ABSTRACT
Biocatalysis using whole cells, crude cell extracts, or purified enzymes has become an important tool in the production of numerous chemical compounds including food additives, agrochemicals, cosmetics, flavors, and, particularly, Pharmaceuticals. The steadily increasing demand for these compounds results in a pressing need to identify and isolate novel biocatalysts. Unfortunately, natural biocatalysts often do not meet the requirements of the chemical industries because they have been optimized by natural evolution to catalyze specific reactions inside living cells. Therefore, molecular biologists have developed a variety of different methods that allow the engineering of enzymes for specific needs. The classical, but time-consuming and cost-intensive, approach includes structure-based predictions to design site-directed mutagenesis experiments and subsequent biochemical characterization of the resulting enzyme variants. Recently, directed evolution has been introduced as a new and powerful method to optimize the properties of a given biocatalyst without requiring knowledge of its structure, or the catalytic mechanism (1-6). Biocatalyst properties that have successfully been optimized by directed evolution include substrate specificity (7), thermal stability (8), and organic solvent resistance (9), but also more sophisticated traits such as cofactor dependence (10) and enantioselectivity (3, 11-15).
