title: Fluorescence spectroscopic analysis of the structure and dynamics of Bacillus subtilis lipase A governing its activity profile under alkaline conditions.
abstract: Because of their vast diversity of substrate specificity and reaction conditions, lipases are versatile materials for biocatalysis. Lipase A from Bacillus subtilis (BSLA) is the smallest lipase yet discovered. It has the typical α/β hydrolase fold but lacks a lid covering the substrate cleft. In this study, the pH-dependence of the activity, stability, structure, and dynamics of BSLA was investigated by fluorescence spectroscopy. By use of a fluorogenic substrate it was revealed that the optimum pH for BSLA activity is 8.5 whereas thermodynamic and kinetic stability are maximum at pH 10. The origin of this behavior was clarified by investigation of ANS (8-anilino-1-naphthalenesulfonic acid) binding and fluorescence quenching of the two single tryptophan mutants W31F and W42F. Variations in segmental dynamics were investigated by use of time-resolved fluorescence anisotropy. This analysis showed that the activity maximum is governed by high surface hydrophobicity and high segmental mobility of surface loops whereas the stability optimum is a result of low segmental mobility and surface hydrophobicity.
authors.affiliation: Department of Chemistry, Niederrhein University of Applied Sciences, Adlerstr. 32, 47798, Krefeld, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kübler
authors.affiliation: Department of Chemistry, Niederrhein University of Applied Sciences, Adlerstr. 32, 47798, Krefeld, Germany.
authors.forename: Kim N
authors.initials: KN
authors.lastname: Ingenbosch
authors.affiliation: Department of Chemistry, Niederrhein University of Applied Sciences, Adlerstr. 32, 47798, Krefeld, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Bergmann
authors.affiliation: Department of Chemistry, Niederrhein University of Applied Sciences, Adlerstr. 32, 47798, Krefeld, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Weidmann
authors.affiliation: Department of Chemistry, Niederrhein University of Applied Sciences, Adlerstr. 32, 47798, Krefeld, Germany. kerstin.hoffmann-jacobsen@hsnr.de.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Hoffmann-Jacobsen
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D013816:Thermodynamics
chemical_list: C515594:8-anilino-1-naphthalenesulfonic acid; D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D008049:Lipase
keywords: Activity; Bacillus subtilis; Fluorescence anisotropy; Fluorescence spectroscopy; Lipase; pH