first-order model with k1 =0·35h(-1) fit the data better (R(2) =0·99) for urea concentrations up to 330mmoll(-1) . Cell concentrations in the range tested (1×10(7) -2×10(8) CFUml(-1) ) were linearly correlated with ureolysis rate (cell dependent Vmax'=6·4×10(-9) mmolCFU(-1) h(-1) ). Neither pH (6-9) nor ammonium concentrations up to 0·19moll(-1) had significant effects on the ureolysis rate and are not necessary in kinetic modelling of ureolysis. Thus, we conclude that first-order kinetics with respect to urea and cell concentrations are likely sufficient to describe urea hydrolysis rates at most relevant concentrations. These results can be used in simulations of ureolysis driven processes such as microbially induced mineral precipitation and they verify that under the stated conditions, a simplified first-order rate for ureolysis can be employed. The study shows that the kinetic models developed for enzyme kinetics of urease do not apply to whole cells of S.pasteurii.

Kinetics of Soil Urease in Four Agricultural Soils Affected by Urease Inhibitor PPD at Contrasting Moisture Regimes

An incubation test was conducted on four agricultural soils to investigate effects of phenyl phosphorodiamidate (PPD) on soil urease kinetics at contrasting moisture regimes with contrasting fertility levels. The PPD made the Michaelis constant (Km) increase and the maximum enzyme reaction velocity (Vmax) decrease, behaving as a mixed inhibitor. With incubation time, Km first increased and then decreased under saturation condition, whereas it decreased under field-moist condition, the opposite of the changes of Vmax. Compared with black soil and albic soils, brown and cinnamon soils had larger Km and lower Vmax and Vmax/Km ratios, indicating that the effectiveness of PPD was greater in soils with low fertility. Compared with brown soil and cinnamon soil, black soil and albic soil showed more increases in Km and decreases in Vmax. To apply PPD under waterlogged, saturated, and low-fertility conditions could be a reasonable way to increase fertilizer nitrogen (N)–use efficiency.

A New Metal-Chelated Cryogel for Reversible Immobilization of Urease

Poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] cryogel was synthesized by cryopolymerization technique at frozen temperature. Iminodiacetic acid (IDA) was then attached covalently to the cryogel as a chelating agent. Then, poly(HEMA-GMA)-IDA cryogel was chelated with Ni(II) ions and this novel metal affinity support was used for adsorption of urease from its aqueous solution. Urease adsorption experiments were carried out in a continuous system by using a peristaltic pump. Maximum urease adsorption onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was found to be 11.30 mg/g cryogel at pH 5.0 acetate buffer and in 25 °C medium temperature. Urease adsorption capacity decreased with increasing ionic strength and increasing chromatographic flow rate. Adsorption kinetics of urease onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was also investigated and it was found that Langmuir adsorption model is applicable for this adsorption study. This novel immobilized metal affinity chromatography support was used 10 times without any decrease at their adsorption capacity. It was also observed that urease enzyme was repeatedly adsorbed and desorbed without significant lost in enzymatic activity.

Kinetics of urease mediated calcite precipitation and permeability reduction of porous media evidenced by magnetic resonance imaging

The enzyme urease drives the hydrolysis of urea leading to the release of ammonium ions and bicar- bonate; in the presence of calcium, the rise in pH leads to increased calcium carbonate saturation and the subsequent precipitation of calcite. Although such alkalinizing ureol- ysis is widespread in nature, most studies have focussed on bacteria (i.e. indigenous communities or urease-active Sporosarcina pasteurii) for calcite precipitation technolo- gies. In this study, urease-active jack bean meal (from the legume Canavalia ensiformis) was used to drive calcite precipitation. The rates of ureolysis (kurea), determined from measured NH4 ? , enabled a direct comparison to microbial ureolysis rates reported in literature. It is also demonstrated that a simple single reaction model approach can simulate calcite precipitation very effectively (3-6 % normalised root-mean-square deviation). To investigate the reduction of permeability in porous media, jack bean meal (0.5 g L -1 ) and solutions (400 mM urea and CaCl2) were simultaneously pumped into a borosilicate bead column. One-dimensional magnetic resonance profiling techniques were used, non-invasively, for the first time to quantify the porosity changes following calcite precipitation. In addi- tion, two-dimensional slice selective magnetic resonance images (resolution of *0.5 9 1.0 mm) revealed that the exact location of calcite deposition was within the first 10 mm of the column. Column sacrifice and acid digestion also confirmed that 91.5 % of calcite was located within the first 14 mm of the column. These results have impor- tant implications for the design of future calcite precipi- tation technologies and present a possible alternative to the well known bacterial approaches.

