Inactivation Of Urease Research Articles

Drying and Urease Inactivation Models of Soybean Using Two-dimensional Spouted Bed Technique

The most important factors for examining the quality of soybeans in the animal feed industry are moisture and urease inactivation. Mathematical models for soybean drying and urease inactivation using two-dimensional spouted bed are developed and validated with experimental data. The influences of initial moisture content, inlet air temperature, air recycle on the drying rate, urease inactivation, and energy consumption are also studied. The results revealed that the increase in initial moisture content slightly affected the drying rate and urease inactivation while the inlet air temperature caused a significant effect. To obtain a high drying rate and save energy consumption, the recycle air ratio should be kept in a range of 80–90%.

Quinone-induced inhibition of urease: Elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5′-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme–inhibitor equilibrium was achieved in no longer than 10 min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ ≈ 2,5-DM-1,4-BQ < 1,4-BQ < TC-1,4-BQ.

Flavonoids as effective protectors of urease from ultrasonic inactivation in solutions

Inactivation of soybean urease in aqueous solution at pH 5.4, 36 degrees C, and high-frequency sonication (2.64 MHz, 1.0 W/cm2) is substantially reduced in the presence of seven structurally different flavonoids. A comparative kinetic study of the effect of these flavonoids on the effective first-order rate constants that characterize the total (thermal and ultrasonic) inactivation k(i), thermal inactivation k(i)*, and ultrasonic inactivation k(i)(US) of 25 nM enzyme solution was carried out. The dependences of the three inactivation rate constants of the urease on the concentrations of flavonoids within the range from 10(-11) to 10(-4) M were obtained. The following order of the efficiency of the flavonoids used in respect of the urease protection from ultrasonic inactivation was found: astragalin > silybin > naringin > hesperidin > quercetin > kaempferol > morin. The results confirm a significant role in the inactivation of the urease of HO* and HO2*, free radicals, which are formed in the ultrasonic cavitation field.

Production of functional protein hydrolysates from Egyptian breeds of soybean and lupin seeds

Enzymatic hydrolysis is an agro-processing aid that can be utilized in order to improve nutritional quality of protein extracts from many sources. In this study, protein extracts from ungerminated and/or germinated local Egyptian soybean and lupin flours were hydrolyzed using the enzyme papain. The hydrolysis processes were carried out for 2 h and aliquots were withdrawn at different time intervals. We have analysed the protein hydrolysate after 30 min hydrolysis as an example of a partially hydrolyzed protein, and after 120 min as an example of greatly hydrolyzed protein. The hydrolysate (2 h treatment at 80°C and pH 7.4) from both soybean and lupin flour contained significantly decreased trypsin inhibitor activity and urease activity, and a reduced phytate content, which improved the overall protein quality. Hydrolysis caused almost complete inactivation of urease in all soybean and lupin samples regardless if the seeds were germinated or not. High protein content, nitrogen solubility and in vitro protein digestibility was shown after hydrolysis. Total protein content (in g/100 g extract) increased in hydrolyzed samples from 48.1 to 51-60 for soybean (dependent on pre-treatment) and from 36.8 to 39.9-48.6 for lupin. Total essential amino acid content was also increased in papain hydrolyzed samples, compared to that in raw and germinated legumes. More specifically, the amount of lysine, sulphur amino acids, histidine, and to a certain extent isoleucine and threonine increased in samples from both legume species. All soybean samples exhibited antioxidant activity while in lupin samples, only those subjected to hydrolysis showed activity. Generally, it was clearly observed that the longer the duration of enzymatic hydrolysis (within the time frame of the experiment), the higher the improvement of the nutritional quality

Heating process of soybean using hot-air and superheated-steam fluidized-bed dryers

The present work investigated drying characteristics and inactivation of urease in soybean dried by superheated-steam and hot-air fluidized beds. The value of effective diffusion coefficient, which was determined by a method of slopes, was increased with increased drying temperature and increased moisture content. Furthermore, it depended on the type of heating medium, with higher moisture diffusion for soybean dried by hot air. Inactivation of the urease enzyme in both media showed difference in rate, in which the enzymatic inactivation was faster for soybean dried in superheated steam than in hot air. For the individual heating medium, the modified first-order reaction was adequately fitted to experimental data. The rate of inactivation was found to increase as the temperature and moisture content were increased. The urease enzyme was inactivated, along with maintaining protein solubility and lysine content being in standard range, as soybean was treated at a temperature between 135 and 150 °C for the hot air and the treatment temperature could be reduced to be lower than 135 °C by using superheated steam.

