Directed Evolution of an Enantioselective Enzyme through Combinatorial Multiple-Cassette Mutagenesis

Abstract

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.