title: Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol.
abstract: Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution. Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.
authors.affiliation: Faculdade de Engenharia dos Recursos Naturais, Universidade do Algarve, Campus de Gambelas, 8000-117 Faro, Portugal. emelo@ualg.pt
authors.forename: Eduardo P
authors.initials: EP
authors.lastname: Melo
authors.forename: Sílvia M B
authors.initials: SM
authors.lastname: Costa
authors.forename: Joaquim M S
authors.initials: JM
authors.lastname: Cabral
authors.forename: Peter
authors.initials: P
authors.lastname: Fojan
authors.forename: Steffen B
authors.initials: SB
authors.lastname: Petersen
mesh_terms: D016880:Anisotropy; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D002942:Circular Dichroism; D004058:Diffusion; D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry*; D005670:Fusarium / Q000201:enzymology; D000441:Hexanols / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D008823:Micelles; D011487:Protein Conformation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
chemical_list: D000441:Hexanols; D008823:Micelles; D004143:Dioctyl Sulfosuccinic Acid; C036260:1-hexanol; D002265:Carboxylic Ester Hydrolases; C019952:cutinase