Effective Diffusivity and Kinetics of Urease Inactivation and Color Change During Processing of Soybeans with Superheated-Steam Fluidized Bed

Influences of temperature and moisture content on diffusional transport property and kinetics of both urease inactivation and color change during soybean treatment with superheated steam in the temperatures of 120–150°C were investigated. The experimental results have shown that the effective diffusivity and apparent rates of such reactions are related to temperature in a way that the rates of water transport, urease inactivation, and brown pigment formation are higher with higher temperature. In addition, these rates are also enhanced by increasing moisture content, except for the browning reaction that is inhibited by this factor due to the dilution effect. The reduction in urease activity is reasonably described by modified first-order reaction and the color change of soybeans is characterized by three Hunter parameters in which the changes of L-, a- and b-values are followed according to zero-order, modified Monod and first-order reactions, respectively. The kinetics of inactivation and color change, along with the experimental data of protein solubility and lysine content, have been suggested that the soybean should be treated with superheated steam at the temperature ranging from 120 to 135°C. The superheated-steam fluidized-bed technique can simultaneously be applied for both drying and inactivating such components in soybeans by a single operation when the initial moisture content falls within a suitable range, approximately lower than 20%d.b. At elevated initial moisture contents, two-stage drying technique is recommended in order to avoid excessive cooked soybeans.

Urease-glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples.

A screen-printed three-electrode amperometric biosensor based on urease and the nicotinamide adenine dinucleotide hydrogen (NADH)-glutamic dehydrogenase system was developed and applied to the screening of heavy metals in environmental samples. The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction catalysed by urease to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the conversion of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically at 0.3 V (vs. Ag/AgCl) after urease immobilization onto the screen-printed three-electrode configuration. Immobilization of urease on the surface of screen-printed electrodes was performed by entrapment in alginate gel and adsorption on the electrode in a nafion film. Low sensitivity to inactivation by metals was recorded after urease entrapment in alginate gel with detection limits of 2.9 and 29.8 mg L(-1) for Hg(II) and Cu(II), respectively. The use of the negatively charged nafion film created a more concentrated environment of cations in proximity to the enzyme, thus enhancing the urease inhibition when compared to gel entrapment. The calculated detection limits were 63.6 and 55.3 microg L(-1) for Hg(II) and Cu(II), respectively, and 4.3 mg L(-1) for Cd(II). A significant urease inactivation was recorded in the presence of trace amounts of metals (microg L(-1)) when the enzyme was used free in solution. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity according to their metal contents. The obtained results were in agreement with the standard methods employed for sample analysis. Nevertheless, the use of the amperometric assay (with free urease) proved more feasible for the screening of trace amounts of metals in polluted samples.

Comparative study of heating processes for full-fat soybeans

Heating is very important to inactivate anti-nutritional factors and enhance quality of soybean products. Four heating techniques i.e. extrusion, fluidized bed, spouted bed and infrared radiation have been investigated along with their performances, regarding moisture reduction, urease inactivation, protein solubility and lysine. The rate of moisture diffusion for soybean kernel in the spouted bed and infrared dryer was described by a semi-empirical drying equation for which the drying constant was determined using non-linear regression. The experimental results have shown that the water mobility is more rapidly accelerated by infrared radiation. Every technique has a potential to reduce the urease activity to the standard range, with the quantity of remaining lysine being insignificantly different ( p<0.05) under the testing conditions. The extent of urease inactivation in each technique depends on the moisture content, time and temperature, the latter being the most important factor. High moisture content induces rapid urease inactivation for the soybeans treated by the convection and radiation-type equipments. Conversely, inhibited inactivation is encountered with the extruded soybeans due to the strong cohesive forces amongst ground soybeans at a higher moisture content of 24% d.b., inducing poor mixing behaviour in the barrel. The results from the lab- and commercial-scale equipment are shown to have higher soluble protein content in 0.2% KOH for the infrared-treated soybeans, comparing to the other techniques. Extrusion consumed the largest quantity of energy followed by the fluidized bed with no recycled air and the micronizer becomes the most effective energy utilization.

