Urease-producing species of intestinal anaerobes and their activities.

Urease activities of anaerobic bacteria that constituted predominant gut flora were examined. It was demonstrated that some strains of Eubacterium aerofaciens, E. lentum, and Peptostreptococcus products produced urease. They were the most numerous species in human feces. All strains of Bifidobacterium infantis and some strains of Bacteroides multiacidus, B. bifidum, Clostridium symbiosum, Fusobacterium necrophorum, F. varium, Lactobacillus fermentum, Peptococcus asaccharolyticus, and P. prevotii produced urease. The optimum pH of the Lactobacillus urease was found to be 4.0, whereas the pH value of B. multiacidus urease was 8.0.     

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.     

Urease-producing species of intestinal anaerobes and their activities.

Urease activities of anaerobic bacteria that constituted predominant gut flora were examined. It was demonstrated that some strains of Eubacterium aerofaciens, E. lentum, and Peptostreptococcus products produced urease. They were the most numerous species in human feces. All strains of Bifidobacterium infantis and some strains of Bacteroides multiacidus, B. bifidum, Clostridium symbiosum, Fusobacterium necrophorum, F. varium, Lactobacillus fermentum, Peptococcus asaccharolyticus, and P. prevotii produced urease. The optimum pH of the Lactobacillus urease was found to be 4.0, whereas the pH value of B. multiacidus urease was 8.0.     

Urease biosynthesis and isolation in Staphylococcus saprophyticus L-1

Experiments were carried out to investigate the effect of organic components of the medium and cultivation conditions on the multiplication rate and urease biosynthesis by Staphylococcus saprophyticus L-1 cells isolated from natural sources. The yeast enzymic hydrolyzate and corn extract were found to be an adequate substitute for the costly organic components--peptone and yeast extract. The substitutes ensured a high level of urease biosynthesis and biomass accumulation. The biomass accumulation was maximum at pH 6.0-7.0 and the urease activity reached maximum at pH 6.0-6.5. The optimum temperature of cultivation was 37 degrees C. Enhanced aeration and constant pH during microbial cultivation in 250 1 fermenters did not increase the biomass accumulation or urease biosynthesis as compared to flask cultivation. The study of urease isolation from the cell extract showed that the ratio of 3 volumes of ethanol to 1 volume of homogenate was optimum and provided the best precipitation of the enzyme. Preliminary thermal treatment of the cell extract increased the urease activity by 2.5 times. In this situation the activity yield was close to 100%.     

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.     

Aluminium rhizotoxicity in maize grown in solutions with Al3+ or Al(OH)-4 as predominant solution Al species

The rhizotoxicity of aluminium at low-pH with Al(3+) and at high pH with Al(OH)-(4) as the main Al species was studied. Aluminium reduced root growth to similar levels at pH 8.0 and pH 4.3, although the mononuclear Al concentration at pH 8.0 was three times lower than at pH 4.3. Al contents of root apices were much higher at pH 8 than at pH 4.3. Callose was induced only marginally at pH 8 and the formation was confined to the epidermis, whereas it proceeded through the cortex with time at pH 4.3. Well-documented genotypical differences in callose formation and Al accumulation could not be found at pH 8. The largest fraction of the root-tip Al was recovered in the cell-wall fraction independent of the solution pH. A sequential extraction of isolated cell walls suggests that most of the cell-wall Al was precipitated Al(OH)(3) at pH 8.0. This can be explained by a drastic pH reduction in the root apoplastic sap to 6.2, whereas at bulk solution pH 4.3 it rose to 5.6. Al precipitation was also confirmed by the microscopic localization of Al. At pH 8, Al could mostly be found in the epidermis, but in the apoplast of the outer cortex at pH 4.3. It is proposed here that at pH 4.3, Al(3+) inhibits root growth through binding to sensitive binding sites in the apoplast of the epidermis and the outer cortex. At pH 8, Al(OH)(3) precipitation in the epidermis causes a mechanical barrier thus impairing the root-growth control of the epidermis.     

