Directed evolution of an enantioselective lipase

Background: The biocatalytic production of enantiopure compounds is of stea dily increasing importance to the chemical and biotechnological industry. I n most cases, however, it is impossible to identify an enzyme that possesse s the desired enantioselectivity. Therefore, there is a strong need to crea te by molecular biological methods novel enzymes which display high enantio selectivity. Results: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved t o catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of r andom mutagenesis by ep-PCR and saturation mutagenesis resulted in an incre ase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions. The recently solved three-dimensional structure of PAL allowed us to analyze the struct ural consequences of these substitutions. Conclusions: A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme. Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby open ing UP a large area of novel applications in biotechnology.