Effective Diffusivity and Kinetics of Urease Inactivation and Color Change During Processing of Soybeans with Superheated-Steam Fluidized Bed

Effective Diffusivity and Kinetics of Urease Inactivation and Color Change During Processing of Soybeans with Superheated-Steam Fluidized Bed

Soybean Drying by Two-Dimensional Spouted Bed

Urease activity, cracking, and breakage are important factors in considering the quality of raw soybean for feed meal industries. A two-dimensional spouted bed dryer was investigated to determine its capability for thermally inactivating the urease enzyme and maintaining its other qualities. The experimental results have shown that the drying kinetics of soybean in a two-dimensional spouted bed dryer are of the form described in the thin layer drying. The expression for the model parameter in Newton's law of cooling equation accounting for the moisture contents and inlet air temperatures was developed. The initial moisture content and inlet air temperature conditions cause cracks in the kernels. The strong collision between kernels and deflector because of high superficial velocity leads to high percentage of broken soybeans in the spout region. However, the velocity of 15.9 m/s can reduce the breakage below 5%. The inactivation of urease at low-to-moderate moisture content is suitably described by the first order kinetics. The modified Monod equation is applied when the moisture content is higher than 26% dry basis due to the inhibitory effect of water content on the inactivation rate. To complete urease inactivation and maintain protein quality, the temperatures of 150°C should be used.

Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples

An amperometric assay based on urease inactivation has been developed for the screening of heavy metals in environmental samples. The enzyme urease catalyses the hydrolysis of urea and the formation of NH 4 + is determined using a NADH-glutamate dehydrogenase coupled reaction system. NADH consumption is monitored amperometrically using screen-printed three electrode configuration and its oxidation current is then correlated to urease activity. The presence of heavy metals in the samples inhibits the urease activity, resulting in a lower NH 4 + production and therefore a decrease in NADH oxidation. The use of metallised carbon electrodes gave a decrease in NADH oxidation potential from +300 mV versus Ag/AgCl compared with >+600 mV for bare carbon electrodes, and thus minimised interferences from oxidizable species present in the samples. Electrodes fouling and possible contamination after reuse and cleaning was also eliminated by using screen-printed disposable electrodes. The linear range obtained for Hg(II) and Cu(II) was 10–100 μg l −1 with a detection limit of 7.2 μg l −1 and 8.5 μg l −1, respectively. Cd(II) and Zn(II) produced enzyme inhibition in the range 1–30 mg l −1, with limits of detection of 0.3 mg l −1 for Cd(II) and 0.2 mg l −1 for Zn(II). Pb(II) did not inactivate the urease enzyme significantly at the studied range (up to 50 mg l −1). Coefficients of variation (CV) values were 6–9% in all cases. Application of the assay system to leachate samples gave reliable and accurate toxicity assessments when compared to atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectroscopy (ICP-MS) analysis. This approach provides to be a simple and rapid (15 min, including enzyme inhibition time) method for metal ions detection.

Inactivation of Jack Bean Urease by Allicin

Allicin—diallyl thiosulfinate—is the main biologically active component of freshly crushed garlic. Allicin was synthesized as described elsewhere and was tested for its inhibitory ability against jack bean urease in 20 mM phosphate buffer, pH 7.0 at 22°C. The results indicate that allicin is an enzymatic inactivator. The loss of urease activity was irreversible, time- and concentration dependent and the kinetics of the inactivation was biphasic; each phase, obeyed pseudo-first-order kinetics. The rate constants for inactivation were measured for the fast and slow phases and for several concentrations of allicin. Thiol reagents, and competitive inhibitor (boric acid) protected the enzyme from loss of enzymatic activity. The studies demonstrate that urease inactivation results from the reaction between allicin and the SH-group, situated in the urease active site (Cys592).

