Ureolytic Activity Research Articles

Ureolytic Activity of Soybean and Corn Residue Extracts

The enzyme urease is responsible for the rapid hydrolysis of urea in agroecosystems where more than 50% of the applied nitrogen (N) can be lost via ammonia volatilization. The objectives of the study were to (1) extract urease from corn (Zea mays L.) and soybean (Glycine max L.) and (2) compare the urease activity from soybean and corn residues to Jackbean (Canavalia ensiformis) urease using Fourier transform infrared spectroscopy (FTI-R). Concentrations of urea and sodium bicarbonate ranging from 0 to 200 mM were analyzed with FTI-R to determine the correlation to peak height. Bicarbonate produced the most responsive peaks to concentration at 1,362 cm−1. Urease extracted from soybean residue was active, producing a bicarbonate peak at 1362 cm−1, whereas no urease activity was observed in corn residue. Variation among urease activities in crop residues could suggest a need for more precise nitrogen management in conservation tillage agroecosystems to reduce ammonia volatilization.

Microbially induced calcite precipitation-based sequestration of strontium by Sporosarcina pasteurii WJ-2.

Contamination by radioactive strontium ((90)Sr) is a significant environmental problem. Ureolytically driven calcium carbonate precipitation has been proposed for use in geotechnical engineering for soil remediation applications. In this study, 68 ureolytic bacterial strains were newly isolated from various environments. Of these, 19 strains were selected based on ureolytic activity shown when cultured on urea agar plates and identified through 16S rRNA gene sequencing. From these selected strains, Sporosarcina pasteurii WJ-2 (WJ-2) was selected for subsequent study. A simple method was developed to determine the effectiveness of microbially induced calcite precipitation (MICP). Unlike any other methods, it does not require advanced skills and sophisticated tools. Using this method, we were able to determine the ability of the bioconsolidated sand to retard the flow of crystal violet through the 25-mL column. Also, MICP by WJ-2 was evaluated for its potential to counteract Sr contamination in column experiments using natural sand. WJ-2-induced precipitation led to successful sequestration of approximately 80 % of the Sr from the soluble fraction of the sand. The utility of MICP in bioremediation was further confirmed through X-ray diffraction, scanning electron microscopy, and inductively coupled plasma mass spectrometry.

Soybean Ureases, but Not That of Bradyrhizobium japonicum, Are Involved in the Process of Soybean Root Nodulation

Ureases are abundant in plants, bacteria, and in the soil, but their role in signaling between soybean and soil microorganisms has not been investigated. The bacterium Bradyrhizobium japonicum forms nitrogen-fixing nodules on soybean roots. Here, we evaluated the role(s) of ureases in the process of soybean nodulation. Chemotaxis assays demonstrated that soybean and jack bean ureases were more chemotactic toward bacterial cells than the corresponding plant lectins. The eu1-a,eu4 soybean, deficient in urease isoforms, formed fewer but larger nodules than the wild-type, regardless of the bacterial urease phenotype. Leghemoglobin production in wild-type plants was higher and peaked earlier than in urease-deficient plants. Inhibition of urease activity in wild-type plants did not result in the alterations seen in mutated plants. We conclude that soybean urease(s) play(s) a role in the soybean-B. japonicum symbiosis, which is independent of its ureolytic activity. Bacterial urease does not play a role in nodulation.

Dental plaque pH and ureolytic activity in children and adults of a low caries population

Objectives. The aim of this study was to evaluate the plaque pH level and ureolytic activity among children and adults of Karen Hill tribes. Methods. Thirty-four children aged 6–10 years and 46 adults aged 20–38 years were interviewed regarding oral hygiene practices, sucrose intake and betel chewing. Caries experience (DMFT and DT), calculus, bleeding on probing (BoP) and Plaque index (PlI) were registered. Ureolytic activity in supragingival plaque was tested at two interproximal sites (11/12 and 41/42) with the rapid urease test (RUT). Registration of plaque pH was performed at two interproximal sites (15/16 and 31/41) before, during and 30 min after rinsing with an urea solution (0.25%). Four interproximal plaque samples (one from each quadrant) per individual were collected to test the bacterial composition using the checkerboard technique. Results. Children and adults had similarly low DMFT and DT values. Children had a higher baseline pH and a higher ureolytic activity in the maxilla (p < 0.05) compared with adults. A significant correlation (r2 = 0.63) was found between baseline pH and urease activity in the mandibular anterior teeth. Caries-free individuals had a higher baseline pH compared with caries active individuals in the anterior mandibular region (p < 0.01). The microbiological composition was characterized by an anaerobic low acidiogenic microbiota. Conclusions. Dental plaque pH is related to the ureolytic activity, which explains the low acidogenic plaque microflora and the low caries levels in the Karen population.

