Directed Evolution as a Means to Engineer Enantioselective Enzymes

Directed Evolution as a Means to Engineer Enantioselective Enzymes 

In each cycle, the library of mutated genes is first inserted in a standard bacterial host such as Escherichia coli or Bacillus subtilis. Subsequently, bacterial colonies are plated out on agar plates and harvested individually by a colony picker. Each colony is placed in a separate well of a microtiter plate containing nutrient broth, so that the bacteria grow and produce the protein of interest. Because each colony originates 

Bacteria producing mutant enzymes grown in nutrient broth 

Recently, a method called iterative saturation mutagenesis (ISM) has been proposed and experimentally implemented, which appears to be exceptionally efficient [23,24], 

The reason(s) for the success of ISM has to do with the fact that sequence space has been confined to defined locations in the protein that are most likely to respond positively in an additive or cooperative manner. In sharp contrast, when performing several rounds of epPCR (4—8 are typical ), the whole protein is addressed repeatedly although only a fraction of the amino acid positions are important. Owing to statistical reasons, a given improved mutant (hit) evolved by ISM is not 

---

Reetz MT (2007) Directed evolution as a means to engineer enantioselective enzymes. In Gotor V (ed) Asymmetric organic synthesis with enzymes. Wiley-VCH, Weinheim, in press... 

---