Enzymes analytical applications

The study of anion coordination has implications for a number of areas in chemistry and biochemistry. These include analytical applications concerned with anion sensing or separation as well as model studies for anion-specific biochemical systems. With respect to the latter, it is of interest that the majority of enzymic systems so far characterized bind substrates which are anionic. 

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. 

The most popular system in mechanistic and model studies as well as in analytical applications (clinical, food, environmental) appears to be that of firefly luciferin and luciferin-type-related model luminescence [3, 5,23, 57], The luciferase from Photinus pyralis, Photinus luciferin 4-monooxygenase (ATP-hydrolyzing), EC 1.13, 12.7, is a hydrophobic enzyme that catalyzes the air oxidation of luciferin in the presence of ATP and magnesium ions to yield light emission ... 

Cypridina luciferin analogs are widely used for several analytical applications (determination of substrates, enzymes, active oxygen species such as superoxide), but they are mainly related to CL [241, 242],... 

In this chapter we report recent analytical applications of CL imaging for the detection of biospecific reactions in macrosamples such as microtiter plates of different format (96 or 384 wells), filter membranes and irregular surfaces represented by specimens related to the cultural heritage, and results obtained when the CCD detector is coupled with optical microscopy for enzyme localization, immunohistochemical reactions, and complementary DNA (cDNA) detection (Table 1). 

Dendrimers are being used as host molecules, catalysts, self-assembling nanostructures analogs of proteins, enzymes, and viruses and in analytical applications including in ion-exchange displacement chromatography and electrokinetic chromatography. 

Enzyme-sensitive supramolecular polymers also hold promise in analytical applications such as the screening of enzyme inhibitors. A simple visual assay based on the hydrogelation of small molecules has been developed for screening the activities of inhibitors of enzymes like acid phosphatase. A number of inhibitors for... 

Enzymes as analytical reagents. Although the technological applications are quantitatively the more important, many of the analytical applications of enzymes are unique and are becoming increasingly important. 

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

The applications of enzymes can be classified into three major categories industrial enzymes, analytical enzymes, and medical enzymes. In this chapter, we review several industrial processes, utilizing industrial enzymes such as starch conversion and enzymatic hydrolysis of celluloses. Before we discuss the enzymatic hydrolysis of starch and cellulose, we review the organic chemistry of carbohydrates. 

Biosensors based on enzyme inhibition are still limited in analytical applications since these sensor technologies are not usually able to discriminate various toxic compounds in the same sample. 

In this paper we report the electrochemical polymerization of the PPy-GOD film on the glassy carbon (GC) electrode in enzyme solution without other supporting electrolytes and the electrochemical behavior of the synthesized PPy-GOD film electrode. Because the GOD enzyme molecules were doped into the polymer, the film electrode showed a different cyclic voltammetric behavior from that of a polypyrrole film doped with small anions. The film electrode has a good catalytic behavior to glucose, which is dependent on the film thickness and pH. The interesting result observed is that the thin PPy-GOD film electrode shows selectivity to some hydrophilic pharmaceutical drugs which may result in a new analytical application of the enzyme electrode. 

Oxidoreductases comprise a large class of enzymes that catalyze biological oxidation/reduction reactions. Because so many chemical transformation processes involve oxidation/reduction processes, the idea of developing practical applications of oxidoreductase enzymes has been a very attractive, but quite elusive, goal for many years [83], Applications have been sought for the production of pharmaceuticals, synthesis and modification of polymers, and the development of biosensors for a variety of clinical and analytical applications [83], In recent years, the use of oxido-reductive enzymes to catalyze the removal of aromatic compounds from... 

Finally, this chapter discusses the use of HPLC itself as an aid in the purification of an enzyme activity. Applications are not restricted to the final stages of a purification. Its use of small sample volumes, its sensitivity, and its speed of separation make HPLC an ideal analytical tool to monitor the efficiency of other steps and procedures that are used during a purification. 

These considerations are important in the context of analytical applications since they dictate the overall operational half-life of the immobilized system. In fact, an immobilized enzyme is particularly vulnerable to deactivation because it is unlikely to be used for monitoring a pure substance. Exposure to a physiological fluid or fermentor medium exposes the catalyst to inhibitors or extraneous matter. For example, the half-life of the immobilized lactase decreased from 89 days to 7 days when the substrate changed from 5% lactose solution to acid whey (40). 

Thus far, few analytical applications of enzymes in a nonaqueous solvent are available. One example is the use of oxidases to oxidize hydrophobic substrates. 

Recent reviews have provided systematic coverage of the enzymatic microreactors used in chemical analysis [4]. Considering that the focus of this chapter is biocatalytic synthesis, it does not consider the analytical applications and the reader is referred to the cited literature ([4] and references given therein). The use of microreactors for high-throughput kinetic characterization of enzymes is another very interesting application of the technology [8], which, for reasons of limited space, is not discussed herein. 

Nature has required the evolution of usefully selective hosts, and proteins (in the forms of enzymes, receptors, and antibodies) provide them. However, no individual protein molecule lasts in a cell for very long. All proteins are constantly anabolized and catabolized, with constant concentrations achieved via homeostasis. Nature never demanded permanence of its molecular recognition machinery. When we utilize biotic receptors for one-time, batch analytical applications, the receptors clearly meet the useful criterion. However, if a receptor must have an extended lifetime in a sensing device, then we propose that biotic receptors represent the easiest place in which to search instead of the right place to search. If a biotic receptor cannot reasonably be made stable enough to survive weeks of service, then it will not be useful for a sensing application no matter how avid or selective. 

The enzymes used for this type of digestion in Analytical Chemistry are mainly hydrolytic enzymes, the catalytic effect of which is based on the insertion of water at a specific bond of the substrate. The hydrolytic enzymes used in analytical applications include lipases (which hydrolyse fats into long-chain fatty acids and glycerol) amylases (which hydrolyse starch and glycogen to maltose and to residual polysaccharides) and proteases (which attack the peptide bonds of proteins and peptides themselves). 

Chemiluminescence. Chemiluminescence (262—265) is the emission of light during an exothermic chemical reaction, generaUy as fluorescence. It often occurs in oxidation processes, and enzyme-mediated bioluminescence has important analytical applications (241,262). Chemiluminescence analysis is highly specific and can reach ppb detection limits with relatively simple instrumentation. Nitric oxide has been so analyzed from reaction with ozone (266—268), and ozone can be detected by the emission at 585 nm from reaction with ethylene. 

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