Commercial Urease Research Articles

Efficacy of soybean-derived crude extract in enzyme-induced carbonate precipitation as soil-improvement technique

Calcium carbonate precipitation using a urease enzyme, referred to herein as Enzyme-Induced Carbonate Precipitation (EICP) is a technique for soil improvement. In this technique, a mixed solution composed of reagents and the urease enzyme, which produces calcite, is utilized as the grouting material. Recently, alternative materials to the urease enzyme have been examined to resolve the cost issue of using the urease enzyme. In this study, several tests were conducted to compare commercial urease and soybean-derived crude urease. A comparison of their hydrolysis rates was done through urease activity tests. The microscopic structures and mineralogy of the precipitated materials, produced during various loading periods, were investigated through SEM and XRD analyses. Moreover, the reinforcing effect of the grouting solutions on the treated soil specimens was evaluated by measuring the unconfined compressive strength (UCS) of the treated samples. Interestingly, the precipitated CaCO3 was vaterite and calcite when using soybean urease, while it was mostly calcite when using the urease enzyme. Higher UCS values were obtained with the soybean urease samples because the precipitated CaCO3 seemed to be concentrated at the inter-grain contacts. It was concluded that soybean powder shows great efficacy as a replacement for commercially produced enzyme urease in soil-improvement techniques mediated by carbonate precipitation.

The effect of metal oxide coating of urea on mineralization in two contrasting soils

AbstractUrease inhibitors are used to delay the conversion of N supplied to soil as urea to better match the mineral N supply to crop demand. Using soil in petri dishes, we compared the spatial and temporal mineral N dynamics close to the site of urea addition for two soils of contrasting texture—sandy soil (2% clay) and clay soil (51% clay)—treated with N fertilizer products that had the potential to inhibit urease. The treatments included trace metal oxide coatings (ZnO, NiO, and CuO) of granular urea fertilizer, N‐(n‐butyl)thiophosphoric triamide (NBPT; a commercial urease inhibitor) coated urea and uncoated urea. In the sandy soil, NBPT inhibited urea hydrolysis, whereas the metal oxide–coated urea had little effect. Unlike in the sandy soil, the most striking result for the clay soil was the “disappearance” of mineral N in the latter part of the incubation. However, a delay in the disappearance of mineral N, hence a higher recovery, for the NBPT‐urea and CuO‐urea was observed at day 14 in the clay soil presumably due to the slower hydrolysis of urea compared to other treatments. Overall, the study of both spatial and temporal mineral N dynamics in soils supplied with urea fertilizers suggests that CuO‐coated urea could have properties that delay the hydrolysis of urea in certain soils. Given the strongly contrasting outcomes for the two soils, further experiments with soils that span the range of texture between these two extremes are warranted.

Effect of urease enrichment degree of multiple sources of urease on bio-cementation efficacy via enzyme-induced carbonate precipitation

This study aims to investigate the effect of the urease enrichment degree of high-purity commercial urease and plant-derived crude ureases from sword bean (SWCU), soybean (SCU), and pigeon pea (PCU) on bio-cementation efficacy via enzyme-induced carbonate precipitation. The urease enrichment degree is defined as the urease activity per gram of organic matter in the urease solution. Bio-cementation efficacy was evaluated by the distribution and morphology of the precipitates and unconfined compressive strength. The results show that the urease enrichment degrees of the high-purity commercial urease, SWCU, PCU, and SCU are 1.08, 1.05, 0.57, and 0.31 mmol/min/g, respectively. The urease enrichment degree is the dominant factor influencing the pattern of CaCO3 distribution by affecting the organic matter distribution. The high-purity commercial urease-treated sand has the smallest calcite crystals (13–23 µm) and the lowest strength (172 kPa). For the plant-derived crude urease, with the decrease of the urease enrichment degree, the generated crystals become smaller, the CaCO3 distribution becomes less uniform, and the soil strength decreases. The SWCU-treated sand exhibits the best bio-cementation efficacy and is recommended to enhance soil strength.

Enzyme Purification Improves the Enzyme Loading, Self-Propulsion, and Endurance Performance of Micromotors.

