abstract
- Aerosol OT, bis(2-ethylhexyl)sodium sulfosuccinate/isooctane reverse micelles were used to investigate the dependence of the lipolytic activity of Rhizopus arrhizus lipase on surfactant concentration. Kinetic constants for the lipolytic reaction were measured in parallel with structural studies using protein fluorescence and circular dichroism (CD) spectroscopy. Km values remained constant throughout the range of AOT concentrations studied. The kcat values decreased with increasing surfactant concentration at constant water-to-surfactant ratio (wo = 11) from 50 mM to 100 mM AOT, but remained constant from 100 to 200 mM AOT. These data suggested an association of the lipase with the micellar membrane. An inflection in the time-course of the reaction was found to be a function of both surfactant and substrate concentrations and was likely an indication of the interfacial nature of the hydrolysis reaction. Structure prediction based on far-UV CD spectral data demonstrated structural reorganization of R. arrhizus lipase upon incorporation into reverse micelles which was characterized by a dramatic increase in beta-sheet and overall accountable secondary structure. Other spectral changes of the lipase upon incorporation into reverse micelles included appearance of fine structure in the near-UV CD spectrum and a blue shift in the fluorescence emission maximum from 336 to 326 nm.