docusate-sodium and 4-nitrophenyl-butyrate

In the water-in-[Bmim][PF(6)] microemulsion stabilized by both AOT and Triton X-100, the lipase-catalyzed hydrolysis of 4-nitrophenyl butyrate (p-NPB) was investigated to evaluate the catalytic efficiency of lipase in this novel microemulsion. The structural parameters of the microemulsion and the conditions of the enzymatic reaction affect the catalytic activity of lipase, especially the concentration of Tris-HCl buffer. Under optimum conditions, the catalytic activity of lipase in the present microemulsion is much higher than that in H(2)O saturated [Bmim][PF(6)]. When the partitioning of the substrate in the microemulsion is taken into account, the catalytic efficiency of lipase in this novel microemulsion is 14.3 times that in H(2)O saturated [Bmim][PF(6)] due to the significant decrease of the Michaelis constant in the microemulsion. Due to the large interface, high water activity, and probably the activating effect of the imidazolium cation in the water pool, the present microemulsion is demonstrated to be a promising medium for the lipase-catalyzed hydrolytic reaction. To demonstrate an important biocatalytic application in the IL-based microemulsion, the lipase-catalyzed synthesis of the flavoring agent benzyl acetate via transesterification of vinyl acetate with benzyl alcohol was also studied in the medium. Due to the high dispersion of lipase, large interface and removal of the byproduct, a maximum yield of 94% was obtained, indicating that the novel microemulsion is really important and useful.

Topics: Absorption; Benzyl Compounds; Biocatalysis; Buffers; Butyrates; Candida; Emulsions; Hydrogen-Ion Concentration; Hydrolysis; Imidazoles; Kinetics; Lipase; Octoxynol; Spectrum Analysis; Succinates; Surface Tension; Surface-Active Agents; Time Factors; Water

The reactivity, stability and unfolding of wild-type (WT) Fusarium solani pisi cutinase and L153Q, S54D and T179C variants were studied in the absence and presence of the dioctyl sulfosuccinate sodium salt (AOT) surfactant. In the absence of surfactant the S54D variant catalytic activity is similar to that of the WT cutinase, whereas L153Q and T179C variants show a lower activity. AOT addition induces an activity reduction for WT cutinase and its variants, although for low AOT concentrations a small increase of activity was observed for S54D and T179C. The enzyme deactivation in the presence of 0.5 mM AOT is relatively slow for the S54D and T179C variants when compared to wild-type cutinase and L153Q variant. These results were correlated with secondary and tertiary structure changes assessed by the CD spectrum and fluorescence of the single tryptophan and the six tyrosine residues. The WT cutinase and S54D variant have similar secondary and tertiary structures that differ from those of T179C and L153Q variants. L153Q, S54D and T179C mutations prevent the formation of hydrophobic crevices responsible for the unfolding by anionic surfactants, with the consequent decrease of the AOT-cutinase interactions.

Topics: Amino Acid Substitution; Anilino Naphthalenesulfonates; Butyrates; Carboxylic Ester Hydrolases; Circular Dichroism; Enzyme Stability; Fungal Proteins; Fusarium; Hydrolysis; Models, Molecular; Mutagenesis, Site-Directed; Recombinant Proteins; Spectrometry, Fluorescence; Succinates; Surface-Active Agents; Thermodynamics