Enzyme-powered micro- and nanomotors make use of biocatalysis to self-propel in aqueous media and hold immense promise for active and targeted drug delivery. Most (if not all) of these micro- and nanomotors described to date are fabricated using a commercially available enzyme, despite claims that some commercial preparations may not have a sufficiently high degree of purity for downstream applications. In this study, the purity of a commercial urease, an enzyme frequently used to power the motion of micro- and nanomotors, was evaluated and found to be impure. After separating the hexameric urease from the protein impurities by size-exclusion chromatography, the hexameric urease was subsequently characterized and used to functionalize hollow silica microcapsules. Micromotors loaded with purified urease were found to be 2.5 times more motile than the same micromotors loaded with unpurified urease, reaching average speeds of 5.5 μm/s. After comparing a number of parameters, such as enzyme distribution, protein loading, and motor reusability, between micromotors functionalized with purified vs unpurified urease, it was concluded that protein purification was essential for optimal performance of the enzyme-powered micromotor.

Interaction and inhibition mechanism of urease in vitro and soil system by a natural benzylisothiocyanate isolated from Moringa oleifera

Urease catalyzes urea hydrolysis in NH3 and CO2 in the soil, leading to N-urea losses by volatilization (30–70%); thus, urease inhibitor addition is an alternative that reduces these losses. In this sense, biophysical studies are important to evaluate potential urease inhibitors and biotechnological applications since currently, the N-(n-butyl)thiophosphoric triamide (NBPT) is the only commercial urease inhibitor for agriculture purposes. Thus, this work focused on evaluating the mechanism of urease inhibition by 4-(4′-O-acetyl-α-L-rhamnosyloxy)benzylisothiocyanate (MFC) isolated from Moringa oleifera L. (Moringaceae). The experiment consisted of spectroscopic studies of enzyme kinetics and activity (urease 12 mU and MFC 0–800 µM); assays to evaluate conformational changes in the enzyme using ultraviolet–visible molecular absorption and molecular fluorescence (5 µM urease and MFC 0–200 µM); the evaluation of thiols and amino/guanidine groups using 5,5′-dithiobis(2-nitrobenzoic acid) (50 µM) and fluorescamine (100 µM). Inhibition of soil ureases using Ferralsol soil samples was performed with MFC compared with thiourea, hydroxyurea, and NBPT (0.5 mM for all). The results of enzymatic studies indicated that MFC is a mixed inhibitor with concentration necessary to produce 50% inhibition (IC50) of 487 μM. From the spectrofluorimetric titration were obtained Ksv (1.30 ×103 M−1) and Kb (1.80 ×102 M−1), which reflect the magnitude of fluorescence suppression and the interaction between urease and MFC, while the n value 0.77 indicates that the stoichiometry of the complex is 1:1. Besides, the interaction process MFC-urease changes protein structure, especially tryptophan (Trp) residues' microenvironment, highlighting MFC action as a mixed inhibitor since the Trp495 and Trp648 residues are far away from the urease catalytic site. The monitoring of the residues containing thiol and amino/guanidine groups indicated covalent interaction of isothiocyanate with these groups forming dithiocarbamates and thiourea (urease inhibitor group) was confirmed as majority products by mass spectrometry. Finally, MFC was applied to the soil samples, and the residual activity of soil ureases was determined, with inhibition of 49.4% (soil A) and 32.0% (soil B), noting that soil inhibition is like NBPT (54.4%, soil A) and can be considered a potential compound for biotechnology application.

Potentially of Soybean as Bio-Catalyst in Calcite Precipitation Methods for Improving the Strength of Sandy Soil

The biocementation-based soil improvement technique that has been recently developed is enzyme-induced calcite precipitation (EICP). Previous studies on the EICP technique used pure commercial urease enzyme as a reaction catalyst to precipitate calcite from urea and calcium chloride. However, pure commercial urease enzyme is inefficient for application on a large scale due to its high price. Therefore, studies reported that soybean is a potential alternative for pure commercial urease enzymes. This study evaluates the strength of sandy soil by treatment using EICP grouting solution with soybean extracts as the bio-catalyst. The extracts used in this study were obtained by filtration and centrifugation treatment. Based on the calcite mass quantification and precipitation ratio, 20 g/L soybean extract concentration in both treatments is used for treating the solution. Then, the unconfined compressive strength (UCS) tests were conducted to evaluate the increase of the strength of sandy soil. The maximum UCS value was obtained at the 28 days of curing time in both treatments. The centrifugation treatment has a higher UCS value with 65.6 kPa than the filtration treatment with 53.6 kPa. Thus, this study elucidated that soybean extract has a great potential to be a bio-catalyst in the calcite precipitation method.