Polyphenolic antioxidants efficiently protect urease from inactivation by ultrasonic cavitation

Inactivation of urease (25 nM) in aqueous solutions (pH 5.0-6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 Wt/cm2, 36-56 degrees C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 Wt/cm2, 36 or 56 degrees C) has been characterized quantitatively, using first-order rate constants: kin, aggregate inactivation; kin*, thermal inactivation; and kin* (US), ultrasonic inactivation. Within the range from 1 nM to 10 microM, propyl gallate (PG) decreases approximately threefold the rate of LFUS-induced inactivation of urease (56 degrees C), whereas resorcinol poly-2-disulfide prevents this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36 degrees C) or 10 nM (56 degrees C). At 0.2-10 microM, human serum albumin (HSA) increases the resistance of urease (at 56 degrees C) treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS-HSA), formed by conjugation of 1.0 nM PGDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56 degrees C).

Kinetics of Thermal Inactivation and Molecular Asymmetry of Urease from Dehusked Pigeongea (Cajanus cajan L.) Seeds

The purified urease from pigeonpea was moderately stable at −10°C. The shelf-life of the enzyme on storage in 0.1 M Tris-acetate buffer, pH 6.5, at −10°C showed a single exponential decay with a t1/2 of approx. 30 days. In the presence of additives like 5mM dithiothreitol the t1/2 increased to 223 days at the same temperature, in a single exponential decay. The Arrhenius plot of the kinetics of the pigeonpea urease catalysed urea hydrolysis over the temperature range 27 to 77°C, was linear. The Q10 value was found to be 1.46. The energy of activation calculated from the Arrhenius equation was found to be 5.1 kcal/mole active site. The thermal denaturation of pigeopea urease at 65 and 70°C was found to obey biphasic kinetics in which half of the activity is destroyed faster than the remaining half. The time course of thermal inactivation can be described by the following equation, consisting of two first order terms: At = Afast.e-kfast + Aslow.e-kslow.t where, At is the residual activity at time t, Afast and Aslow, kfast and kslow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. The data suggests the existence of site-site heterogeneity in oligomeric urease molecule from pigeonpea.

Evidence for a potential-dependent reversible inactivation of urease adsorbed on a gold electrode

The potentiometric collection mode of the scanning electrochemical microscope has been applied to a kinetic study of changes in immobilised enzyme activity. Urease adsorbs spontaneously onto gold surfaces from aqueous solution and the immobilised enzyme was shown to retain activity as a catalyst for the hydrolysis of urea to ammonium and bicarbonate ions. Urease activity was monitored using an ion-selective potentiometric tip electrode to detect a product (NH4+) of the enzymatic reaction and the use of a 50 µm radius gold electrode to immobilise the enzyme results in a steady state diffusion field for the reaction products and enables quantitative interpretation of the data. The ammonium ion flux determined from the tip potential decreased by approximately 40% when the potential of the gold was scanned or stepped across the range from 0.0 to+0.4 V vs. Ag/AgCl. The decrease in flux is reversible and the original value almost completely recovered after the potential was returned to 0 V. The flux decrease is attributed to inactivation of the enzyme. The timescale of the inactivation is limited by the establishment of a steady state concentration profile of ammonium ions at the 50 µm radius source. The kinetics of the reactivation are significantly slower with an apparent first order rate constant of 0.022 s-1. No faradaic processes were observed at the gold electrode over the potential range from 0.0 to +0.4 V. We therefore suggest that changes in the enzyme quaternary structure due to the changing electric field and ionic composition at the interface are responsible.

A study of inhibition of urease from seeds of the water melon (Citrullus vulgaris).

Urease from seeds of the water melon was found to be inhibited by various salts of sodium. Sodium fluoride strongly inhibited the activity in the low urea concentration range. The enzyme was also inhibited by a high concentration of urea which was completely abolished in the presence of 10 mM sodium fluoride. Time-dependent inactivation of urease with iodoacetic acid, N-ethylmaleimide and p-hydroxymercuribenzoate exhibited biphasic kinetics in which half of the initial activity was lost in the fast phase and the remainder in a slow phase. Each phase exhibited first-order kinetics. These observations are suggestive of the existence of half-and-half distribution of sites.

