Enzymes, enantioselective, directed evolution

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

The chapter by Reetz includes many examples and explanations of methods illustrating how directed evolution has been applied to prepare new enzyme catalysts. Directed evolution of enantioselective enzymes has emerged as a fundamentally new approach to asymmetric catalysis. It involves the combination... 

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

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].

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids [4]. Scientists at Diversa expanded the collection of nitrilases by metagenome panning [56]. Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity, limited activity (substrate acceptance), and/or product inhibition. Diversa also reported the first example of the directed evolution of an enantioselective nitrilase [20]. An additional limitation had to be overcome, which is sometimes ignored, when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/or decreased enantioselectivity at high substrate concentrations [20]. 

Several reports regarding the directed evolution of enantioselective epoxide hydrolases (EHs) have appeared [23,57-59]. These enzymes constitute important catalysts in synthetic organic chemistry [4,60]. The first two reported studies concern the Aspergillus niger epoxide hydrolase (ANEH) [57,58]. Initial attempts were made to enhance the enantioselectivity of the AN E H -catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-19). The WT leads to an Evalue of only 4.6 in favor of (S)-20 (see Scheme 2.4) [58]. 

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