Applications of Isolated Enzymes

In one-step biotransformations particularly, isolated enzymes have distinctive advantages, since they can be used simply, almost like any chemical reagent, and - importantly for isotopic labeling - they do not result in any isotope dilution. Since a large number of enzymes are commercially available, many types of reactions can be carried out. 

Examples of the use of isolated enzymes in isotopic labeling include N- or O-acylations, hydrolyses, redox reactions and group transfer reactions. Most of them aim for applications such as racemate resolution, catalysis of reactions and synthesis of enantiomerically pure molecules. 

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Highly enantioselective reduction of ethyl 6-benzyloxy-3,5-dioxohexanoate by ADH of Acinetohacter calcoaceticus has been reported (97 to >99% ee) [6]. Regi-oselectivity was not encountered, however, as was the case in the reduction of a variety of 3,5-dioxohexanoates A with baker s yeast [7]. The application of isolated enzymes in an anticipated regio- and enantioselective reduction of diketo esters A seemed most promising to us. 

The application of isolated enzymes in biotransformations is often superior to the use of cell-systems, since the various enzymes existing in living cells may result in the formation of by-products and thus cause side-reactions of the substrates and impair the yield of the desired products. 

The application of isolated enzymes to preparative organic synthesis on an industrial scale is a matter of active research worldwide. Since the late sixties, immobilized enzymes have been used in amino acid production in continuous processes on a large scaled 2. In the late seventies, the use of soluble enzymes, especially in membrane reactors, broadened the scope of enzyme technology13, 41 and opened the way to simultaneous use of more than one enzyme for complex conversions -especially coenzyme-dependent biotransformations[5-7. In the early 1980s the use of enzymes was extended further to involve organic solvents[8, 9 ... 

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. 

Here we will focus on the biochemical aspects. The techniques of isolating enzymes, the process of enzyme immobilisation and the behaviour of immobilised enzyme reactors are discussed in detail in the BIOTOL text Technological Applications of Biocatalysts", so will not deal with these aspects in detail here. In outline, however, once the desired enzyme is isolated, it is attached to a carrier material. In order to ascertain sufficient accessibility of the enzyme, a bifunctional spacer molecule is attached to the carrier ... 

Although genetic studies have identified a number of putative aromatic PK aromatases and/or cyclases, in vitro characterization of these enzymes is usually difficult since the postulated Hnear or monocyclic poly 3-ketone templates are often unstable to be isolated or synthesized. For these reasons, the generality and synthetic applications of these enzymes have not been studied in most cases. However, there is plenty of evidence to suggest that enzymatic aromatization and cyclization take place for a variety of linear precursors with various chain lengths [32]. 

A related assay was later reported which makes use of a different and more convenient indicator (bromothymol blue) 78). However, it is emphasized that all of the assays based on pH change reported so far refer to the use of isolated enzymes. In real applications, supernatants are likely to be used, as in directed evolution studies. In supernatants, however, pH variations may occur. Therefore, an optimized assay was later developed in which supernatants are employed (79). Thus, the pH of the buffer is adjusted to the acidity of the medium, enabling about 4000 samples in kinetic resolution investigations to be screened per day. 

Application of this enzyme variant in the bioreactor allows far easier product isolation. Protein engineering turns out to be the most efficient method and also... 

A particularly useful laboratory application of restriction enzymes is in the epidemiologic investigation of bacterial and plasmid isolates to see if patient isolates reflect personnel- or equipment-based sources of infection (43). A fruitful approach to this issue has been to use restriction enzymes and probes to study the fingerprint pattern of ribosomal rRNA genes, which because of their Icirge number will demonstrate characteristic identifying patterns even within strains. 

The use of isolated enzymes, therefore, has great advantages. However, for the preparative applications of isolated redox enzymes, especially for continuous processes, effective and simple methods for the continuous recycling of the active cofactors have to be available. In addition, such systems have to be stable over long times and the separation of the product must be simple to render technical processes economically feasible. Until now, this problem has been generally solved by coupling of the synthetic enzyme with a... 

The use of baker s yeast for selective reductions has a long history, while the use of isolated enzymes is more recent. Dehydrogenases and reductases require a nicotinamide cofactor (NADH or NADPH), from which a hydride is transferred to the substrate carbonyl. Enzymes from different species have been classified according to their selectivity (hydride transfer to si- or re-face of the carbonyl) [14]. The cofactors to be used together with isolated enzymes are commercially available (e.g. from Sigma-Aldrich), but are for most applications too costly to use in stoichiometric amounts. However, cofactor in situ regeneration can be... 

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