Urease-based biosensor for mercuric ions determination

Urease immobilized into Nafion film on the surface of an ion sensitive field effect transistor (ISFET) has been used for heavy metal salt measurement. Enzyme immobilization into a negatively charged polymer seems to cause an increase in the effect of heavy metal inhibition due to cation accumulation in the polymeric matrix. Fifty percent urease inactivation was observed with 0.2 μM Ag (I), 1.5 μM Hg (II) or 5 μM Cu (II). To make the urease-based biosensor sensitive only to mercuric ions, it is proposed to add small amounts (up to 100 μM) of NaI into the sample (to suppress sensitivity to Ag ions) and to rewash the sensor in 100 mM solution of EDTA for 5 min to remove Cu ions. Restoration of enzyme activity after Hg (II) inhibition can be obtained by sensor rewashing in a 300 mM solution of NaI for 5 min.

Solute modulation of urease inactivation kinetics

The effect of miscible solvents and salts on the sigmoidal grace period inactivation behavior of the enzyme urease was examined at 50°C. Methanol, glycerol, and Na 2SO 4 stabilized whereas acetone and salts such as KNO 3 and LiCl accelerated the rate of inactivation. The initial dissociation of the native urease hexamer into two trimers appears to be the rate-limiting step of the inactivation pathway under the conditions examined. Salts and solvents appear to influence dissociation kinetics by altering the hydrophobic forces that stabilize urease quaternary structure. This hypothesis is supported by the effect of the various salts on the dissociation rate followed the Hofmeister series. Aggregation of the trimer species, the second step in the inactivation pathway, was very rapid and appeared to not be strongly affected by the presence of solutes.

Kinectic Studies of Helicobacter pylori Urease Inhibition by a Novel Proton Pump Inhibitor, Rabeprazole.

Urease is an important virulence factor of pathogenicity of gastric Helicobacter pylori. The inhibition of H. pylori urease by the novel proton pump inhibitor, rabeprazole, was investigated kinetically. It was found to act as an irreversible noncompetitive inhibitor of the enzyme. The inhibitory potency of rabeprazole was dependent on the pH of reaction mixture and its Ki values were 0.14 microM (pH 5.0), 0.34 microM (pH 7.0) and 6.10 microM (pH 8.5). Progressive inactivation of urease by rabeprazole initially proceeded according to pseudo-first-order kinetics with respect to the remaining enzymatic activity at pH 7.0 and 37 degrees C, with a second-order rate constant of 0.0017 microM-1 s-1. When the inactivation half-life was plotted versus the reciprocal of the rabeprazole concentration, a straight line was obtained with a slope of -3.12. From an Arrhenius-plot of the temperature-dependence of the inactivation (over the range of 5-37 degrees C), an activation energy of 13.2 kcal/mol was calculated. Recovery of activity was incomplete for H. pylori urease inhibited by rabeprazole, suggesting that the rabeprazole-urease complex is very stable.

Essential Role of Urease in Germination of Nitrogen-Limited Arabidopsis thaliana Seeds

In Arabidopsis thaliana, urease transcript levels increased sharply between 2 and 4 d after germination (DAG) and were maintained at maximal levels until at least 8 DAG. Seed urease specific activity declined upon germination but began to increase in seedlings 2 DAG, reaching approximately 75% of seed activity by 8 DAG. Urea levels showed a small transient increase 1 DAG and then approximately paralleled urease activity, reaching maximal levels at approximately 9 DAG. Urease inhibition with phenylphosphorodiamidate resulted in a 2- to 4-fold increase in urea levels throughout seedling development. Arginine pools (0-8 DAG) changed approximately in parallel with the urea pool. Consistent with arginine being a major source of urea, arginase activity increased 10-fold in the interval 0 to 6 DAG. Allopurinol, a xanthine dehydrogenase inhibitor, had no effect on urea levels up to 3 DAG but reduced the urea pool by 30 to 40% during the interval 5 to 8 DAG, suggesting that purine degradation contributed to the urea pool well after germination, if at all. in aged Arabidopsis seeds, there was correlation between phenylphosphorodiamidate inactivation of urease and germination inhibition, the latter overcome by NH4NO3 or amino acids. Since urease activity, urea precursor, and urea increase in young seedlings, and since urease inactivation results in a nitrogen-reversible inhibition of germination, we propose that urease recycles urea-nitrogen in the seedling.