Longevity of Raw and Lyophilized Crude Urease Extracts

The stability (longevity of activity) of three crude urease extracts was evaluated in a laboratory study as part of an effort to reduce the cost of urease for applications that do not require high purity enzyme. A low-cost, stable source of urease will greatly facilitate engineering applications of urease such as biocementation of soil. Inexpensive crude extracts of urease have been shown to be effective at hydrolyzing urea for carbonate precipitation. However, some studies have suggested that the activity of a crude extract may decrease with time, limiting the potential for its mass production for commercial applications. The stability of crude urease extracts shown to be effective for biocementation was studied. The crude extracts were obtained from jack beans via a simple extraction process, stored at room temperature and at 4 ℃, and periodically tested to evaluate their stability. To facilitate storage and transportation of the extracted enzyme, the longevity of the enzyme following freeze drying (lyophilization) to reduce the crude extract to a powder and subsequent re-hydration into an aqueous solution was evaluated. In an attempt to improve the shelf life of the lyophilized extract, dextran and sucrose were added during lyophilization. The stability of purified commercial urease following rehydration was also investigated. Results of the laboratory tests showed that the lyophilized crude extract maintained its activity during storage more effectively than either the crude extract solution or the rehydrated commercial urease. While incorporating 2% dextran (w/v) prior to lyophilization of the crude extract increased the overall enzymatic activity, it did not enhance the stability of the urease during storage.

Application of soybean powder as urease enzyme replacement on EICP method for soil improvement technique

Many researchers have confirmed the application of calcite precipitation method by urease enzyme induction as a potential soil improvement technique. However, this method needs an expensive investment due to the urease enzyme issue. The enzyme itself brings 90% of the total materials cost. It means that finding new inexpensive material for replacing the enzyme is considered essential for the development of this method. In this study, a potential material, which is soybean powder, was evaluated through several experimental tests. A comparison of urease activity value between EICP using soybean powder and commercial urease as a catalyst for hydrolysis of urea compound was made through a series of conductivity tests. It was found that EICP using soybean urease can be a potential alternative to a commercial product in terms of urease activity. The precipitation materials were analysed using a microscale test to analyses the minerals type. Besides, the reinforcing effect of EICP solution on soil specimens was evaluated by conducting unconfined compressive strength (UCS) test and acid leaching test. The results of the UCS test were indicated that the soybean powder is a potential material to be used in soil improvement technique.

EXAMINATION OF CALCITE PRECIPITATION USING PLANT-DERIVED UREASE ENZYME FOR SOIL IMPROVEMENT

The enzyme-mediated calcite precipitation (EMCP) method was established as a soil-improvement technique. This study discussed the possibility of using a plant-derived urease enzyme as a bio-catalyst. The urease enzyme was utilized for the hydrolysis of urea, which causes the supply of calcium ions to precipitate calcium carbonate. Several sources that may contain the urease enzyme were examined. Extracts from cabbage and soy pulp, without purification, were utilized as substitutes for commercial urease enzyme products. Initially, the solution was composed of 15 g/L of crude extract and 1 mol/L each of urea and calcium chloride. The precipitation results using commercial products and crude extracts were compared under the same concentration of reagent solution. A mineralogical analysis of the precipitated materials was also conducted. The mineralogy and morphology of the precipitated materials were examined by Fourier-Transformed Infra-Red (FTIR) spectroscopy, X-Ray Diffraction (XRD), and Scanning Electromagnetic (SEM) tests. The precipitation ratios using the crude extracts as replacements for the urease enzyme were found to be more than 60% of the maximum theoretical value. From the mineralogical and morphological analyses, the calcite mineral was confirmed as the precipitated material. The results of this study indicated that crude extracts from cabbage and soy pulp are potential materials for sources of the urease enzyme

Starch-based C-dots from natural Gadong tuber as pH fluorescence label for optical biosensing of arginine