Isolation, Purification and Partial Characterisation of Urease from Seeds of Water Melon (Citrullus vulgaris)

Urease from seeds of water melon was purified to apparent homogeniety upto a sp act of 3750 units/mg protein with 31% recovery. Enzyme showed single protein band on native PAGE by urease specific staining. The mol wt of the enzyme was 4,70,000 and the preparation was free from bound nucleotides (A₂₈₀/A₂₆₀=1.14). The enzyme exhibited maximum activity in 50 mM Tris-acetate buffer (pH 8.5). The Km for urease was 8 mM. The enzyme was not inhibited by 25 mM of EDTA in 50 mM Tris-acetate buffer (pH 8.0 and 8.5).     

Production of nanocalcite crystal by a urease producing halophilic strain of <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis> and analysis of its properties by XRD and SEM

Cementation of salt-containing soils can be achieved by salt-tolerant or halophilic calcite precipitation bacteria. Therefore, the isolation of calcite-producing bacteria in the presence of salt is the first step in the microbial cementation of saline soils. Urease producing bacteria can cause calcite nano-crystals to precipitate by producing urease in the presence of urea and calcium. The purpose of this study was to isolate urease producing halophilic bacteria in order to make calcite precipitate in saline soil. The calcite and the properties of the strains were further analyzed by X-ray diffraction (XRD) and scanning electron microscope equipped with an energy dispersive X-ray detector. In this study, a total of 110 halophilic strains were isolated, from which 58 isolates proved to have the ability of urease production. Four strains were identified to produce nano-calcite using urease activity in the precipitation medium. The XRD studies showed that the size of these particles was in the range of 40–60 nm. Strain H3 revealed that calcite is mostly produced in the precipitation medium containing 5% salt in comparison with other strains. This strain also produced calcite precipitates in the precipitation medium containing 15% salt. Phylogenetic analysis indicated that these isolates are about 99–100% similar to Staphylococcus saprophyticus.     

Urease activity in microbiologically-induced calcite precipitation.

The role of microbial urease in calcite precipitation was studied utilizing a recombinant Escherichia coli HB101 containing a plasmid, pBU11, that encodes Bacillus pasteurii urease. The calcite precipitation by E. coli HB101 (pBU11) was significant although its precipitation level was not as high as that by B. pasteurii. Addition of low concentrations (5-100 microM) of nickel, the cofactor of urease, to the medium further enhanced calcite precipitation by E. coli (pBU11). Calcite precipitation induced by both B. pasteurii and E. coli (pBU11) was inhibited in the presence of a urease inhibitor, acetohydroxamic acid (AHA). These observations on the recombinant urease have confirmed that urease activity is essential for microbiologically-induced calcite precipitation. Partially purified B. pasteurii urease was immobilized in polyurethane (PU) foam to compare the efficacy of calcite precipitation between the free and immobilized enzymes. The immobilized urease showed higher K(m) and lower V(max) values, which were reflected by a slower overall calcite precipitation. However, scanning electron micrographs (SEM) identified that the calcite precipitation occurred throughout the matrices of polyurethane. Furthermore, PU-immobilized urease retained higher enzymatic activities at high temperatures and in the presence of a high concentration of pronase, indicating that immobilization protects the enzyme activity from environmental changes.     

Strain improvement of <Emphasis Type="Italic">Sporosarcina pasteurii</Emphasis> for enhanced urease and calcite production

Phenotypic mutants of Sporosarcina pasteurii (previously known as Bacillus pasteurii) (MTCC 1761) were developed by UV irradiation to test their ability to enhance urease activity and calcite production. Among the mutants, Bp M-3 was found to be more efficient compared to other mutants and wild-type strain. It produced the highest urease activity and calcite production compared to other isolates. The production of extracellular polymeric substances and biofilm was also higher in this mutant than other isolates. Microbial sand plugging results showed the highest calcite precipitation by Bp M-3 mutant. Scanning electron micrography, energy-dispersive X-ray and X-ray diffraction analyses evidenced the direct involvement of bacteria in CaCO₃ precipitation. This study suggests that calcite production by the mutant through biomineralization processes is highly effective and may provide a useful strategy as a sealing agent for filling the gaps or cracks and fissures in any construction structures.     

