Inverting Enantioselectivity of Burkholderia cepacia KWI-56 Lipase by Combinatorial Mutation and High-throughput Screening Using Single-molecule PCR and In Vitro Expression

The enantioselectivity of lipase from Burkhorderia cepacia KWI-56 has been inverted using a novel in vitro technique for construction and screening of a protein library by single-molecule DNA amplification by PCR followed by in vitro coupled transcription/translation system termed single-molecule-PCR-linked in vitro expression (SIMPLEX). Four amino acid residues (L17, F119, L167, and L266) in the hydrophobic substrate-binding pocket of the lipase were selected for mutation based on a structural model of a substrate–enzyme complex, and a combinatorial mutation library was constructed by SIMPLEX and screened for ( R) and ( S)-configurations of p-nitrophenyl 3-phenylbutyrate. Some combinations of amino acid substitutions in the four positions of the lipase were found as effective for changing the enantiopreference from the ( S)-form substrate to the ( R)-form. Two variants were expressed in the original host cells and purified to homogeneity, showing completely reversed enantioselectivity for the ( R)-form of ethyl 3-phenylbutyrate (selectivity factor E R=38 or 33), whereas the wild-type lipase was ( S)-selective (selectivity factor E S=33). Thus the semi-rational and semi-random combinatorial design of a mutant library followed by a high-throughput screening based on their enzymatic activity should be a powerful tool to engineer the enantioselectivity of enzymes.