A fluorescence bi-enzyme optical arginine biosensor based on novel fluorophore carbon nanodots (C-dots) pH indicator derived from natural Gadong (Dioscorea hispida) tuber was developed. The brightly luminescent pH sensitive carbon-based quantum dots (QDs) exploited from exotic wild yam polysaccharides via aqueous-based synthesis method were immobilized on the acrylic-N-acryloxysuccinimidecopolymer microspheres. Recombinant Glaciozyma antarctica arginase (GaArg) cloned in E. coli expression system and commercial urease were grafted on the acrylic microspheres (AMS) via peptide covalent link. During fluorescence arginine detection, the immobilized arginase catalyzed hydrolysis conversion of arginine to ornithine and urea, and subsequent enzymatic hydrolysis of urea by immobilized urease rendering deprotonation of starch-based C-dots, resulted in the heightening of C-dots’ photoluminescence intensity due to elevated localized pH. Under 380 nm excitation, the C-dots fluorescence intensity at 420 nm increased linearly with arginine over the concentration range of 1–10 mM, with the limit of detection (LOD) estimated at 0.9 mM. The Gadong starch-based C-dots, which prepared from different natural tuber samples showed high reproducible physico-chemical properties, and gave reproducible fluorescence intensity at different pH values with relative standard deviation (RSD) values obtained at <1.0%, which suggests high reproducibility of the synthesis of starch-based luminecent C-dots. The optical enzymatic biosensor doped with C-dots pH label demonstrated high selectivity towards fluorescence biorecognition of arginine compared to some other amino acids normally present in packaged fruit juices. No significant difference was found between C-dots-labeled enzymatic fluorescence microsensor and HPLC standard method for arginine content detection in fruit juice samples of grapes, mangoes and oranges.

Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor

The nanoparticles (NPs) aggregates of commercial urease from jack beans (Canavalia ensiformis) were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine dihydrochloride. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM), UV and Fourier transform infrared (FTIR) spectroscopy. The TEM images of urease NPs showed their size in the range, 18–100nm with an average of 51.2nm. The ENPs were more active and stable with a longer shelf life than native enzyme molecules. The ENPs were immobilized onto chitosan (CHIT) activated nitrocellulose (NC) membrane via glutaraldehyde coupling with 32.22% retention of initial activity of free ureaseNPs with a conjugation yield of 1.63mg/cm2. This NC membrane was mounted at the lower/sensitive end of the ammonium ion selective electrode (AISE) with O-ring and then electrode was connected to a digital pH meter to construct a potentiometric urea biosensor. The biosensor exhibited optimum response within 10s at pH 5.5and 40°C. The biosensor was employed for measurement of potentiometric determination of urea in sera of apparently healthy and persons suffering from kidney disorders. The biosensor displayed a low detection limit of 1µM/L with a wide working range of 2–80µM/L (0.002–0.08mM) and sensitivity of 23mV/decade. The analytical recovery of added urea in serum was 106.33%. The within and between-batch coefficient of variations (CVs) of present biosensor were 0.18% and 0.32% respectively. There was a good correlation (r = 0.99) between sera urea values obtained by reference method (Enzymic colorimetric kit method) and the present biosensor. The biosensor had negligible interference from Na+,K+,NH+4 and Ca2+ but Mg2+,Cu2+ and ascorbic acid but had slight interference, which was overcome by specific ion selective electrode. The ENPs bound NC membrane was used maximally 8–9 times per day over a period of 180 days, when stored in 0.01M sodium acetate buffer pH 5.5 at 4°C.

Fluorometric enzymatic assay of l-arginine

The enzymes of l-arginine (further − Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further – arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415nm under excitation at 360nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100nM to 6μМ with a limit of detection of 34nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

ARGINASE-BASED AMPEROMETRIC BIOSENSOR FOR MANGANESE IONS ANALYSIS

The development of simple cost-effective sensitive enzymatic methods for analysis of toxic metallic ions is an actual problem. Promising tools for elaboration of such methods are Mn2+-dependent enzymes. A novel manganese(II)-sensitive amperometric bi-enzyme biosensor based on of recombinant human arginase I (arginase) isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer with urease and apo-enzyme of arginase was placed onto a polyaniline-Nafion composite platinum electrode. The developed sensor revealed a high sensitivity to Mn2+-ions – 9200±20 A/(M∙m2)with the apparent Michaelis-Menten constant derived from Mn2+-ions calibration curve of 11.5±1.0 µM. A linear concentration range was observed from 1 µM to 6,5 µM MnCl2, a limit of detection being of 0.15 µM and a response time – 2.5 min. The proposed biosensor may be useful to monitor manganese compounds in laboratories of medicine, food industry and environmental control service.

