Enzyme using covalent technique

In summary, enzyme immobilization is extremely important in the scale-up of many biocatalytic processes. The preferred method for pharmaceutical production involves covalent binding through cross-linking or attachment to a support. Noncovalent attachment is less attractive, but it is heavily utihzed owing to the commercial availabihty of industrial quantities of some enzymes immobilized using this technique. 

A difficult problem in utilizing enzymes as catalysts for reactions in a non-cellular environment is their instability. Most enzymes readily denature and become inactive on heating, exposure to air, or in organic solvents. An expensive catalyst that can be used only for one batch is not likely to be economical in an industrial process. Ideally, a catalyst, be it an enzyme or other, should be easily separable from the reaction mixtures and indefinitely reusable. A promising approach to the separation problem is to use the technique of enzyme immobilization. This means that the enzyme is modified by making it insoluble in the reaction medium. If the enzyme is insoluble and still able to manifest its catalytic activity, it can be separated from the reaction medium with minimum loss and reused. Immobilization can be achieved by linking the enzyme covalently to a polymer matrix in the same general manner as is used in solid-phase peptide synthesis (Section 25-7D). 

The support obtained by the sol-gel technique was used to immobilize commercial CRL following three procedures. In the first, the lipase was immobilized on PS by ADS in the second, the enzyme was covalently bonded on the support previously silanized and activated with glutaralde-hyde (SPS) in the absence (CB1) and presence of an additive (CB2) and, in the third, the enzyme was encapsulated in the absence (EN1) and presence of an additive (EN2). 

Homogeneous enzyme immunoassays have also been developed for serum T4 determination. These procedures are rapid and simple to use and have also been applied to several major automated instruments.For example, the enzyme-multiplied immunoassay technique (EMIT) for T4 measurement uses glucose-6-phosphate dehydrogenase covalently hnked to T4 as the enzyme label.Binding of T4 specific antibody to this label reduces enzyme activity, perhaps as a result of steric or allosteric inhibition As the concentration of unlabeled T4 increases, less enzyme-labeled hormone is bound by the antibody. As a result, the catalytic activity of the unbound enzyme conjugate increases in direct proportion to the amount of T4 in the specimen. The indicator reaction involves oxidation of glucose-6-phosphate with simultaneous reduction of nicotinamide-adenine dinu-... 

Although unlike RIA, enzyme immunoassays can be carried out in homogeneous systems without a separation step (based on the change in enzyme activity during the immune reaction), in practice, heterogeneous (enzyme-linked immunosorbent assay) methods are much more frequently used. The antibody, or in the case of the double-antibody method the second antibody, is immobilized, either covalently or by coating enzyme multiplied immunoassay technique (EMIT). This can be done on the walls of microtiter plates. After the immunogenic reaction, the enzyme activity, which is the equivalent of radioactivity in RIA systems, can be measured by suitable photometric methods on the microtiter plates themselves. 

Enzyme-multiplied immunoassay technique Perhaps the best known homogeneous assay format is the enzyme-multiplied immunoassay technique (EMIT), in which the analyte is covalently attached to an enzyme, and the formation of an analyte-antibody complex blocks the active site and inhibits enzyme activity. When this blocked enzyme is mixed with the experimental sample, there is competition between the enzyme-linked analyte and the sample analyte for occupation of the antibody s antigen-binding site. The more of the analyte present in the sample, the more of the enzyme is released from inhibition, and the level of enzyme activity can thus be used to determine the quantity of the analyte. 

Perhaps the most effective method of immobilization for stabilizing a protein is through multipoint covalent bindings between the protein surface and a polymer support. In this way, the protein is covalently attached to a three-dimensional support lattice through multiple points leading to a significant reduction in protein mobility. Enzymes immobilized using this technique far exceed the stability... 

When the reaction product is soluble in water, enzyme regeneration is difficult to achieve, since the enzyme is often lost during isolation of the product. One way to overcome this problem is application of immobilised enzyme systems. The enzyme is either covalently or ionically attached to an insoluble carrier material or is entrapped in a gel. Depending on the size of the particles used, a simple filtration and washing procedure can be used to separate the immobilised enzyme from the dissolved product A well-known example of this technique is the industrial production of 6-APA. 

---