Superior Biocatalysts by Directed Evolution

Useful biocatalysts for organic chemistry can be created by directed evolution.Mutations are introduced into genes encoding biocatalyst proteins of interest by error-prone PCR or other random mutagenesis methods. The mutated genes can be rearranged by recombinative processes like DNA shuffling, thereby significantly enhancing the efficiency with which genes can be evolved. These genes are expressed in suitable microbial hosts leading to the production of functional biocatalysts. Selection or screening procedures serve to identify in a large library of potential candidates the biocatalyst which possesses the desired properties. Examples of applications include subtilisin E with greatly improved catalytic activity and stability in organic solvent, an esterase with 50-fold higher activity in organic solvent, and a β-lactamase conferring a 32,000-fold increased antibiotic resistance. Furthermore, directed evolution of a bacterial lipase resulted in a significant increase in enantioselectivity,thereby demonstrating the enormous potential of this process for organic chemistry.