New enzymatic method for l-arginine determination based on use of human arginase i and urease

It is developed a sensitive and selective method for quantitative analysis of L-arginine with spectrophotometric method of detection of the reaction product (ammonium) using o-Phthalaldehyde. The method is based on the use of highly purified arginase I and commercial urease. The new method is highly sensitive for L-arginine determination, a wide range of linearity and insensitivity to interfering influence of wide range of amino acids

L-Arginine-selective microbial amperometric sensor based on recombinant yeast cells over-producing human liver arginase I

The article provides a description of a microbial l-arginine-selective amperometric biosensor based on recombinant cells of the methylotrophic yeast Hansenula polymorpha. The intact and permeabilized cells (p-cells) co-immobilized with commercial urease on a surface of electrode were used as biorecognizing elements of biosensors. Operational parameters of the bioelectrodes were estimated. The developed p-cells-based sensor revealed a satisfactory selectivity to l-arginine (Arg) and sensitivity (14±1.2A(Mm2)−1) with an apparent Michaelis–Menten constant derived from Arg calibration curve 0.51±0.05mM. The dynamic range is linear till 0.6mM Arg, a detection limit is 0.085mM and a response time 60s. The developed microbial biosensor was used for Arg assay in real samples of wine and juice in comparison with other analytical approaches. The Arg values obtained with different methods demonstrated a strong correlation.

Bi-enzyme l-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode

A novel l-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline–Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to l-arginine. The sensitivity of the biosensor was 110±1.3nA/(mMmm2) with the apparent Michaelis–Menten constant (KMapp) derived from an l-arginine (l-Arg) calibration curve of 1.27±0.29mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038mM and a response time — 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals (“Tivortin”, “Cytrarginine”, “Aminoplazmal 10% E”) for l-Arg testing. The obtained l-Arg-content values correlated well with those declared by producers.

Design, Synthesis, and Biological Evaluation of Phosphoramide Derivatives as Urease Inhibitors

The design, synthesis, and biological evaluation of phosphoramide derivatives as urease inhibitors to reduce the loss of ammonia has been carried out. Forty phosphorus derivatives were synthesized and their inhibitory activities evaluated against that of jack bean urease. In addition, in vivo assays have been carried out. All of the compounds were characterized by IR, (1)H NMR, MS, and elemental microanalysis. In some cases, detailed molecular modeling studies were carried out, and these highlighted the interaction between the enzyme active center and the compounds and also the characteristics related to their activity as urease inhibitors. According to the IC(50) values for in vitro inhibitory activity, 12 compounds showed values below 1 microM and 8 of them represent improvements of activity in comparison to the commercial urease inhibitor N-n-butylthiophosphorictriamide (NBPT) (100 nM) (AGROTAIN). On the basis of the activity results and the conclusions of the molecular modeling study, a structural model for new potential inhibitors has been defined.

Inhibition of bacterial urease by autoxidation of furan C‐18 fatty acid methyl ester products

Autoxidation products of synthetic methyl 9,12‐epoxy‐octadeca‐9,11‐dienoate (MEFA) were investigated by gas chromatography‐mass spectrometry analysis and tested for bacterial urease inhibition. A suspension of oxidized MEFA in 10% Tween 80 was an effective inhibitor for bacterial urease extract fromHelicobacter pylori (I50=1.3 mM) and for commercial urease fromBacillus pasteurii (I50=0.06 mM). The urease inhibitory effect was cancelled by adding cysteine to the reaction mixture. The total content of biologically active oxidized products in the mixture was found to be 6.2%. Dioxo‐ene derivatives of MEFA on the thin‐layer chromatography plate surface were converted into more stable compounds, whose formation in the mixture reduced inhibition ofB. pasteurii to about 2% of the former level. The mechanism of urease inhibition is supposed to involve the interaction of the thiol groups of the enzyme’s active center with the inhibitor molecules.

Purification of enzymes by high-speed gel filtration on TSK-GEL SW columns

The purification of enzymes was investigated by high-speed gel filtration on TSK-GEL G3000SWG columns packed with porous silica gel deactivated by chemically bonded hydrophilic compounds. Crude β-galactosidase from bacterial cells and commercial urease were purified ca. 15-fold in a single gel filtration. These enzymes were eluted within an hour from the column and the recoveries of enzymatic activity were almost 100% although the operation was carried out at room temperature (22°). Samples up to 100 mg could be applied to the column without loss of separation efficiency.

Four commercial urease reagents and a laboratory-prepared reagent compared for analysis of blood urea nitrogen with the Beckman analyzer.

Three laboratory-prepared urease reagents were compared with a commercial preparation supplied for routine use on the Beckman Blood Urea Nitrogen Analyzer. There were discrepancies in results for urea nitrogen among the four urease reagents when matching serum and the corresponding oxalate/fluoride treated plasma were compared as measured with the Beckman Analyzer and continuous-flow (AutoAnalyzer) method. All four urease preparations were affected by fluoride, but to different extents. We believe that an effective laboratory reagent can be prepared in the laboratory at significantly lower cost.