Enantioselectivity of enzymes

Perhaps the biggest impact on the practical utilization of enzymes has been the development of nonaqueous enzymology (11,16,33,35). The use of enzymes in nonaqueous media gready expands the scope of suitable transformations, simplifies thek use, and enhances stabiUty. It also provides an easy means of regulation of the substrate specificity and regio- and enantioselectivity of enzymes by changing the reaction medium. 

Examples of Enhancing the Enantioselectivity of Enzymes by Directed Evolution... 

Effusive beam technique, 157-158 Electron bombardment flow radiolysis, 238 Electrospray ionization and ionic clusters, 168 Enantiomers, separation techniques, 154-155 Enantioselectivity of enzymes, 148 Enthalpy-entropy compensation plots, 261 Enthalpy of activation, and quantum tunneling, 67, 70-71... 

IV. Examples of Directed Evolution as a Means to Control the Enantioselectivity of Enzymes... 

When water molecules interact with an enzyme, it is natural that conformational changes can occur, which in turn can cause changes in the selectivity of the enzyme. Since enantioselectivity of enzymes is of major importance for many applications, it is a common task to investigate how to choose reaction conditions providing the maximal enantioselectivity. As might be expected, because water can interact with enzymes in many ways, it is difficult to generalize the effects. In some studies of lipase-catalyzed esterification reactions, no effects of water activity on enantioselectivity were observed [30]. In a similar study, no effects were observed in most cases, while the enantioselectivity of one lipase-catalyzed reaction decreased... 

Since then, a number of propositions to modify (if possible, to enhance) the enantioselectivity of enzymes (mostly hydrolases) by medium engineering have been put forward [20, 51, 60, 64—69], providing a challenging mix of scientifically and practically interesting questions. 

Figure 2.1 Enantioselectivity of enzyme-catalyzed reactions in organic media plotted as a function of the logP-value of the solvent. Data obtained from the literature 1. (open circles) [81], 2. (open diamonds) [17], 3. (closed triangles) [19], 4. (closed circles) [20], 5. (open squares) [82], 6. (closed squares)...

Figure 2.2 Enantioselectivity of enzyme-catalyzed reactions in a set of organic media. The order and relative separation of the solvents plotted on the abscissa was obtained by assuming a strict correlation between the particular solvent and the E-value for the...

It is known that enantioselectivity of enzymes depends on many different parameters such as temperature, substrate structure, reaction medium, and presence of water. Enantiopreference of enzymes can be greatly affected, even reversed, by changing the reaction solvent. Such an example was reported by Ueji et al. in 1992 for Candida cylindracea lipase-catalyzed esterification of ( )-2-phenoxy propionic acid with 1-butanol [29]. 

Nevertheless, it appears from the literature that no general rules for the effect of solvent on enantioselectivity of enzymes can be established, and that the effect of other parameters having an influence on enantioselectivity (temperature, type of substrates, presence of water, etc.) are linked together and may modify the effect of solvents. 

The advantage of fluorescence-based assays is their high sensitivity. It is therefore perhaps surprising that few such systems have been developed for evaluating the enantioselectivity of enzyme-catalyzed reactions. Fluorescence as a detection method is used in an enzyme-coupled assay [26] (see Section 9.3.4.3) and in the capillary array electrophoresis [25] (see Section 9.3.6.5). If several substrates need to be screened simultaneously, fluorescence-based substrate arrays as enzyme fingerprinting tools can be used, although enantioselectivity still needs to be addressed [26e],... 

Reetz MT (2004a) Controlling the enantioselectivity of enzymes by directed evolution Practical and theoretical ramifications. Proc Natl Acad Sci USA 101 5716-5722... 

This chapter consequently focuses on the application of enzymes for the selective cleavage of esters, amides and nitriles [2], Out of all the reported industrial applications of enzymes these type of hydrolyses constitute more than 40% [3], Enzymatic hydrolyses are often performed because of the enantioselectivity of enzymes, and in particular of the lipases that are used for the production of enantiopure fine chemicals. 

Interestingly, the result of sequencing (Tab. 11.1) shows that the mutation L — G at position 162 is maintained, whereas S155F transforms into S155M. In summary, the experiments described here constitute the first examples of the application of recombinant methods in the quest to improve the enantioselectivity of enzymes. It is apparent that several forms of DNA shuffling are quite successful. 

The examples presented in this chapter demonstrate that a combination of various analytical approaches and the selection of suitable model systems can add valuable information to our knowledge about pathways and enzymes involved in the biosynthesis of chiral volatiles. Some of the techniques need further improvement, e.g. by use of radioactively labeled precursors the detection threshold of metabolites can be lowered significantly addition of precursors in concentrations comparable to those in natural plant or microbial systems would be possible. The investigation of the enantioselectivity of enzymes has to be emphasized, eventually not only enzymes commercially available or easily accessible in microorganisms but also those active in plant systems have to be studied. 

The chemo-, regie- and enantioselectivity of enzymes make them ideal asymmetric catalysts. Most important is the differential recognition of diastereotopic groups of chiral and prochiral substrates. The mild conditions under which most enzymes operate minimize isomerization, epimerization and racemization associated with many other chemical processes.. 8... 

Shafiee, M. Boudoua, V. Griffon, J.F. Pompon, A. Gosselin, G. Eriksson, S. Imbach, J.L. Maury, G. Study of the enantioselectivity of enzymes involved in nucleoside analog metabolism deoxycytidine kinase. Nucleosides Nucleotides, 16, 1767-1770 (1997)... 

The often increased enantioselectivity of enzymes in ionic liquids was also explored by Kamal and Chouhan for the synthesis of enantiomerically pure 1,2-diols in ionic liquids. The kinetic resolution of the did was catalyzed by immobilized lipase... 

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