Morphological changes in Proteus mirabilis O18 biofilm under the influence of a urease inhibitor and a homoserine lactone derivative

Proteus mirabilis is a pathogenic gram-negative bacterium that frequently causes kidney infections, typically established by ascending colonization of the urinary tract. The present study is focused on ureolytic activity and urease inhibition in biofilms generated by P. mirabilis O18 cells. Confocal microscopy revealed morphological alterations in biofilms treated with urea and a urease inhibitor (acetohydroxamic acid, AHA), as some swarmer cells were found to protrude from the biofilm. The presence of a quorum-sensing molecule (N-butanoyl homoserine lactone, BHL) increased biofilm thickness and its ureolytic activity. Laser interferometric determination of diffusion showed that urea easily diffuses through P. mirabilis biofilm, while AHA is blocked. This may suggest that the use of urease inhibitors in CAUTIs may by less effective than in other urease-associated infections. Spectroscopic studies revealed differences between biofilm and planktonic cells indicating that polysaccharides and nucleic acids are involved in extracellular matrix and biofilm formation.

Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation

Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.

Engineered applications of ureolytic biomineralization: a review

Microbially-induced calcium carbonate (CaCO3) precipitation (MICP) is a widely explored and promising technology for use in various engineering applications. In this review, CaCO3 precipitation induced via urea hydrolysis (ureolysis) is examined for improving construction materials, cementing porous media, hydraulic control, and remediating environmental concerns. The control of MICP is explored through the manipulation of three factors: (1) the ureolytic activity (of microorganisms), (2) the reaction and transport rates of substrates, and (3) the saturation conditions of carbonate minerals. Many combinations of these factors have been researched to spatially and temporally control precipitation. This review discusses how optimization of MICP is attempted for different engineering applications in an effort to highlight the key research and development questions necessary to move MICP technologies toward commercial scale applications.

Structural and functional studies on urease from pigeon pea (Cajanus cajan)

Urease is an enzyme that catalyzes the hydrolysis of urea, forming ammonia and carbon dioxide, and is found in plants, microorganisms and invertebrates. Although plant and bacterial ureases are closely related at amino acid and at the structural level, the insecticidal activity is seen only in the plant ureases. In contrast, both plant and bacterial ureases exhibit antifungal activity. These two biological properties are independent of its ureolytic activity. However, till date the mechanism(s) behind the insecticidal and fungicidal activity of ureases are not clearly understood. Here we report the crystal structure of pigeon pea urease (PPU, Cajanus cajan) which is the second structure from the plant source. We have deduced the amino acid sequence of PPU and also report here studies on its stability, insecticidal and antifungal activity. PPU exhibits cellulase activity. Based on the structural analysis of PPU and docking studies with cellopentoase we propose a possible mechanism of antifungal activity of urease.

Factors influencing urease driven struvite precipitation

The concern about phosphorus depletion is driving more efficient phosphorus use and recycling. In this context the precipitation of phosphate as struvite from wastewaters has recently gained considerable traction. This study investigates the influence of different parameters on the ureolytic phosphate precipitation (pH increase by means of urease activity). Wastewater with a high phosphate concentration (>50mgL−1 PO4–P) and low volatile suspended solids (⩽3.0gL−1 VSS) resulted in a high phosphate removal efficiency (87%) and struvite crystals that could be separated from the sludge. An increase in ionic strength resulted in a decrease in ureolytic activity. An increase in Mg2+:PO43- molar ratio increases the degree of supersaturation with respect to struvite formation, which enhances the phosphate removal. Above a Mg2+:PO43- molar ratio of 3, additional Mg2+ dosage did not significantly affect the phosphate removal efficiency. Wastewater containing low phosphate concentrations (<50mgL−1 PO4–P), needed disproportionally large chemical additions and are thus not economically rewarding. For those wastewaters the presence of calcium interfered with the struvite precipitation rendering the precipitation of calcium phosphate a better option.

