StEP in Directed Evolution

Protocol for Individual Steps in Directed Evolution of a Lipase for Desymmetrization of meso-l,4-Diacetoxycyclopentene [33]... 

Process steps in Directed Evolution Creating Diversity and Checking lor Hits... 

Figure 5.2 The individual steps in directed evolution illustrated for the case of enhancing or reversing stereoselectivity [15g, 19].

Figure 3.2 Examples of gene shuffling methods used for DNA library creation in directed evolution, (a) Homology-dependent primer-independent DNA shuffling (b) homology-dependent primer-dependent StEP (c) homology-independent SHIPREC...

The use of molecular biology methods, described in Section 5.3 seems to be especially worthwhile as it offers novel possibilities of optimization on process adjustment. Directed evolution leads to the formation of new biocatalysts with improved characteristics (selectivity, activity, stability, etc.). Incorporation ofnon-proteinogenic amino acids makes it possible to reach beyond the repertoire of building blocks used by nature. The prospect of bioconjugate preparation offers the possibility to form functional clusters of enzymes and to perform multiple synthetic steps in one pot. 

Decarboxylases are one of the members of the enolase superfamily. The most important and interesting point of this class of enzymes is that they are mechanistically diverse and catalyze different overall reactions. However, each enzyme shares a partial reaction in which an active site base abstracts a proton to form a nucleophile. The intermediates are directed to different products in the different active sites of different members. However, some enzymes of this class exhibit catalytic promiscuity in their natural form. ° This fact is considered to be strongly related to the evolution of enzymes. Reflecting the similarity of the essential step of the total reaction, there are some successful examples of artificial-directed evolution of these enzymes to catalyze distinctly different chemical transformation. The changing of decarboxylase to racemase described in Section 2.5 is also one of these examples. 

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