title: Ionic liquid activated Bacillus subtilis lipase A variants through cooperative surface substitutions.
abstract: The interest in performing enzyme-catalyzed reactions in amphiphilic systems, e.g., imidazolium-based ionic liquids (ILs) or surfactants, has been increased over the past decades. Directed protein evolution has been successful in tailoring enzymes for desired properties. Herein, nine IL-resistant Bacillus subtilis lipase A variants, particularly an IL-activated variant M1 (M134N/N138S/L140S), were identified by directed evolution. For instance, variant M2 (M134R/L140S) showed almost doubled specific activity (16.9 vs. 9.4 U/mg) and resistance (233% vs. 111%) at 9 vol% 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([C4 mim][TfO]) compared with wild-type. The specific activities and resistance of purified individual single and double variants have been studied in five different IL-aqueous mixtures. The re-activation of lipase variant M1 (not wild-type) at high IL concentration was attributed to the cooperative effect of three surface substitutions (M134N, N138S, L140S) near the substrate-binding cleft. The presence of IL/substrate clusters under assay conditions was likely related to the re-activation effect. This study provides first example of IL-activated lipase variant generated by protein engineering, and helps to better understand the protein-IL interaction.
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Ning
authors.initials: N
authors.lastname: Jia
authors.affiliation: Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany.
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany. u.schwaneberg@biotec.rwth-aachen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D019020:Directed Molecular Evolution; D020536:Enzyme Activators / Q000378:metabolism*; D052578:Ionic Liquids / Q000378:metabolism*; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D020536:Enzyme Activators; D052578:Ionic Liquids; D050505:Mutant Proteins; D008049:Lipase
keywords: clusters; cooperative effects; directed evolution; ionic liquids; lipase; protein engineering