Soil response to chemicals used in a field experiment

Abstract The effect of chemicals (Reglone 200 SL and Elastiq 550 EC) on soil microorganisms and their enzymatic activity was estimated. The study was conducted in a field experiment which was set up in the split-block design and comprised three treatments. Soil samples were taken six times, twice in each year of study. The results showed that the application of chemicals generally had no negative effect on the number of soil microorganisms. The application of Reglone 200 SL caused an increase of proteolytic and ureolytic activity and affected the activity of dehydrogenases, acid and alkaline phosphatases in the soil. The soil subjected of Elastiq 550 EC was characterized by lower activity of dehydrogenases, protease, urease and alkaline phosphatase.

Nickel affects xylem Sap RNase a and converts RNase A to a urease

BackgroundNickel (Ni) is an essential micronutrient; however, its metabolic or physiological functions in plants and animals are largely uncharacterized. The ribonucleases (RNase, e.g., RNase A) are a large family of hydrolases found in one form or many forms facilitating nitrogen (N) cycling. It is currently unknown how either a deficiency or excess of Ni influences the functionality of ribonucleases, like RNase A. This is especially true for perennial crops possessing relatively high Ni requirements.ResultsWe report that the 'rising’ xylem sap of pecan [Carya illinoinensis (Wangenh.) K. Koch, a long-lived tree] at bud break contains a 14 kDa RNase A (aka, RNase 1), which amount has a 33% greater in Ni-deficient as in Ni-sufficient trees when exposed to Ni ions exhibits ureolytic activity. The homologous 13.4 kDa bovine pancreatic RNase A likewise exhibits ureolytic activity upon exposure to Ni ions. Ni therefore affects enzymatic function of a typically non-metalloenzyme, such as it transforms to an enzyme capable of hydrolyzing a linear amide; thus, converting an endonuclease esterase into a urease.ConclusionsWe conclude that Ni potentially affects the level and activity of RNase A present in the spring xylem sap of pecan trees, and probably in other crops, it has the same influence. The catalytic property of RNase A appears to shift from a nuclease to a urease relying on Ni exposure. This is suggestive that RNase A might possess novel metabolic functionality regarding N-metabolism in perennial plants. The ability of Ni to convert the activity of plant and animal RNase A from that of a ribonuclease to a urease indicates a possible unrecognized beneficial metabolic function of Ni in organisms, while also identifying a potential detrimental effect of excessive Ni on N related metabolic activity if there is sufficient disruption of Ni homeostasis.

Bacterial Urease and its Role in Long-Lasting Human Diseases

Urease is a virulence factor found in various pathogenic bacteria. It is essential in colonization of a host organism and in maintenance of bacterial cells in tissues. Due to its enzymatic activity, urease has a toxic effect on human cells. The presence of ureolytic activity is an important marker of a number of bacterial infections. Urease is also an immunogenic protein and is recognized by antibodies present in human sera. The presence of such antibodies is connected with progress of several long-lasting diseases, like rheumatoid arthritis, atherosclerosis or urinary tract infections. In bacterial ureases, motives with a sequence and/or structure similar to human proteins may occur. This phenomenon, known as molecular mimicry, leads to the appearance of autoantibodies, which take part in host molecules destruction. Detection of antibodies-binding motives (epitopes) in bacterial proteins is a complex process. However, organic chemistry tools, such as synthetic peptide libraries, are helpful in both, epitope mapping as well as in serologic investigations. In this review, we present a synthetic report on a molecular organization of bacterial ureases - genetic as well as structural. We characterize methods used in detecting urease and ureolytic activity, including techniques applied in disease diagnostic processes and in chemical synthesis of urease epitopes. The review also provides a summary of knowledge about a toxic effect of bacterial ureases on human body and about occurrence of anti-urease antibodies in long-lasting diseases.

Inhibition of Sporosarcina pasteurii under Anoxic Conditions: Implications for Subsurface Carbonate Precipitation and Remediation via Ureolysis

The use of Sporosarcina pasteurii to precipitate calcium carbonate in the anoxic subsurface via ureolysis has been proposed for reducing porosity and sealing fractures in rocks. Here we show that S. pasteurii is unable to grow anaerobically and that the ureolytic activity previously shown under anoxic conditions is a consequence of the urease enzyme already present in the cells of the aerobically grown inoculum. The implications are discussed, suggesting that de novo synthesis of urease under anoxic conditions is not possible and that ureolysis may decline over time without repeated injection of S. pasteurii as the urease enzyme degrades and/or becomes inhibited. Augmentation with a different ureolytic species that is able to grow anaerobically or stimulation of natural communities may be preferable for carbonate precipitation over the long term.

