Detergents are commonly applied in lipase assays to solubilize sparingly soluble model substrates. However, detergents affect lipases as well as substrates in multiple ways. The effect of detergents on lipase activity is commonly attributed to conformational changes in the lid region. This study deals with the effect of the nonionic detergent, poly(ethylene glycol) dodecyl ether, on a lipase that does not contain a lid sequence, lipase A from Bacillus subtilis (BSLA). We show that BSLA activity depends strongly on the detergent concentration and the dependency profile changes with pH. The interaction of BSLA with detergent monomers and micelles is studied using fluorescence correlation spectroscopy, time-resolved anisotropy decay, and temperature-induced unfolding. Detergent-dependent hydrolysis kinetics of two different substrates at two pH values are fitted with a microkinetic model. This analysis shows that the mechanism of interfacial lipase catalysis is strongly affected by the detergent. It reveals an activation mechanism by monomeric detergent that does not result from structural changes of the lipase. Instead, we propose that interfacial diffusion of the lipase is enhanced by detergent binding.
Title: Fluorescence spectroscopic analysis of the structure and dynamics of Bacillus subtilis lipase A governing its activity profile under alkaline conditions Kubler D, Ingenbosch KN, Bergmann A, Weidmann M, Hoffmann-Jacobsen K Ref: Eur Biophysical Journal, 44:655, 2015 : PubMed
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 alpha/beta 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.
