Directed evolution
As directed evolution (English: directed evolution ) is called the optimization and modification of proteins, enzymes and nucleic acids in vitro on an evolutionary way, ie by means of a random-based mutagenesis and subsequent selection and identification of improved mutants. It is increasingly used mainly in white biotechnology and biocatalysis to improve the suitability of enzymes for industrial applications.
Principle
The random -based mutagenesis is usually performed with PCR-based methods such as error - prone PCR. There are also methods, such as DNA shuffling recombinative, Structure -Based Combinatorial Protein Engineering ( scope) or RACHITT, wherein a chimeric gene from two or more related genes is generated.
Crucial to the success of directed evolution is to identify improved mutants of the protein. Often provides the analysis of successful mutants surprising results such as mutations that significantly affect the activity of the enzyme, even though they are very far outside the active site of the enzyme. Directed evolution adds so much to the understanding of the mode of action of enzymes.
A quote from F. Arnold is this: " You only get what you screen for". The proper choice of fitness function is critical to the success of directed evolution.
As is necessary ( for enzymes, the reaction mechanism ) in the rational protein design, that is, the change of a protein through directed mutagenesis in its gene, exact knowledge of the structure of the protein, and its mechanism of action, only a selection of the desired mutants, is required for the directed evolution. For properties of enzymes such as the solvent or the temperature stability, the molecular causes are still not fully understood, therefore, directed evolution is the drug of choice. Other applications are changing the regioselectivity, chemoselectivity and enantioselectivity of enzymes and substrate specificity, in particular towards unnatural substrates. Directed evolution of nucleic acids is mainly used in the aptamer technology ( SELEX ) and for the optimization of ribozymes.