Analytical Applications of Immobilized Enzymes

Techniques such as potentiometry, polarography, and microcalorimetry have been chosen in exploiting the benefits of immobilized enzymes (see Chapter 4). Enzymes incorporated into membranes form part of enzyme electrodes. The surface of an ion-sensitive electrode is coated with a layer of porous gel in which an enzyme has been polymerized. When the electrode is immersed in a solution of the appropriate substrate, the action of the enzyme produces ions to which the electrode is sensitive. For example, an oxygen electrode coated with a layer containing glucose oxidase can be used to determine glucose by the amount of oxygen consumed m the reaction, and urea can be estimated by the combination of a selective ammonium ion-sensitive electrode and a urease membrane. 

A number of analytical techniques have been used to measure isoenzymes or isoforms. They include electrophoresis (see Chapter 5), chromatography (see Chapter 6), chemical inactivation, and differences in catalytic properties, but the most common routine methods are now based on immunochemical methods. 

Isoelectric focusing (see Chapter 5), has been used successfully to separate isoenzymes that differ in the amount of covalently bound sugar residues, such as sialic acid. 

Ion-exchange chromatography makes use of differences in net molecular charge at a given pH to separate isoenzymes. A typical ion-exchange material is DEAE cellulose, in which ionizable DEAE groups are attached to an inert cellulose 

Other forms of chromatography that have been appfied to firactionation of isoenzyme mixtures include high-performance liquid chromatography (HPLC) and affinity chromatography. The latter makes use of differences between isoenzymes in their affinities for a specific ligand that is attached to an inert insoluble support used as the stationary phase in a chromatography column or in a batch technique. 

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L Gorton, G Marko-Varga. In S Lam, G Malikin, eds. Analytical Applications of Immobilized Enzyme Reactors. London Blackie Academic Professional, Chapman Hall, 1994, pp 1-21. 

Wingard LB, Katchalski E, Goldstein L (eds) (1981) Analytical applications of immobilized enzymes and cells applied biochemistry and bioengineering, vol 3. Academic, New York... 

L. B. Wingard, Jr., E. Katchalski-Katzir, and L. Goldstein, Eds., Applied Biochemistry and Bioengineering, Volume 3 Analytical Applications of Immobilized Enzymes and Cells, Academic Press, New York, 1981. 

Lam, S., Mallikin, G., Eds. Analytical Applications of Immobilized Enzyme Reactors Blackie Academic and Professional Glasgow, U.K., 1994. 

Whereas in traditional enzymatic analysis spectrophotometric methods dominate, test strips and biospecific electrodes are at the leading edge in the analytical application of immobilized enzymes. This may be expected to continue at least until the mid-90s. 

Analytical applications of immobilized enzyme sequences in conjunction... 

Immobilized enzymes and whole cells have found well-documented applications in industry, medicine, and analytical chemistry. Theoretically, it should be possible to carry out any enzymatic reaction with the help of the respective immobilized enzyme or whole cell containing the enzyme. The technique of using an immobilized enzyme for a chemical transformation is not basically different from using the soluble enzymes. In commercial applications, the immobilized enzymes can be used in a continuous-flow reactor. However, the optimum conditions for a specific reaction will have to be redetermined before maximum turn-over can be achieved. Thus, proteolytic enzymes such as trypsin, when immobilized on an anionic matrix such as cofpolyethylene-maleic anhydride), require a much lower pH for reaction than in solution. Some typical applications of immobilized enzymes that are currently being made, or are in the process of development, are mentioned in Table 15-1. 

In addition to the analytical applications, there was sporadic work on the employment of flow calorimetry for the investigation of enzyme kinetics [23,24]. In 1985 Owusu et al. [25] published the first report on the use of flow microcalorimetry for the study of immobilized enzyme kinetics approaching... 

Analytical usefulness of immobilized bioluminescent assays depends on properties of their immobilized enzymes. The most popular application of immobilized bioluminescent systems is for analysis and monitoring of chemical and biochemical analytes and environmental pollutants. The wide range of analytes measured and monitored by immobilized bioluminescent systems has been reviewed. Stability, sensitivity, precision, and effects of interfering substances and the microenvironment are also discussed. 

Immobilized enzymes are defined as biocatalysts that are restrained or localized in a microenvironment yet retain their catalytic properties. Immobilization often increases stability and makes the reuse of the enzyme preparation very simple. The repeated analysis that can be performed by the use of enzymes in an immobilized form reduces the cost of the analysis. The ideal immobilization procedure for a given enzyme is one which permits a high turnover rate of the enzyme yet also retains a high catalytic activity over time. There are two major applications for immobilized enzymes in analytical systems. In one, the enzyme is immobilized onto a particulate solid support matrix, which is then packed into a small column and incorporated into a flow system. The other involves immobilization within or on the surface of an electrode, whereby the electrochemical transduction of enzymatic product is monitored. 

This technique of immobilized enzymes combines the unique features of both forms of catalysis the specificity of the enzymes with the stability and ease of handling and storing of supported heterogeneous catalysts. Furthermore, immobilized enzymes can be reused and are applicable to flow systems. Consequently, they find increasing applications in several analytical areas and in medicine. 

After application of the enzyme a membrane (size exclusion dialysis membrane) is put over the electrode surface and prevents the diffusion of the protein and/or other components into the test solution. This way seems the simplest method for immobilization of enzymes and has been frequently employed.Nevertheless, any membrane at the surface hinders the mass transport of the analyte to some extent resulting in decreased signals and longer response times. 

We have recently begun studies geared at the analytical application of enzymes immobilized on microelectrodes. We had previously demonstrated that 4-amino phenyl phosphate could be employed as a substrate for the determination of the enzymatic activity of alkaline phosphatase by following the oxidation of 4-amino-phenol which is the product of the enzymatic reaction [28]. In the present case, the interest was in determining whetiier such an approach could be transposed to a microelectrode and to determine if the immobilization process gave rise to an enzymatically active interface. 

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