Liebeton_2000_Chem.Biol_7_709

BACKGROUND: The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry. In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity. Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity.
RESULTS: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase 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 structural 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 opening up a large area of novel applications in biotechnology.