Evolved enzyme ups selectivity

Directed evolution of an epoxide hydrolase from the common fungus Aspergillus niger has led to mutant enzymes that have up to 25 times higher enantioselectivities than the wild-type enzyme (Angew. Chem. Int. Ed., published online Jan. 13, dx.doi.org/10.1002/anie.200502746). Manfred T. Reetz and coworkers at Max Planck Institute for Coal Research, in Mülheim, Germany, applied their combinatorial active-site saturation test (CAST) iteratively to improve the enzyme's properties. Instead of randomly introducing mutations into the entire gene and amino acid sequence, as is done in DNA shuffling or error-prone PCR (epPCR) methods, CAST generates focused libraries of mutants by simultaneously randomizing just a few amino acid sites next to the binding pocket. Reetz and coworkers created a set of libraries based on three sites; the gene of the best hit was then used as a template for subsequent CASTing iterations. The top hydrolase catalyzes the hydrolytic kinetic resolution of a chiral epoxide with a selectivity of 115 in favor of the S-enantiomer compared with 4.6 for the wild-type enzyme and 10.8 for an epPCR-derived enzyme.