A procedure for purifying jack bean urease for clinical use

Urease with a purity meeting the requirements of analytical use was purified from jack bean meal through steps consisting of 20% acetone extraction, heat treatment, acid precipitation, and lyophilization. For extraction of urease, one part of bean meal was mixed with 5 parts of 20% acetone containing 1 mM EDTA and 1 mM 2-mercaptoethanol, and stirred at 20 degrees C for 5 min. Milky substances in the extract were removed by heat treatment. Urease in the clear yellow supernatant was precipitated by adjusting the pH of the solution to 5.4 with citric acid. The acid precipitated urease was neutralized by dissolving in 0.015 M phosphate buffer, pH 8.5 (final pH 6.8 to 7.0) and then lyophilized. By this procedure, the purity of the enzyme was increase 14.7 fold, the recovery of activity was 63%, and the yield was 6.75 g from 1 kg of bean seeds. The specific activity of the preparation was 411 units/mg protein (240 units/mg solid), and the free ammonia content was less than 0.01 microgram per unit. Some other proteins were present in the urease preparation as examined by gel filtration and gradient polyacrylamide gel electrophoresis. The molecular weight of the enzyme estimated by gel filtration was 480,000. However, two urease activity bands with molecular weight of 230,000 and 480,000 were observed in the polyacrylamide gel electrophoregram. From the result of determination of blood urea nitrogen (BUN), this simple purification procedure could be used for practical preparation of urease from jack bean meal for clinical analysis.     

Kinetic properties of Helicobacter pylori urease compared with jack bean urease

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.     

Field detection of urease and carbonic anhydrase activity using rapid and economical tests to assess microbially induced carbonate precipitation

Microbial precipitation of calcium carbonate is a widespread environmental phenomenon that has diverse engineering applications, from building and soil restoration to carbon sequestration. Urease-mediated ureolysis and CO₂ (de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate minerals, yet many environmental microbial community studies rely on marker gene or metagenomic approaches that are unable to determine in situ activity. Here, we developed fast and cost-effective tests for the field detection of urease and CA activity using pH-sensitive strips inside microcentrifuge tubes that change colour in response to the reaction products of urease (NH₃) and CA (CO₂). The urease assay proved sensitive and useful in the field to detect in situ activity in biofilms from a saline lake, a series of calcareous fens, and ferrous springs, finding relatively high urease activity in lake samples. Incubations of lake microbes with urea resulted in significantly higher CaCO₃ precipitation compared to incubations with a urease inhibitor, showing that the rapid assay indicated an on-site active metabolism potentially mediating carbonate precipitation. The CA assay, however, showed less sensitivity compared to the urease test. While its sensitivity limits its utility, the assay may still be useful as a preliminary indicator given the paucity of other means for detecting CA activity in the field. Field urease, and potentially CA, activity assays complement molecular approaches and facilitate the search for carbonate-precipitating microbes and their in situ activity, which could be applied toward agriculture, engineering and carbon sequestration technologies.     

