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.

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.

An enzymatically controlled one-step synthesis of a non-isocyanate polyurethane monomer from sustainable resources is feasible under mild reaction conditions.

Singlet oxygen is a reactive oxygen species undesired in living cells but a rare and valuable reagent in chemical synthesis. We present a fluorescence spectroscopic analysis of the singletoxygen formation activity of commercial peroxidases and novel peroxygenases. Singlet-oxygen sensor green (SOSG) is used as fluorogenic singlet oxygen trap. Establishing a kinetic model for the reaction cascade to the fluorescent SOSG endoperoxide permits a kinetic analysis of enzymatic singlet-oxygen formation. All peroxidases and peroxygenases show singlet-oxygen formation. No singlet oxygen activity could be found for any catalase under investigation. Substrate inhibition is observed for all reactive enzymes. The commercial dye-decolorizing peroxidase industrially used for dairy bleaching shows the highest singlet-oxygen activity and the lowest inhibition. This enzyme was immobilized on a textile carrier and successfully applied for a chemical synthesis. Here, ascaridole was synthesized via enzymatically produced singlet oxygen.