Immobilized urease

Ayhan, F., Yousefi Rad, A., and Ayhan, H. (2002) Biocompatibility investigation and urea removal from blood by urease-immobilized HEMA incorporated poly(ethyleneglycol dimethacrylate) microbeads. J. Biomed. Mater. Res. B Appl. Biomat. 64B(1), 13-18. 

An unusual type of derivative is the complex that forms between urease and bentonite in acid medium (61). The adsorbed form was found catalytically active. Similarly, urease immobilized in a polyacrylamide gel matrix has been used to prepare a urea-specific enzyme electrode (62). Yet another active water-insoluble derivative has been prepared (63) by allowing p-chloromercuribenzoate-treated urease to react with a diazotized copolymer of p-amino-D,L-Phe and L-Leu. Urease has been found to retain about 20% of its original activity when encapsulated in 100 n microcapsules of benzalkonium-heparin in collodion (64). 

Using the negatively charged nafion film, Rodriguez et al. have improved the sensitivity of urease immobilized reaching a limit of 64 and 55 pgl 1 of mercury and copper, respectively, instead of 2.9 and 29.8 mgl 1 using the alginate gel alone [14],... 

Liang ZP, Feng YQ, Meng SX et al (2005) Preparation and properties of urease immobilized onto glutaraldehyde cross-linked chitosan beads. Chin Chem Lett 16 135-138... 

A monolithic monofunctional enzymatically coupled FET sensitive to urea was first fabricated by Kuriyama and Kimura s group (44). Their integrated FET chip has two hydrogen ion-sensitive FET elements. One FET element has a urease-immobilized membrane, working as an enzyme FET, and the other has a ultraviolet (UV) light-inactivated, urease-immobilized membrane (see Section 3), working as a reference FET. 

An electrode for measuring urea has been described (Gll), consisting of a thin film of urease, immobilized in acrylamide gel, on the surface of a glass electrode responsive to NH. Conditions are carefully selected to ensure stability of the enzyme, and the potential developed is proportional to the logarithm of the urea concentration. Blood glucose and lactate have been determined with a membrane electrode in which the enzyme (glucose oxidase or lactate dehydrogenase) is trapped in a porous or jellied layer at the membrane surface (W20). 

Of course, insulin release is not the unique target in the dehvery field. Indeed, for example, Ishihara [47] matches the problem of urea release using a non-erodible membrane. The system is constituted by a pH-sensitive membrane (copolymerization of 4-carboxyacrylanilide and methacrylate) sandwiched among a membrane containing urease immobilized in free radically cross-linked A, A -methylenebisacrylamide. System permeability with respect to a model dmg ((1,4-bis-(2-hydroxyethoxy)benzene) varies with urea concentration in the external environment. 

The first investigation of the influence of particle mass transfer on the reaction kinetics in a flow microcalorimeter, dealing with properties of urease immobilized on controlled pore glass, was published in 1985 [25]. More recently, the evaluation of microcalorimetric data in the case of particle-diffusion limitation was improved and simplified by introducing the principle of the differential bed [28,29]. 

Example Applications. Previous work has mostly been concerned with testing the theoretical models and obtaining proof of concept (29,39). Antimony pH tips were used to image the activity of urease immobilized in a disk of glutaraldehyde/BSA gel and to quantify the total flux of H+ in the presence of a saturating concentration of urea (29). Wei et al. (39) showed that urease kinetics can also be quantified using an ammonium-selective neutral carrier-based tip to determine the concentration profile of NHj produced by the hydrolytic reaction... 

Urease, immobilized on Eupergit C Origin jack bean Fluka Lab... 

The activity of the Sweetzyme pellets co-immobilized with urease depends on many factors [29]. These factors include the concentration of urea and the pH in the bulk solution and the activity of urease immobilized in the outer layer (Zone 1) of the pellet. These factors influence the production of ammonia and the neutralization of the diffusing hydrogen ions and hence the size of the active XI zone (Zone 2). 

