Recombinant methods work exceedingly well in the directed evolution of an enantioselective enzyme. For the kinetic resolution of the ester rac‐1 by a lipase [Eq. (a)] three steps were applied: 1) Generation of mutants by the error‐prone polymerase chain reaction (epPCR), 2) identification of “hot spots” in the enzyme by epPCR and simplified combinatorial multiple‐cassette mutagenesis (CMCM), and 3) extension of the process of CMCM to cover a defined region of protein sequence space. From lass then 40 000 enzyme varients generated, one was found which catalyzes the reaction with almost 50‐times higher enantioselectivity than the wild‐type enzyme.

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