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%.     

pH-dependent denaturation of thrombin-activated porcine factor VIII

Thrombin-activated porcine factor VIII (fVIIIaIIa) is a stable, active, 160-kDa heterotrimer at concentrations exceeding 2 x 10(-7) M in 0.7 M NaCl, 0.01 M histidine Cl, 5 mM CaCl2, pH 6.0, at 4 degrees C or 20 degrees C. Two of the subunits, fVIIIA1 and fVIIIA2, are derived from the heavy chain of the plasma-derived, heterodimeric fVIII precursor. The third subunit, fVIIIA3-C1-C2, is derived from the fVIII light chain. We now find that fVIIIaIIa undergoes a sharp decline in coagulant activity between pH 7 and 8. At pH 7.5, the activity of fVIIIaIIa at 3 x 10(-7) M decays within a few hours to a stable level that is approximately 70% of the value at pH 6.0, whereas at pH 8.0, greater than 99% of the activity is lost. The activity cannot be restored by readjusting the pH to 6.0. The loss of activity at pH 8.0 coincides with dissociation of the fVIIIA2 subunit since an inactive fVIIIA1/A3-C1-C2 heterodimer can be isolated by Mono S high performance liquid chromatography. After prolonged incubation at pH 8.0, the fVIIIA1 subunit also dissociates. The free fVIIIA2 fragment appears to be poorly soluble which may explain the irreversible loss of activity. Analytical velocity sedimentation of the pH-inactivated fVIIIaIIa preparation also is consistent with dissociation and precipitation of the fVIIIA2 fragment. We propose that denaturation of fVIIIaIIa by pH-dependent subunit dissociation may provide a major mechanism of inactivation of fVIIIaIIa under physiologic conditions.     

Jack been urease (EC 3.5.1.5). II. The relationship between nickel, enzymatic activity, and the "abnormal" ultraviolet spectrum. The nickel content of jack beans

At low pH, EDTA promotes the loss of the tightly bound nickel ions from jack bean urease. The specific activity of soluble enzyme after partial EDTA-promoted inactivation is a linear function of the nickel content. The results are consistent with the presence of 2.0 nickel ions per 97 000-dalton subunit in pure urease. The time scale for loss of enzymatic activity and nickel under these conditions is similar to that for loss of the "abnormal" tail absorption in the ultraviolet and visible absorption spectrum of urease (including the shoulder at approximately 420 nm). This indicates that nickel in urease is essential for enzymatic activity and establishes that the metal ions are in part responsible for the tail absorption in the ultraviolet spectrum of urease. After partial inactivation in the presence of EDTA either at low pH or in 2.5 M guanidinium chloride at neutral pH, urease did not regain activity in the presence of Ni2+. As yet apourease has not been produced reversibly. Jack bean seeds grown hydroponically without added nickel were low in both urease activity and nickel (10 and 6%, respectively, of parent seeds). Several other metal ions were readily available. This result suggests that metal ions other than nickel cannot substitute for nickel in the formation of normally active urease.     

Soybean Leaf Urease: A Seed Enzyme?

The soybean (Glycine max L. [Merrill]) var Itachi has 0.2 to 0.3% the urease activity found in developing embryos of a normal line, Prize. The hydroxyurea sensitivity and pH preference of this basal seed urease indicate that it represents a unique enzyme rather than an unusually low level of the normal seed urease. Itachi's seed urease is less sensitive to hydroxyurea inhibition (65-80% inhibition) than Prize seed urease (85-95% inhibition) and is more active at pH 6.1 and 8.8 than at 7.4, whereas the normal seed urease is least active at pH 8.8. Both properties of the basal seed urease are in agreement with the behavior of the leaf urease in extracts of Prize and Itachi leaves.

Neither the leaf urease nor the Itachi seed urease is immuneprecipitated by affinity-purified seed urease antibodies. However, when antibody is in excess, Staphylococcus aureus (Cowan) cell walls containing protein A can precipitate soluble antibody-urease complexes (47-68% of total enzyme) from both leaf (Itachi and Prize) and Itachi seed extracts. Under identical conditions, greater than 90% of Prize seed urease is precipitated. At a 100-fold dilution of antibody, 60% of Prize seed urease is still antibody-complexed while the antibody recognition of the leaf or Itachi seed urease is reduced to 2 to 24%.