Darcy‐scale modeling of microbially induced carbonate mineral precipitation in sand columns

This investigation focuses on the use of microbially induced calcium carbonate precipitation (MICP) to set up subsurface hydraulic barriers to potentially increase storage security near wellbores of CO2 storage sites. A numerical model is developed, capable of accounting for carbonate precipitation due to ureolytic bacterial activity as well as the flow of two fluid phases in the subsurface. The model is compared to experiments involving saturated flow through sand‐packed columns to understand and optimize the processes involved as well as to validate the numerical model. It is then used to predict the effect of dense‐phase CO2 and CO2‐saturated water on carbonate precipitates in a porous medium.

Diatomaceous earth as a protective vehicle for bacteria applied for self-healing concrete

Crack repair is crucial since cracks are the main cause for the decreased service life of concrete structures. An original and promising way to repair cracks is to pre-incorporate healing agents inside the concrete matrix to heal cracks the moment they appear. Thus, the concrete obtains self-healing properties. The goal of our research is to apply bacterially precipitated CaCO₃ to heal cracks in concrete since the microbial calcium carbonate is more compatible with the concrete matrix and more environmentally friendly relative to the normally used polymeric materials. Diatomaceous earth (DE) was used in this study to protect bacteria from the high-pH environment of concrete. The experimental results showed that DE had a very good protective effect for bacteria. DE immobilized bacteria had much higher ureolytic activity (12-17 g/l urea was decomposed within 3 days) than that of un-immobilized bacteria (less than 1 g/l urea was decomposed within the same time span) in cement slurry. The optimal concentration of DE for immobilization was 60% (w/v, weight of DE/volume of bacterial suspension). Self-healing in cracked specimens was visualized under light microscopy. The images showed that cracks with a width ranging from 0.15 to 0.17 mm in the specimens containing DE immobilized bacteria were completely filled by the precipitation. Scanning electron microscopy (SEM) and energy dispersive spectrometry (EDS) were used to characterize the precipitation around the crack wall, which was confirmed to be calcium carbonate. The result from a capillary water absorption test showed that the specimens with DE immobilized bacteria had the lowest water absorption (30% of the reference ones), which indicated that the precipitation inside the cracks increased the water penetration resistance of the cracked specimens.

Influence of temperature on the effectiveness of a biogenic carbonate surface treatment for limestone conservation

So far, most studies on microbiologically induced carbonate precipitation for limestone conservation have been performed at temperatures optimal for the activity of the calcinogenic bacteria (i.e., 20-28°C). Successful application in practice, however, requires adequate performance in a wide range of environmental conditions. Therefore, the aim of this study was to select microorganisms that are most suited for biodeposition at temperatures relevant for practice. In a first step, ureolytic microorganisms were screened for their growth and ureolytic activity at different temperatures (10, 20, 28, and 37°C). Large differences in calcinogenic activity could be observed between experiments performed on agar plates and those performed in solution and in limestone. In a second step, the influence of temperature on the performance of the biodeposition treatment with different ureolytic microorganisms was evaluated, both on the consolidative and protective effect of the treatment. In contrast with the experiments on agar plates, the Sporosarcina psychrophila strains failed to produce significant amounts of calcium carbonate on limestone in conditions relevant for practice, even at 10°C. This resulted in a poor performance of the treatment. From experiments performed on limestone prisms, it appeared that the mesophilic Bacillus sphaericus produced the highest amount of carbonate in the shortest amount of time at all temperatures tested. As a result, compared to the untreated specimens, the highest consolidative (64% lower weight loss upon sonication) and protective effect (46% decreased sorptivity) were observed for the treatments with this species. From this study, it appears that among all ureolytic strains tested, B. sphaericus is most suited for biodeposition applications in practice.

Ubiquitous urease affects soybean susceptibility to fungi

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9894-1) contains supplementary material, which is available to authorized users.

