Urease characteristics

A.P. Soldatkin, V. Volotovsky, A.V. Elskaya, N. Jaffrezic-Renault, and C. Martelet, Improvement of urease based biosensor characteristics using additional layers of charged polymers. Anal. Chim. Acta 403,... 

The gas-sensing electrodes also are used for the potentiometric measurement of biologically important species. An enzyme is immobilized at or near the gas probe. The gas sensor measures the amount of characteristic gas produced by the reaction of the analyzed substance with the enzyme. For example, an enzyme electrode for urea [NH2C(0)NH2] determination is constructed by the immobilization of urease onto the surface of an ammonia-selective electrode. When the electrode is inserted into a solution that contains urea, the enzyme catalyzes its conversion to ammonia ... 

Life as we know it would be impossible without the astonishing characteristics of enzymic catalysis. This catalysis is not only highly efficient, so that reactions may proceed at low temperature and at neutral pH with the speed required by living cells, but it exhibits also a remarkable specificity. Let us cite two typical examples First, the enzyme urease catalyzes the hydrolysis of urea but of no other compound (1). Second, the catalytic action is frequently restricted to one of the antipodes of optically active substrates. Thus, chymotrypsin will catalyze the hydrolysis of acylated L-tyrosinamides, but will not catalyze the reaction of the corresponding derivatives of D-tyrosine (2). 

Due to their limited stability, the use of immobilized enzymes might be problematic for monitoring enzyme production (e. g., urease for arginase monitoring). Optical methods which do not need immobilized biocompounds should be more profitable in long-term procedures. Nevertheless, special dyes or substrates with varying optical characteristics after an enzymatic conversion must have at one s disposal. 

The best-known enzyme electrode is that used to analyze for urea in blood. The enzyme urease is immobilized in a polyacrylamide hydrophilic gel and fixed at the bottom of a glass electrode whose characteristics make it an NH4 ISE. Alternatively, the ISE can be a composite system designed to detect NH3. In the presence of the enzyme, urea is hydrolyzed according to the reaction... 

Table 1. General characteristics of representative urease, hydrogenase, and CODHs...

Table 1 summarizes the general characteristics of representative urease, hydrogenase and CODHs. As it will be further discussed below, the X-ray structures of only two Ni-containing proteins, urease and hydrogenase, are known [16, 17]. The former has the well known triose phosphate isomerase (TIM) barrel topology (Fig. 1) whereas the latter displays a so far unique folding (Fig. 2). The next challenge will be the elucidation of the crystal structures of the CODH/ACS enzyme of Clostridium thermoaceticum and of the simpler CODH from Rhodospirillum rubrum. 

Entrapment in polyacrylamide gel Active immobilized enzyme study of the characteristics and overall reaction rate of unbuffered gel-immobilized urease particles 817... 

Table 19.1 Characteristic quantities of the enzymes urease and catalase (from Voet D, Voet JG, Pratt CW (2002) Lehrbuch der Biochemie, 2nd edn. Wiley VCH, Weinheim).

The quotient k JK represents the apparent rate coefficient of a second-order reaction whose rate density is determined by the frequency of (effective) collisions between enzyme and substrate molecules. For the same concentrations of enzyme and substrate, respectively, the catalytic efficiency can be described by the quotient koJKM- With the help of the values of k /K, we can investigate which among several substrates the enzyme prefers is therefore a measure of the substrate specificity of an enzyme where high values indicate high specificity. Typical values lie between 10 and 10 mol L s . Table 19.1 is a compilation of characteristic quantities of the enzymes urease and catalase. 

Enzyme specificity is a second characteristic that is important in life processes. Unlike other catalysts, enzymes are often quite specific in the type of reaction they catalyze and even the particular substance that will be involved in the reaction. For example, strong acids catalyze the hydrolysis of any amide, the dehydration of any alcohol, and a variety of other processes. However, the enzyme urease catalyzes only the hydrolysis of a single amide, urea (Reaction 10.2 and I Figure 10.3) ... 

Figure 2. Urease genes amplified from seven unique, pure bacterial cultures isolated from Snake River Plain Aquifer groundwater. Lanes A through G show the characteristic PCR product (ca. 400 base pair length) amplified from these...

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. 

Because of a build-up of product in the enzyme membrane, enzyme electrodes require washing prior to contact with the next sample. The washing time varies from just 20 s for urease in conjvmction with an ammonia electrode to as long as 10 min for urease with a pH electrode. The washing time increases with enzyme membrane thickness, as also observed for additional membranes discussed above. It will also depend on the enzyme used and on the characteristic of the base sensor itself, and will be affected by diffusion and kinetic effects. The use of flow injection analysis simplifies the procedures, since the carrier stream serves to wash out between samples. 

Tamura M, Sugihara M, Uragami T (1979), Ultrafiltration, hydrolysis, and adsorption characteristics of membranes from cellulose nitrate, urease stylite, activated charcoal , Augeiv Makromol Chem, 79,67-77. 

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