Extended urease

In extended X-ray absorption fine structure (EXAFS) studies of urease, Hasnain, Piggott, and co-workers (33, 34) demonstrated that spectra were similar to those of benzimidazole complexes, consistent... 

Horseradish peroxidase (and urease) played an important role in the development of the modern concept of the nature of an enzyme and the role of metal ions (Sumner and Somers, 1943 Willstatter, 1965). The species now known as compound II (HRP-II) formed as a result of the reaction of HRP with H202, was discovered in 1937 (Keilen and Mann, 1937). Later compound I (HRP-I), formed prior to HRP-II was identified (Theorell, 1941). The spectra of HRP-I and HRP-II in the 400 nm (Soret band) region have been determined (Chance, 1949 a, b) and measurements have also been extended to the visible region (Chance, 1952). Formation of HRP-I is first order in H202 and HRP (Chance, 1943) and the -OOH group is essential for the oxidation of HRP by peroxide. The enzymatic cycle can be summarised by the following equations (George, 1952),... 

Activity assays of enzymes bound to solid phases in EIA systems have previously been limited to fixed-time spectrophotometric methods following incubation of substrate and solid phase for extended periods of time. Kinetic assays of enzyme activity have not been used to date because of the difficulty in directly monitoring initial rates of enzyme reactions in a turbid solid phase suspension. With urease as the label, an ammonia gas sensing electrode can be used to directly quantitate the amount of urease-labeled antigen or hapten bound to a double-antibody solid phase by continuously measuring the initial rate of ammonia produced from urea as a substrate. 

The rate portion of curve, expressed in millivolts per minute, can be calculated by graphically extending the straight line portion of the curve. This rate can then be plotted vs urease concentration, and a curve, shown in Fig. 3, is obtained. At lower enzyme concentration the correlation is linear, and at higher levels nonlinearity occurs owing to the time response... 

In vitro studies using slurries of cattle manure and urine indicated that addition of the urease inhibitor N-(ft-butyl) thiophosphoric triamide (NBPT) can extend the survival of coliform bacteria in the manure (Varel et ah, 2007a). However, coliform bacteria were rapidly eliminated when the plant oil thymol was used in combination with NBPT in the manure slurries (Varel et ah, 2007a). Similarly, the application of NBPT with the plant oil extracts of linalool and pine oil on surfaces of feedlot pens... 

Plots of phase angle difference in the interferometer arms vs. time are related to heat-production vs. time, and this in turn is related to the concentration of the species responsible for heat production. Typical instrument output for the urea/urease system is shown in Figure 3. Calibration curves can be constructed as shown in Figure The system is quite stable, and reasonably sensitive. Minimum detectable levels of urea are 5 mM, compared to the 0.1-5 mM limits for traditional detectors. Over extended time periods (7 days) the relative standard deviation at 5 mM concentrations is better than 5 /.. The optimum FIA conditions were around 1.0 ml/min flow rate, with a sample loop of 0.1-0.25 ml. 

For this technique to be of more general use it must also be applicable to other enzymes. We have began by extending the procedure to urease, an enzyme whose entrapment in polypyrrole, as well as use in a conductometric biosensor for urea has already been reported (35, 36). We have found that urease was readily electrodeposited and that the activity of the deposited enzyme was easily seen by admittance measurements (Figure. 4). The apparent Km for the immobilis enzyme was calculated fi om the double-reciprical plot (y = 0.17 + O.SOx, regression coefficient = 0.9994, n = 9) to be 2.9 mM, which can be compared to the range 3-5.1 mM for the soluble enzyme (36). A control electrode produced at the same time displayed negligible response to urea. 

The above results demonstrate the use of electrochemical and photolithographic methods for the spatially controlled deposition of enzymatic and anti-interference layers in a controlled way upon the surface of miniature electrodes. Although the results concentrate upon ucose measurement, the example of urease indicates that the electrochemical method should be extendible to other enzymes and we are currently investigating this. We beUeve the measurements made using the electrodes with the thin anti-interference layer are particularly interesting and show that the modified electrodes can be used in complex matrixes such as complex yeast extract medium. 

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