title: Towards understanding directed evolution: more than half of all amino acid positions contribute to ionic liquid resistance of Bacillus subtilis lipase A.
abstract: Ionic liquids (ILs) are attractive (co-)solvents for biocatalysis. However, in high concentration (>10 % IL), enzymes usually show decreased activity. No general principles have been discovered to improve IL resistance of enzymes by protein engineering. We present a systematic study to elucidate general engineering principles by site saturation mutagenesis on the complete gene bsla. Screening in presence of four [BMIM]-based ILs revealed two unexpected lessons on directed evolution: 1) resistance improvement was obtainable at 50-69 % of all amino acid positions, thus explaining the success of small sized random mutant libraries; 2) 6-13 % of substitutions led to improved resistance. Among these, 66-95 % were substitutions by chemically different amino acids (e.g., aromatic to polar/aliphatic/charged amino acids), thus indicating that mutagenesis methods introducing such changes should, at least for lipases like BSLA, be favored to improve IL resistance.
authors: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074 Aachen (Germany).
authors: Victorine Josiane
authors: VJ
authors: Frauenkron-Machedjou
authors: Alexander
authors: A
authors: Fulton
authors: Leilei
authors: L
authors: Zhu
authors: Carolin
authors: C
authors: Anker
authors: Marco
authors: M
authors: Bocola
authors: Karl-Erich
authors: KE
authors: Jaeger
authors: Ulrich
authors: U
authors: Schwaneberg
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology*; D019020:Directed Molecular Evolution; D052578:Ionic Liquids / Q000494:pharmacology*; D008049:Lipase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis
chemical_list: D052578:Ionic Liquids; D008049:Lipase
keywords: BSLA; directed evolution; ionic liquids; protein engineering; site saturation mutagenesis