Superior biocatalysts by directed evolution

Useful biocatalysts for organic chemistry can be created by directed evolut ion. Mutations are introduced into genes encoding biocatalyst proteins of i nterest by error-prone PCR or other random mutagenesis methods. The mutated genes can be rearranged by recombinative processes like DNA shuffling, the reby significantly enhancing the efficiency with which genes can be evolved . These genes are expressed in suitable microbial hosts leading to the prod uction of functional biocatalysts. Selection or screening procedures serve to identify in a large library of potential candidates the biocatalyst whic h possesses the desired properties. Examples of applications include subtil isin E with greatly improved catalytic activity and stability in organic so lvent, an esterase with 50-fold higher activity in organic solvent, and a b eta-lactamase conferring a 32,000-fold increased antibiotic resistance. Fur thermore, directed evolution of a bacterial lipase resulted in a significan t increase in enantioselectivity, thereby demonstrating the enormous potent ial of this process for organic chemistry.