Enzyme enantioselective

Directed Evolution as a Means to Engineer Enantioselective Enzymes... 

Figure 2.1 Strategy for directed evolution of an enantioselective enzyme [6,8].

Figure 2.2 The experimental stages of directed evolution of enantioselective enzymes [6,8].

Another approach for the synthesis of enantiopure amino acids or amino alcohols is the enantioselective enzyme-catalyzed hydrolysis of hydantoins. As discussed above, hydantoins are very easily racemized in weak alkaline solutions via keto enol tautomerism. Sugai et al. have reported the DKR of the hydantoin prepared from DL-phenylalanine. DKR took place smoothly by the use of D-hydantoinase at a pH of 9 employing a borate buffer (Figure 4.17) [42]. 

Persson, M., Costes, D., Wehtje, E. and Adlercreutz, P. (2002) Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity. Enzyme and Microbial Technology, 30, 916-923. 

If kinetic resolution is being studied, the ratio of pseudo-e nantiomers can be measured by MS, allowing for the determination of ee-values (and/or of selectivity factors E). The same applies to the reaction of pseudo prochiral compounds. This system has been used successfully in the directed evolution of enantioselective enzymes. However, it should work equally well in the case of asymmetric transition metal catalyzed reactions. In the original version about 1,000 ee-deter-minations were possible per day (Figure 6).94 The second-generation version based on an 8-channel multiplexed spray system enables about 10,000 samples to be handled per day, the sensitivity being 2% ee.96... 

Reetz MT, Wang LW, Bocola M (2006) Directed evolution of enantioselective enzymes iterative cycles of CASTing for probing protein-sequence space. Angew Chem Int Ed 45 1236-1241... 

The reversibility of hydrogen transfer reactions has been exploited for the racemi-zation of alcohols and amines. By coupling the racemization process with an enantioselective enzyme-catalyzed acylation reaction, it has been possible to achieve dynamic kinetic resolution reactions. The combination of lipases or... 

The lipase-catalyzed resolutions usually are performed with racemic secondary alcohols in the presence of an acyl donor in hydrophobic organic solvents such as toluene and tert-butyl methyl ether (Scheme 1.3). In case the enzyme is highly enantioselective E = 200 or greater), the resolution reaction in general is stopped at nearly 50% conversion to obtain both unreacted enantiomers and acylated enantiomers in enantiomerically enriched forms. With a moderately enantioselective enzyme E = 20-50), the reaction carries to well over 50% conversion to get unreacted enantiomer of high optical purity at the cost of acylated enantiomer of lower optical purity. The enantioselectivity of lipase is largely dependent on the structure of substrate as formulated by Kazlauskas [6] most lipases show... 

Directed Evolution of Enantioselective Enzymes as Catalysts for Organic Synthesis... 

Enantioselective enzyme-catalyzed reactions may involve the transformation of a prochiral substrate into a chiral product, in which case the selectivity is measured by the enantiomeric excess (ee). The transformations can also involve kinetic resolution of racemic substrates, in which case enantioselectivity is measured by the selectivity factor E reflecting the relative rates of reaction of the R)- and (5)-enantiomer. 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

In what appears to be a particularly irmovative development in the area of UV/ Vis-based ee screening systems, the determination of the enantiomeric purity of chiral alcohols 9 is based on a new concept of using two enantioselective enzymes to modify the product (84). The method allows the determination of ee values independent of the concentration, which may be of significant advantage in directed evolution projects. It can be used in three different biocatalytic processes, namely biohydroxylation of alkanes, reductase-catalyzed reduction of ketones, and lipase-or esterase-catalyzed ester hydrolysis. 

A number of other ee assays relying on the use of fluorescence have been described, although their general utility in the high-throughput evaluation of enantioselective enzymes remains to be demonstrated in most cases (66-70). A high-throughput ee... 

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