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.
Koga
Yuichi
Y
New Energy and Industrial Technology Development Organization, Sunshine 60 Bldg, 3-1-1 Higashi Ikebukuro, Toshima-ku, Tokyo 170-6028, Japan.
Kato
Katsuya
K
Nakano
Hideo
H
Yamane
Tsuneo
T
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D015723:Gene Library; D057927:Hydrophobic and Hydrophilic Interactions; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics*; D010654:Phenylbutyrates / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity
D010654:Phenylbutyrates; D008049:Lipase