Preparation and characterization of kappa-carrageenan immobilized urease

Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity.     

Modification and characterization of cellulosic cotton fibers for efficient immobilization of urease

Cotton fibers were first grafted by polyacrylonitril in the presence of KMnO(4) and oxalic acid as a combined redox initiator. Moreover, modification of the grafted cotton fibers was done by changing the nitrile group (-CN) into hydrazidine group through the reaction with hydrazine hydrate, then the fibers were activated by glutaraldehyde to introduce free aldehyde groups which were able to react with amino groups of urease to form Schiff's base, and result in cotton fibers immobilized urease. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of enzyme before and after the immobilization of urease. The results showed that the optimum temperature of immobilized urease was 35°C, which was higher than that of the free enzyme (30°C), and the immobilized urease exhibited a higher relative activity than that of free urease over 35°C. The optimal pH for immobilized urease was 6.5, which was lower than that of the free urease (pH 7.0), and the immobilization resulted in stabilization of enzyme over a wider pH range. The kinetic constant value (K(m)) of immobilized urease was higher than that of the free urease. However, the thermal and operational stabilities of immobilized urease have been improved greatly.     

Immobilization of urease on vermiculite

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses.     

Immobilization of urease onto chemically modified acrylonitrile copolymer membranes

Poly (acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to seven different chemical modifications. The amounts of new groups incorporated in the membranes with the modifications were determined. Urease was covalently immobilized on the modified membranes. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity was found for urease bound to membranes modified with hydroxylammonium sulfate (68%) and hydrazinium sulfate (67%). Optimum pH of free urease was determined to be 5.8. For positively charged membranes, pH optimum was shifted to higher values, while for negatively charged membranes-to lower pH. The charge of the matrix affected also the rate of the enzyme reaction. The highest rate was measured with urease immobilized on membranes modified with hydroxylammonium sulfate and hydrazinium sulfate. The major part of the immobilized enzyme on different modified membranes remained stable-only ca. 20% of enzyme activity was lost for 4 h at 70 degrees C while the free enzyme was totally inactivated.     

Covalent immobilization of urease on polysiloxane matrices containing 3-aminopropyl and 3-mercaptopropyl groups

A sol-gel method of covalent immobilization of urease on polysiloxane matrices is developed which uses glutaraldehyde or Ellman’s reagent as binding agents. We show that the urease covalently bound to the poly(3-mercaptopropyl)siloxane matrix retains 67–84% of its activity and is stable, losing only 10% of its activity after 300 days. The urease adsorbed on the poly(3-mercaptopropyl)siloxane matrix was more active than the native urease. On the basis of the literature, we suggest that, in this case, the 3-mercaptopropyl groups of the polysiloxane matrix are brought into close proximity to the active site of urease, where they possibly act as proton donors, which results in an accelerated enzymatic reaction. Covalent immobilization of urease on the polysiloxane matrix containing 3-aminopropyl was less efficient, because the immobilized enzyme was significantly less active. At the same time, the urease adsorbed on the same matrix showed high activity (60–86%).     

Study of immobilization and properties of urease for creation of a biosensor based on semiconductor structures]

Many-sided investigations of urease immobilization methods were carried out to create the biosensor devices on the base of semiconductor structures. Special attention was concentrated on the biomembrane formation by means of urease and bovine serum albumin (BSA) cross-linking by gaseous glutaraldehyde. Optimal conditions for the formation process were selected which preserve about 20% of total urease activity after the cross-linking. The properties of enzyme immobilized by the above-mentioned method have been comprehensively studied. They included the urease activity dependence on pH, ionic strength, incubation buffer capacity as well as the enzyme stability during its functioning, storing and thermoinactivation. As was shown, for immobilized ureas Km value for urea at pH 7.0 and 20 degrees C is 1.65 time less than for free enzyme. In the presence of EDTA (1 mM) the enzyme activity in the biomembrane is practically unchanged under a month storing. Biomembrane possesses good adhesion to silicon surface and its swelling level under different conditions does not exceed 35%. The conclusion is made about the prospects of the used method of biomembrane formation for biosensor technology based on semiconductor structures.     