Activatory Effect of Germination on Catalytic Capacity of Urease Extracted from Beans Samples (Effect of Germination on the Kinetics of Urease from Beans)

Activatory Effect of Germination on Catalytic Capacity of Urease Extracted from Beans Samples (Effect of Germination on the Kinetics of Urease from Beans)

Kinetics and mechanism of jack bean urease inhibition by Hg2+.

BackgroundJack bean urease (EC 3.5.1.5) is a metalloenzyme, which catalyzes the hydrolysis of urea to produce ammonia and carbon dioxide. The heavy metal ions are common inhibitors to control the rate of the enzymatic urea hydrolysis, which take the Hg2+ as the representative. Hg2+ affects the enzyme activity causing loss of the biological function of the enzyme, which threatens the survival of many microorganism and plants. However, inhibitory kinetics of urease by the low concentration Hg2+ has not been explored fully. In this study, the inhibitory effect of the low concentration Hg2+ on jack bean urease was investigated in order to elucidate the mechanism of Hg2+ inhibition.ResultsAccording to the kinetic parameters for the enzyme obtained from Lineweaver–Burk plot, it is shown that the Km is equal to 4.6±0.3 mM and Vm is equal to 29.8±1.7 μmol NH3/min mg. The results show that the inhibition of jack bean urease by Hg2+ at low concentration is a reversible reaction. Equilibrium constants have been determined for Hg2+ binding with the enzyme or the enzyme-substrate complexes (Ki =0.012 μM). The results show that the Hg2+ is a noncompetitive inhibitor. In addition, the kinetics of enzyme inhibition by the low concentration Hg2+ has been studied using the kinetic method of the substrate reaction. The results suggest that the enzyme first reversibly and quickly binds Hg2+ and then undergoes a slow reversible course to inactivation. Furthermore, the rate constant of the forward reactions (k+0) is much larger than the rate constant of the reverse reactions (k-0). By combining with the fact that the enzyme activity is almost completely lost at high concentration, the enzyme is completely inactivated when the Hg2+ concentration is high enough.ConclusionsThese results suggest that Hg2+ has great impacts on the urease activity and the established inhibition kinetics model is suitable.

Potentiometric Study of Urease Kinetics over pH 5.36–8.21

AbstractTo meet the demand for a convenient method of urease assay we present the potentiometric measurement of urease activity with use of an ammonium ion‐selective electrode. The membrane of the electrode was plasticized poly(vinyl chloride) containing physically immobilized nonactine as an NH4+‐sensitive ionophore and a lipophilic salt as an anionic additive. The calibration in buffers covering pH 5.36–9.97 showed that the electrode had a Nernstian response at pH <9.17 down to about 0.06 mM NH4+ and that for each buffer the detection limit grew with an increase in pH, concentration and the presence of Na2EDTA. The response time of the electrode was 15 s. In the study of urease kinetics performed with use of the electrode over pH 5.36–8.21 we have shown that the Michaelis constant of the enzyme does not significantly change with pH and that the maximum reaction rate‐pH profile, unlike those hitherto reported, has two maxima located at a short distance of 0.7 pH unit (7.2 and 6.5). Such a profile may pave the way for a new interpretation of the acid‐base mechanism of urease action. The potentiometric method presented can be helpful in screening for urease inhibitors, the substances of importance in therapies of diseases induced by ureolytic bacteria and in agriculture for proper nitrogen management in soil.