The cell culture urease also resembles leaf urease by the criteria of pH preference, hydroxyurea sensitivity, and recognition by seed urease antibodies. In the presence of cycloheximide, nickel stimulates cell culture urease levels (14- or 35-fold depending on assay pH) indicating that cell cultures make a preponderance of apourease under nickel-limiting conditions.

Inasmuch as the ureases of leaf, cell culture, and Itachi seeds are more closely related to each other than they are to the abundant (Prize) seed urease, suggests that the three tissues either contain an identical urease or related tissue-specific isozymes. This second form of urease may have an assimilatory role since it is found in both leaf and seed sink tissues and is required for urea assimilation in cell culture (Polacco 1977 Plant Physiol 59: 827-830).

     

Amidase and urease activities in plants

Summary Foliar fertilization has received considerable attention in recent years. Because of the importance of amides and urea as N sources, this work was carried out to study the enzymes that catalyze the hydrolysis of these compounds in plant leaves. The methods developed for assay of these enzymes in plants involve determination by steam distillation of the NH₄ ⁺–N produced by amidase or urease activity when plant materials are incubated at 37°C with buffered (0.1M THAM pH 8.0) amide solution or buffered (0.1M THAM pH 7.5) urea solution, respectively. Amidase and urease were detected in 21 diverse plants in the families of Gramineae and Leguminosae. Results showed that amidase and urease have optimum activities at buffer pH values of 8.0 and 7.5, respectively. Both amidase and urease activities were decreased significantly upon freezing or air-drying of plant samples before enzyme assay. These differences were proportional to the original activities of fresh plant materials. Studies on the effect of temperature on amidase and urease activities showed that these enzymes are inactivated at temperatures above 60 and 70°C, respectively. The energy of activation of the reaction catalyzed by amidase and urease in plants, expressed in kJ·mole^–1, ranged from 44.0 to 51.2 (avg.=47.1) and from 43.1 to 56.5 (avg.=51.2) when formamide and urea were used as substrates, respectively. The apparent K_m constants of these enzymes varied among the plant samples studied. By using the Lineweaver-Burk plot, the K_m values for amidase when formamide was used as a substrate ranged from 2.0 to 9.4 (avg.=5.8 mM) and for urease ranged from 0.4 to 1.6 (avg.=0.8 mM). The V_max values of 7 plant samples, expressed in g of NH₄ ⁺–N produced/0.1 g of plant materials/2h, ranged from 137 to 514 for amidase and from 29 to 123 for urease. The importance of these enzymes in application of amides and urea to plant leaves is discussed.     

A simple method to determine urea concentration using intact Helicobacter pylori and BromoCresol Purple as a pH indicator

A modified method for urea quantification, by measuring the ammonia formed by urease, used the urease-positive Helicobacter pylori in place of purified urease with a pH indicator dye, BromoCresol Purple, to provide a color change. The color formed was stable for 20-min and could be read at 588-nm for urea quantification. Using this method, urea standard curves were linear up to 8.3-mM. As there was no need for centrifugation or precipitation, the assay was developed for use with 96-well microplates.     

Physical and chemical studies of a low-molecular-weight form of urease

1. A new form of enzymically active jack-bean [Canavalia ensiformis (L.) DC] urease corresponding to an S_20,w value of 11·8s and a molecular weight of 260000 was investigated. 2. Conversion of 18s urease (EC 3.5.1.5) into the 12s form depends on both low protein concentration and pH. Above pH5·3 urease exists in the 18s form and below pH4·8 in the 12s form; between these two pH values a 12s–18s rapid-equilibrium process is observed. 3. Comparison of the properties of 18s and 12s urease indicated no major differences. 4. A survey of other good urease sources revealed that the 12s form can also be obtained from soya bean (Glycine soja Sieb. and Zucc. cultivar Biloxi) and the bacterium Bacillus pasteurii (Miguel) Migula, but not from watermelon (Citrullus vulgaris Schrad. cultivar Congo). 5. The 12s forms from jack bean and Bacillus pasteurii did not hybridize.