Encapsulated urease

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

Lvov et al. studied the influence of solvent ethanolby encapsulating urease using poly(styrenesulfonate, sodium salt) and poly(allylamine hydrochloride sodium salt) in 1 1 ethanol water mixture. In the presence of ethanol, urease activity was lower. Ethanol causes segregation of polyion network due to partial removal of the hydration water between the polyelectrolytes (Figure 56.12). ° ... 

FIGURE 56.12 Schematic (top row) and CLSM images illustrating permeation and encapsulation of urease-fluorescein isothiocyanate (FITC) into polyion multilayer capsules. Left, in water middle, in water/ethanol mixture 1 1 right, the capsule with encapsulated urease again in the water. (Reprinted with permission from Nano Lett., 1(3), Lvov, Y., Antipov, A., Mamedov, A., Mohwald, H., and Sukhorukov, G.B., Urease encapsulation in nanoorganized microshells, 125. Copyright 2001 American Chemical Society.)... 

We also investigated the influence of proteolytic. Figure 12.6 shows the dependences of the activity of free (curve 1 ) and encapsulated (curve 2 ) urease on the time of incubation with proteinase K at 37 °C. For comparison, the dependences obtained in the absence of proteinase K (curves 1, 2) are also shown in Fig. 12.6. Urease was encapsulated into the (PSS/PAA) 3 PSS shell. As can be seen in Fig. 12.7, the activity of free urease in the presence of the proteolytic enzyme steeply decreases to zero, whereas encapsulated urease retains the ability to decompose urea in the presence of proteinase. Since the enzymes in PEMC are not degraded by proteinase K, these microcapsules can be used for quantitative analysis of low-molecular... 

Abstract. The urea-urease system is a pH dependent enzymatic reaction that was proposed as a convenient model to study pH oscillations in vitro here, in order to determine the best conditions for oscillations, a two-variable model is used in which acid and substrate, urea, are supplied at rates kh and ks from an external medium to an enzyme-containing compartment. Oscillations were observed between pH 4 and 8. Thus the reaction appears a good candidate for the observation of oscillations in experiments, providing the necessary condition that kh > ks is met. In order to match these conditions, we devised an experimental system where we can ensure the fast transport of acid to the encapsulated urease, compared to that of urea. In particular, by means of the droplet transfer method, we encapsulate the enzyme, together with a suitable pH indicator, in a l-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid membrane, where differential diffusion of H+ and urea is ensured by the different permeability (Pm) of membranes to the two species. Here we present preliminary tests for the stability of the enzymatic reaction in the presence of lipids and also the successful encapsulation of the enzyme into lipid vesicles. 

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). 

Lvov Y, Antipov AA, Mamedov A et al (2001) Urease encapsulation in nanoorganized microshells. Nano Lett 1 125-128... 

Y. Lvov, A.A. Antipov, A. Mamedov, H. Mbhwald, G.B. Sukhorukov, Urease Encapsulation in Nanoorganized MicrosheUs , Nano Lett., 1, 125 (2001)... 

Yu AM, Gentle 1, Lu GQ, et td. (2006) Nanoassembly of biocompatible microcapsules for urease encapsulation and their use as biomimetic reactors. Chem Common 2150-2152... 

Lvov, Y, Antipov, A., Mamedov, A., Mohwald, H., Sukhorukov, G.B. Urease encapsulation in nanoor-ganized microshells. Nano Lett. 2001,1 (3), 125—128. 

Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARE-1 into polyelectrolyte multilayer capsules. The co-precipitation of calcium caibonate, urease, and dextran followed up by multilayer film coating and Ca- extracting by EDTA resulted in formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10 to 10 M. Sensitivity to urea in concentration range of 10 to 10 M was monitored on capsule assemblies (suspension) and on single capsule measurements. Urea presence can be monitored on single capsule level as illustrated by confocal fluorescent microscopy. 

In order to verify a feasibility of fluorescence based nrea sensing on two component co-encapsulation we fabricated two polyelectrolyte capsnle samples of the (PSS/ PAHj PSS shell architecture with different content of dye and mease. The first sample Sample I) contained in average 0.6 pg SNARF-1 dextran per capsule, while the content of the SNARF-1 dextran in the other sample was 0.2 pg per capsifle Sample II). The concentration of active urease in samples was opposite 0.2 and 0.6 pg/capsule respectively what gives an average SNARF-1 dextran/urease ratio of 3 1 and 1 3 in these investigated samples. 

Spectrofluoremetric studies were carried out to determine the correlation between the fluorescence intensity of the SNARF-1 dextran/urease capsules and the pH of the medium. Both the capsule samples were stored for 10 min in the 0.05 M TRIS-maleate buffer at pH in the range 5.5-9. The excitation wavelength was 540 nm. The capsules fluorescence spectra of the first sample are shown in Figure 2. The spectra obtained for both samples were similar. The encapsulated dye is capable to provide information of the medium acidity in a reasonably wide range of pH. It is seen that fluorescence... 

In this study we have demonstrated a particular example of a sensor system, which combines catalytic activity for the substrate (urea) and at the same time enabling to monitor the enzymatic reaction by co-encapsulated pH sensitive dye. Substrate sensitive enzyme urease was co-encapsulated together with SNARF-1 coupled to dextran in multilayer tnicrocapsules. Enzymatic activity was recorded by fluorescent changes caused by increasing of pH in course of enzymatic cleavage of urea as measured on... 

In order to determine the catalytic characteristics of the encapsulated enzymes, we obtained the dependences of the stationary rate of substrate conversion on the substrate concentration. As an example, the curves of saturation of urease with urea in the reaction of urea decomposition are depicted in Figure 5. It can be seen that the dependences for urease in microcapsules, are generally similar to those for free enzyme, except small differences in the affinity constants. In particular, the Michaelis constant AM with respect to urea is 7.1 D2.2 mM for urease in microcapsules of eleven and seven layers, whereas the AM for free urease is 2.5 DO. mM. The maximal rate Umax for urease in microcapsules of eleven layers is 20% lower than that for urease in microcapsules of seven layers. The AM with respect to pyruvate for microcapsules containing LDH was not different from for free enzyme. 

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