Preparation and characterization of urease immobilized onto porous chitosan beads for urea hydrolysis

Urease was covalently immobilized onto porous chitosan beads via primary amine groups connected to the backbone via a six-carbon linear alkyl spacer. The optimum conditions for enzyme immobilization are activating the beads with 1%(w/w) glutaraldehyde, reacting the activated beads in pH 7 buffer with the enzyme, using an enzyme to bead weight ratio of 25, and without lyophilization. Chitosan-bound urease was found to fully retain its specific activity. Properties of the immobilized urease were characterized under batch and flow conditions. Increased optimum reaction temperature, enhanced thermal stability and storage stability, and excellent reusability were found after enzyme immobilization. Continuous hydrolysis of urea solution was studied in a column packed with the enzyme-containing beads for its possible application in regenerating dialysate solution during hemodialysis.     

Enzymatic detection of mercuric ions in ground-water from vegetable wastes by immobilizing pumpkin (Cucumis melo) urease in calcium alginate beads

Present report describes a quick and simple test based on enzyme inhibition for the detection of mercury in aqueous medium by urease immobilized in alginate beads. Urease was extracted from the discarded seeds of pumpkin (Cucumis melo) and was purified to apparent homogeneity (5.2-fold) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation (Sp activity 353 U/mg protein, A(280)/A(260)=1.12) was immobilized in 3.5% alginate leading to 86% immobilization. Effect of mercuric ion on the activity of soluble as well as immobilized enzyme was investigated. Hg(2+) exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The alginate immobilized enzyme showed less inhibition. There was no leaching of the enzyme over a period of 15 days at 4 degrees C. The inhibition was non-competitive and the K(i) was found to be 1.26x10(-1)microM. Time-dependent interaction of urease with Hg(2+) exhibited a biphasic inhibition behavior in which approximately half of the initial activity was lost rapidly (within 10 min) and reminder in a slow phase. Binding of Hg(2+) with the enzyme was largely irreversible, as the activity could not be restored by dialysis. The significance of the observations is discussed.     

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.     

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.     

Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.     

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%.     

Immobilization imparts stability to watermelon urease to work in water miscible organic media

The behaviour of alginate immobilized and soluble watermelon (Citrullus vulgaris) urease in water miscible organic solvents like, acetonitrile, dimethylformamide (DMF), ethanol, methanol, and propanol is described. The organic solvents exhibited a concentration dependent inhibitory effect on both the immobilized and the soluble urease in the presence of urea. Pretreatment of soluble enzyme preparations with organic solvents in the absence of substrate for 10 min at 30°C led to rapid loss in the activity, while similar pretreatment of immobilized urease with 50% (v/v) of ethanol, propanol, and acetonitrile was ineffective. Time-dependent inactivation of immobilized urease, both in the presence and in the absence of urea, revealed stability for longer duration of time even at very high concentration of organic solvents. The soluble enzyme, on the other hand, was rapidly inactivated even at fairly lower concentrations. The results suggest that the immobilization of watermelon urease in calcium alginate make it suitable for its application in organic media. the observations are discussed.     

Inhibition of chitosan-immobilized urease by slow-binding inhibitors: Ni, F and acetohydroxamic acid

The inhibitions by Ni²⁺ and F⁻ ions and by acetohydroxamic acid of jack bean urease covalently immobilized on chitosan membrane was studied (pH 7.0, 25°C) and compared with those of the native enzyme. The reaction progress curves of the immobilized urease-catalyzed hydrolysis of urea were recorded in the absence and presence of the inhibitors. They revealed that the inhibitions are of the competitive slow-binding type similar to those of native urease. The immobilization weakened the inhibitory effect of the inhibitors on urease as measured by the inhibition constants K_i^*. The increase in their values: 17.9-fold for Ni²⁺, 26.5-fold for F⁻ and 1.7-fold for acetohydroxamic acid, was accounted for by environmental effects generated by heterogeneity of the urease–chitosan system: (1) mass transfer limitations imposed on substrate and reaction product in the external solution, and (2) the increase in local pH on the membrane produced by both the enzymatic reaction and the electric charge of the support. By relating the K_M/K_i^* ratio to the electrostatic potential of chitosan it was found that while the reduced Ni²⁺ inhibition is mainly brought about by the potential, inhibition by acetohydroxamic acid is independent of the potential, and the acid inhibits urease in its non-ionic form. The reduction in F⁻ inhibition was ascribed to the increased pH in the local environment of the immobilized enzyme.     