With a gelatin membrane entrapped between two dialysis membranes and containing 46 U/cm of enzyme, the H2O2 formation corresponds to only 110 mU/cm, ie, less than 1% of the initial enzyme activity (Figure 14-19). This indicates a large excess of enzyme in the membrane. Consequently, the membrane is diffusion controlled. The low apparent activity may be attributed mainly to the diffusion resistance of the dialysis membrane for glucose. On the other hand, the measured activity of the membrane containing 46 U/cm is already about 70% of the activity used for immobilization. This value approaches that expected for pure kinetic control of the process [287], The apparent activity of urease immobilized in a cellulose triacetate membrane was found to be 66% of the initially applied enzyme activity [288]. 

Ayhan, F. et al., Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters, Biores. Technol., 81, 131, 2002. 

Mullerschulte, D. and Daschek, W., Application of radiation grafted media for lectin affinity separation and urease immobilization—A novel-approach to tumor-therapy and renal-disease diagnosis, Radiat. Phys. Chem., 46, 1043, 1995. 

Figure 13.17 Cyclic voltammograms for the electropolymerization of urease entrapped PPy onto carbon paper obtained from different scanning rates and the detected potential changes of 1.0 mM urea concentration from the urease immobilized. Permission awaited...

Fig. 19 Effect of reaction time and number of cycles on the enzymatic activity of urease immobilized in PNIPAAm cryogel and PNIPAAm hydrogel, incubated in 10 mL urea solution (3.2 g/L) at 50 °C. Reprinted from [34] with permission from John WUey Sons...

Guilbault and Montalvo were the first, in 1969, to detail a potentiometric enzyme electrode. They described a urea biosensor based on urease immobilized at an ammonium-selective liquid membrane electrode. Since then, over hundreds of different applications have appeared in the literature, due to the significant development of ion-selective electrodes (ISEs) observed during the last 30 years. The electrodes used to assemble a potentiometric biosensor include glass electrodes for the measurement of pH or monovalent ions, ISEs sensitive to anions or cations, gas electrodes such as the CO2 or the NH3 probes, and metal electrodes able to detect redox species some of these electrodes useful in the construction of potentiometric enzyme electrodes are listed in Table 1. 

Fig. 15. Lineweaver - Burk plots for urease immobilized on ClCMC (a) and for ADH coupled on 4-aminobenzoylcellulose (b).

Krajewska, B., M. Leszko, and W. Zaborska. 1990. Urease Immobilized on Chitosan Membrane Preperation and Properties. Journal of Chemical Technology and Biotechnology 48 337-350. 

The amount of active urease immobilized into the polyelectrolyte microcapsules was determined under assumption that the enzyme retains its activity while encapsulated. Free urease had 100 U/mg according to the data sheets. The activities of free and encapsulated enzyme were determined from the decomposition of urea into two ammonia molecules and CO using a pH-sensitive d e Bromocresol purple [24], The urease aliquot solutions were added to a reaction mixture contained a necessary amount of urea and 0.015 mM Bromocresol, whose pH was aprioiy brought up to 6.2. The reaction kinetics was recorded as a change in the optical absorption of the dye at 588 run to obtain the linear calibration plot. Then, the known number of microcapsules containing urease and SNARF-1 dextran was added to the reaction solution. The revealed activity of enzyme was compared with amount of free urease. 

YAM 06] Yamada K., Iizawa Y., Yamada J.-I. et al., Retention of activity of urease immobilized on grafted polymer films , Journal of Applied Polymer Science, vol. 102, pp. 4886 896, 2006. 

In the case of urease immobilized gel, the addition of urea, a substrate of... 

A urea sensor has conventionally consisted of a urease-immobilized membrane and electrode for the determination of the metabolites electrodes for ammonium ion (20), pH (27), ammonia gas (22), and carbon dioxide (23) have been used. These sensors were all potentiometric ones to give Nernstian responses, the linear concentration range of which was typically between 10" and 10 M of urea. If one wishes to detect urea with using the ion- or gas-selective electrodes, urease is indispensable not only for the specific recognition based on the bioaffinity interaction but also for chemical conversion of urea to detectable species, since urea is essentially electro-inactive. On the contrary, the polymer-modified electrode described here does not rely upon enzymes the concentration of urea can be determined directly. 

First potentiometric biosensor Urease immobilized on an ammonia electrode to detect urea... 

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