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Directed Evolution of Bacillus subtilis Lipase A by Use of Enantiomeric Phosphonate Inhibitors: Crystal Structures and Phage Display Selection
Author(s) -
Dröge Melloney J.,
Boersma Ykelien L.,
van Pouderoyen Gertie,
Vrenken Titia E.,
Rüggeberg Carsten J.,
Reetz Manfred T.,
Dijkstra Bauke W.,
Quax Wim J.
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500308
Subject(s) - bacillus subtilis , lipase , directed evolution , phosphonate , saturated mutagenesis , mutant , biochemistry , chemistry , protein engineering , stereochemistry , enzyme , enantiomer , mutagenesis , cutinase , site directed mutagenesis , dna shuffling , combinatorial chemistry , biology , gene , bacteria , genetics
Phage display can be used as a protein‐engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2‐ O ‐isopropylidene‐ sn ‐glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with ( S )‐(+)‐ and ( R )‐(−)‐IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis–Menten parameters. The 3D structures of the Sc and Rc inhibitor–lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.