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.     

Polyaniline as a support for urease immobilization

Polyaniline synthesized by chemical oxidative polymerization was used as an immobilization support for jack bean urease. Such immobilized enzyme has a good catalytic activity, storage stability, and reusability. Properties of free and immobilized urease were compared. Blends of polystyrene, cellulose acetate and poly(methyl methacrylate) with polyaniline were used for urease immobilization as well.     

Urease immobilized on an aminated polysulphone membrane – inhibition by boric acid

An enzymatic membrane for application in the processes of decomposition and removal of urea from aqueous solutions was prepared: jack bean urease was immobilized on an aminated polysulphone membrane by adsorption. The inhibition of the system by boric acid was studied using procedures based on the MICHAELIS-MENTEN integrated equation (non-linear regression, and the linear transformations of WALKER and SCHMIDT, JENNINGS and NIEMANN, and BOOMAN and NIEMANN). The reaction was carried out in a 100 mM phosphate buffer of pH 7.0, containing 2 mM EDTA, obtained by neutralization of orthophosphoric acid with NaOH, at an initial urea concentration of 10 mM, and a temperature of 25 °C. The reaction was initiated by the addition of the enzyme to the urea solution, and was monitored by removing samples of the reaction mixture for NH₃ determinations by the phenol-hypochlorite method until the urea was exhausted. The results were compared with those obtained earlier under the same reaction conditions for free urease and urease covalently immobilized on chitosan. The inhibition was found to be competitive, similar to that of the free enzyme and urease immobilized on chitosan, with inhibition constants K_i equal to 0.36, 0.19 and 0.60 mM. The results show that adsorption of the enzyme on a polysulphone membrane changed the enzyme to a lesser degree than covalent immobilization of the enzyme on a chitosan membrane.     

Properties of urease immobilized on the functional organic silica surface

The paper deals with kinetics of the urea hydrolysis by microbial-origin urease dissolved and immobilized on the organic silica surface. It is shown that hydrolysis kinetics for soluble urease is described by the Michaelis-Menten equation until the concentration of urea reaches 1 M. Two fractions differing in the Michaelis constant are revealed for silochrome immobilized urease. The rate of urea hydrolysis by native and immobilized urease was studied depending on the pH value in presence of the substrate in the 1 M and 5 mM concentration. The hydrolysis rate of 1 M urea in the buffer-free solution by silochrome-immobilized urease is practically independent of pH within 4.5-6.5. Application of a 2.5 mM phosphate-citrate buffer as a solvent causes an increase in the hydrolysis rate within this pH range. For a soluble urease the 1 M urea hydrolysis rate dependence on pH is ordinary at pH 5.8-6.0. If the substrate concentration is 5 mM, the pH-dependences for the rate of the urea hydrolysis by silochrome- and aerosil-immobilized urease are close and at pH above 6.0 coincide with those for a soluble enzyme. The found differences in the properties of soluble and immobilized ureases are explained by the substrate and reaction products diffusion.     

Polysaccharide derivatives as coats for nylon tube urease

Urease was immobilized on O-alkylated nylon tubes coated with polyaminated derivatives of starch or dextran. The specific activity of the enzyme coil and the relative stability of the immobilized enzyme, compared with immobilized urease derived from other nylon tube modifications, were enhanced. Also, the nonspecific binding of urease to O-alkylated nylon tubes was virtually eliminated by the coating process.     

Immobilization of urease on gelatin — poly (HEMA) copolymer preparation and characterization

Urease was immobilized onto gelatin-poly (HEMA) copolymer by covalent linkage. Maximum amount of urease was immobilized onto the support at a pH of 8.5. The optimal pH of the immobilized urease was similar to that of free urease; the optimal temperature showed an increase of 10 °C over the free enzyme. The stability of the immobilized urease for a range of pH, temperature and shelf life was greater than the corresponding values for the free enzyme. The same result was obtained for k _m also.Grateful acknowledgement is made to CSIR, Govt. of India for the research associateship conferred on Dr. M. Chellapandian which helped the progress of this piece of research investigation.