Enzyme retaining

Subsequent to a further washing step, to remove any unbound antibody-enzyme conjugate, the activity of the enzyme retained is quantified by a straightforward enzyme assay. The activity recorded is proportional to the quantity of antigen present in the sample assayed. A series of standard antigen concentrations may be assayed to allow construction of a standard curve. The standard curve facilitates calculation of antigen quantities present in unknown samples. 

The most common of these systems is the enzyme-multiplied immunoassay technique or EMIT, which is particularly suited to the measurement of small molecules (haptens) such as drugs. EMIT is a trade mark of the Syva Corporation of Palo Alto, California. Although it does not involve the separation of bound fraction from free it is nevertheless a competitive assay system. The antigen is labelled with an enzyme in such a way that the enzyme retains its catalytic activity. When the antigen binds to the antibody the enzyme becomes inhibited, probably by an induced conformational change or by steric hindrance of the enzyme active site (Figure 7.15). 

Abu-Soud and coworkers110 were able to remove heme and BH4 from NOS by dialysis with 2 M urea. The remaining enzyme retained the ability to reduce ferricyanide and cytochrome c. The reduction of cytochrome c was about 9 x in the presence of calmodulin. The two sites of calmodulin for influencing the reaction are shown in Figure 14. 

Clearly, it would not be advantageous for synthesis and degradation to occur simultaneously, especially as they occur in the same cell compartment, the cytosol. Reciprocal control, that is when one enzyme is On the other is Off, is achieved via reversible phosphorylation of the key controlling enzyme and allosteric control (see Table 6.2 and Section 3.2.2). However, it is probable that both enzymes retain some residual activity at most times. 

This assumption has since been swept aside and it is now recognized that a broad range of enzymes retain their activity on exposure to organic solvents or organic solvent-water mixtures. The addition of organic solvent allows the coupling of the exquisite selectivities observed from traditional approaches with numerous other advantages, such as ... 

DeBoer E, Plat H, Wever R (1987) Algal vanadium(V)-bromoperoxidase. A halogenating enzyme retaining full activity in apolar solvent systems. In Laane C, Tramper J, Lilly MD (eds) Biocatalysis in organic media. Elsevier, Amsterdam, p 317... 

It has often been found that enzymes retain their activity when suspended in ionic liquids of low hydrogen bond basicity, but they are inactive in those with high hydrogen bond basicity (e.g., Cl , NOi", and acetate-containing ionic liquids) 274). Some recent biocatalytic reactions are discussed below, with an emphasis on the function of the ionic liquids in enzyme catalysis. 

Martinek et al. (1981) also studied stabilization in systems of low water content. Several enzymes have been microencapsulated into reversed micelles formed by surfactants in apolar organic solvents (see Chapter 9). The enzymes retained their catalytic activity and substrate specificity. 

Proteins and enzymes have been successfully entrapped in surfactant-solubilized water pools in organic solvents [268-278]. Furthermore, many reversed-micelle-entrapped enzymes retained their activity and could be used for peptide synthesis [273,274]. That the water pools corresponding to very small w-values exhibited freezing points Mow — 50°C enabled both the enzyme structures and the rates of enzyme-catalyzed reactions to be investigated at low temperatures. These studies much aided the development of cryoenzymology [279, 180],... 

Acid phosphomonoesterase (EC 3.1.3.2). Milk contains an acid phosphatase which has a pH optimum at 4.0 and is very heat stable (LTLT pasteurization causes only 10-20% inactivation and 30 min at 88°C is required for full inactivation). Denaturation of acid phosphatase under UHT conditions follows first-order kinetics. When heated in milk at pH 6.7, the enzyme retains significant activity following HTST pasteurization but does not survive in-bottle sterilization or UHT treatment. The enzyme is not activated by Mg2+ (as is alkaline phosphatase), but it is slightly activated by Mn2+ and is very effectively inhibited by fluoride. The level of acid phosphatase activity in milk is only about 2% that of alkaline phosphatase activity reaches a sharp maximum 5-6 days post-partum, then decreases and remains at a low level to the end of lactation. 

Most commonly used enzyme names have the suffix "-ase" attached to the substrate of the reaction (for example, glucosi-dase, urease, sucrase), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.]... 

Fig. 1. Optical absorption spectra of urease from jack bean (Canavalia ensiformis). (A) Enzyme, 43.3 mg/ml in 1 mM /i-mercaptoethanol/1 mM EDTA (B) with 10 mM acetohydroxamic acid (C) urease, 11.9 mg/ml after acidification, pH 3.8 (C ) after 2 hours at pH 3.8 the latter enzyme retained 6.1% of its original activity. Redrawn, with permission, from Ref. I.

In spite of his heavy professional occupations, Courtois established a very successful career as a researcher. This activity began in 1931 he wrote a university thesis on the adsorption of sugars by metallic hydroxides in 1932. He obtained the Doctorat es Sciences d Etat in 1938, with a thesis devoted to a kinetic study of some plant phosphatases. These enzymes retained his attention for some years, but the carbohydrates, from the chemical as well as the enzymic point of view, quickly became the favorite research topic of Professor Courtois. 

Just one example will be given here.195 Evaporation of water from aqueous solutions of MEEP and the enzyme urease yields films that can be cross-linked by exposure to gamma rays. The cross-linked films absorb water to form hydrogels in which the enzyme molecules are trapped within the interstices of the gel network. Some of the enzyme molecules may also be covalently grafted to the polymer side groups. The immobilized enzyme retained approximately 80% of its activity for the conversion of urea to ammonia. This system can, in principle, be used for the immobilization of a wide variety of enzymes, and for their use in biochemical flow reactors, or in sensors. 

Enzymes need some water to remain catalytically active, but this amount of water is small. Randolph et al. (1988) investigated the use of cholesterol oxidase from Cleocysticum chrysocreas by placing the immobilized enzyme in a packed bed and exposing it to bone-dry carbon dioxide. While the enzyme quickly lost activity, it was rapidly restored with only 1% v/v water in the system. Water acts not only to help the enzyme retain its native conformation, but also it can act as a solubility modifier. Organic solvents and other nonaqueous environments can remove the enzyme s essential water (Zaks and Klibanov 1988). 

The investigation of a PDC1-PDC6 fusion enzyme, retaining only Cys 221... 

The CuZn-SOD activity is completely inhibited by cyanide, and also partly by azide. The Cu2+ and Zn2+ ions can be substituted by other metal ions such as Co2+, Ni2+ etc., but the activity is found only when those substituted enzymes retain Cu2+ in the native site (Table 10.1). 

Enzymes are immobilized by attachment to or confinement in water-insoluble materials (Fig. 1). Enzymes can be immobilized by adsorption on biologically inert carriers like organic polymers, glass, mineral salts, metal oxides, and different silicates. Since enzymes retain their activity for a longer time in an undissolved form, many reactions catalyzed by enzymes can be carried out in continuous systems. Immobilized enzymes can be used in agitated vessels, fluidized or Fixed bed tower reactors40). 

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