Dual Effects of Ionic Strength on Klebsiella aerogenes Urease: pH-Dependent Activation and Inhibition

A combined temperature-pH study of urease kinetics was performed with the primary objective to assign the observed pKa values to the ionizable groups of the enzyme active site involved in the reaction. This was done in view of the fact that the identity of the groups detected by pH studies reported in the literature has never been conclusively resolved. Accordingly, herein the urease kinetic parameters KM and vmax were measured at eight pHs between 5.0 and 8.3 in noninteracting biological buffers (MES and HEPES), at each pH at five temperatures between 15 and 35 °C. The determined values of pKas and of the enthalpies of ionizationΔHiono allowed the ionizable groups to have the pKas assigned. Unlike previously assumed to have pKa ≈ 6.5, the determined acidic pK2 ≈ 5.0 was assigned to a histidine residue (most likely His320 by Klebsiella aerogenes numbering). By contrast, the basic pK1 ≈ 8.8 was assigned to the Ni–Ni bridging hydroxyl ion, a feature that supports the mechanism operative in the urease activity in which this hydroxyl is a general acid. Further, the pH-dependent analysis of the obtained KM and vmax values revealed that even though both the thermodynamic and activation parameters of the urease reaction were little pH-dependent, their values indicated that the most favorable conditions for the substrate binding are at pH 5.5–6.0, and those for the catalytic step, at pH 6.5–7.0. Taken together, the work brings in new mechanistic information of significance to the understanding of the activity of urease.

Flow rate dependent kinetics of urease immobilized onto diverse matrices

A detailed study on the newer methods of coupling urease to diverse matrices have been investigated towards optimizing the efficiency of the immobilized enzyme action and cost effectiveness for the industrial applications. With some of the matrices like collagen, DEAE-cellulose and nylon, even the crude extract of jack bean meal, a major and inexpensive form of urease has been found to be extremely efficient and has indicated that factors other than covalent bonding contribute to the enzyme immobilization. Increased flow rate of the substrate has also resulted in increased reaction kinetics. Apparently, the rapid removal of the product, ammonium ion is largely responsible for this effect. Based on these informations, a large scale column with nylon net has been tested for its efficiency in handling urea effluent from fertilizer plants.

Flow rate dependent kinetics of urease immobilized onto diverse matrices

Flow rate dependent kinetics of urease immobilized onto diverse matrices

Activator effects on urease kinetics

AbstractAn experimental study of the effects of amino acid additives on the rates of urease kinetics showed that the degree of enhancement is sensitive to the relative levels of additives and substrate, and that enhancement can turn to inhibition at especially low concentrations of either arginine, DL‐alanine, or glycine. Kinetic models developed to interpret these and prior literature data showed that all the data are consistent in the framework of the steady state model proposed but contradict the expectations that would follow from an equilibrium based treatment.

Some properties and purifications of urease

1. 1.|The kinetics of urease (urea amidohydrolase, EC 3.5.1.5) catalysed ureolysis in maleic acid-NaOH and Tris-H 2SO 4 buffers have been examined over a range of pH from 5.0 to 9.0 using 10 different preparations of the enzyme, substrate concentrations of between 1 and 10 mM urea and temperature ranging from 1 to 29°. Michaelis-Menten kinetics were followed, but each preparation of urease employed displayed a unique pair of kinetic parameters. 2. 2.|Parallel responses by the Michaelis constants to changes of pH were found and suggested that histidine, α-ammonium and sulphydryl groups are of importance in the reaction. 3. 3.|Temperature dependances of the Michaelis constants were functions of the preparations of enzyme. It is concluded that urease as commonly prepared is not a homogeneous catalytic entity. 4. 4.|Ion-exchange chromatography on XE-64 yielded enzyme with maximum specific activity of 3000 Sumner units per mg. 5. 5.|Urease with specific activity of 5300 Sumner units per mg has been consistently obtained by passage of crude enzyme through molecular sieves of polyacrylamide gel and of Sephadex G-200.