Inverting enantioselectivity of Burkholderia cepacia KWI-56 lipase by combinatorial mutation and high-throughput screening using single-molecule PCR and in vitro expression.

Abstract:

: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.

journal_name

J Mol Biol

authors

Koga Y,Kato K,Nakano H,Yamane T

doi

10.1016/s0022-2836(03)00782-4

keywords:

subject

Has Abstract

pub_date

2003-08-15 00:00:00

pages

585-92

issue

3

eissn

0022-2836

issn

1089-8638

pii

S0022283603007824

journal_volume

331

pub_type

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