Establishment of ruminal enzyme activities and fermentation capacity in dairy calves from birth through weaning

The objectives of this study were to characterize the establishment of ruminal fermentation and enzymatic activities in dairy calves from birth to weaning (d 83). Six Holstein calves, immediately separated from their mother at birth, were fed colostrum for 3 d after birth, and thereafter milk replacer, starter pelleted concentrate, and hay until d 83 of age. Ruminal samples were collected from each calf every day for the first 10 d, and additionally at d 12, 15, 19, 22, 26, 29, 33, 36, 40, 43, 47, 50, 55, 62, 69, and 83. Ruminal samples were collected 1h after milk feeding with a stomach tube. The pH and redox potential (Eh) were immediately measured. Samples were kept for further determination of ammonia nitrogen (NH3-N) and volatile fatty acid (VFA) concentrations, and xylanase, amylase, urease, and protease activities. Ruminal pH averaged 6.69, 5.82, and 6.34, from d 1 to 9, d 10 to 40, and d 43 to 83 of age, respectively. At first day of life, the ruminal Eh value was positive (+224mV). From d 2 to 9, d 10 to 40, and d 43 to 83 of age, ruminal Eh averaged −164, −115, and −141mV, respectively. From d 1 to 3, d 4 to 22, and d 26 to 83 of age, NH3-N concentration averaged 60.1, 179.8, and 58.2 mg/L, respectively. No VFA were detected in ruminal samples collected on d 1 of life of calves. From d 2 to 10 and d 12 to 83 of age, ruminal total VFA concentration averaged 19.5 and 84.4mM, respectively. Neither ruminal xylanase or amylase activities were observed at d 1 of age. From d 5 to 15 and d 19 to 83 of age, the xylanase activity averaged 182.2 and 62.4 μmol of sugar released per hour per gram of ruminal content dry matter (DM), respectively. From d 5 to 83 of age, the amylase activity reached 35.4 μmol of sugar released per hour per gram of ruminal content DM. The ruminal ureolytic activity was observed with an average value of 6.9 μg of NH3-N released per minute per gram of ruminal content DM over the 83-d experimental period. From d 1 to 4 and d 5 to 83 of age, the proteolytic activity was 8.2 and 27.9 optical density units per hour per gram of ruminal content DM, respectively. The fermentative and enzymatic activities were rapidly established in the rumen from d 2 after birth. Most parameters did not evolve further after 1 mo of age.

Short communication: Technological and genotypic comparison between Streptococcus macedonicus and Streptococcus thermophilus strains coming from the same dairy environment

The species Streptococcus thermophilus is widely used for the preparation of several dairy products, and its technological contribution is clear. On the other hand, although Streptococcus macedonicus was first described more than 10 yr ago and, despite the scientific interest around this issue, the exact role of Strep. macedonicus in cheese making has yet to be clarified. In this study, 121 strains belonging to both species and isolated from the same dairy environment were genetically characterized by random amplification of polymorphic DNA (RAPD)-PCR and compared for the main biochemical features of technological interest, such as acid production, galactose utilization, citrate metabolism, exopolysaccharide production, and lipolytic, ureolytic, exocellular proteolytic, and decarboxylasic activities. Analysis by RAPD-PCR highlighted a remarkable genotypic heterogeneity among strains in both species, and, at a similarity level of 78%, all the isolates and reference strains of Strep. thermophilus grouped together and were well separated from the strains of Strep. macedonicus, confirming that these 2 species are different microbial entities. Comparison between genetic and phenotypic or biotechnological data did not reveal any relationships.

Effects of environmental factors on microbial induced calcium carbonate precipitation

To gain an understanding of the environmental factors that affect the growth of the bacterium Sporosarcina pasteurii, the metabolism of the bacterium and the calcium carbonate precipitation induced by this bacterium to optimally implement the biological treatment process, microbial induced calcium carbonate precipitation (MICP), in situ. Soil column and batch tests were used to assess the effect of likely subsurface environmental factors on the MICP treatment process. Microbial growth and mineral precipitation were evaluated in freshwater and seawater. Environmental conditions that may influence the ureolytic activity of the bacteria, such as ammonium concentration and oxygen availability, as well as the ureolytic activities of viable and lysed cells were assessed. Treatment formulation and injection rate, as well as soil particle characteristics are other factors that were evaluated for impact on uniform induction of cementation within the soils. The results of the study presented herein indicate that the biological treatment process is equally robust over a wide range of soil types, concentrations of ammonium chloride and salinities ranging from distilled water to full seawater; on the time scale of an hour, it is not diminished by the absence of oxygen or lysis of cells containing the urease enzyme. This study advances the biological treatment process MICP towards field implementation by addressing key environmental hurdles faced with during the upscaling process.