Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase Al Lecitase (Lecitase Ultra), were encapsulated in AOT/isooctane reverse micelles and evaluated regarding their structure and activity. Preparations were also tested as effective biocatalysts. Small-angle X-ray scattering (SAXS), electronic paramagnetic resonance (EPR), and fluorescence spectroscopy were the techniques applied to assess the effects of enzyme incorporation to a reverse micellar nanostructure. SAXS analysis showed that the radius of gyration (R-g) changed from 16 to 38 A, as the water content (w(0)) increased. Elongated shapes were more commonly observed than spherical shapes after enzyme encapsulation. EPR studies indicated that enzymes do not participate in the interface, being located in the aqueous center. Fluorescence energy transfer showed that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. Enzymatic activity toward a standard esterification reaction endured after the enzyme was incorporated into the micelles. The activity of TLL for systems with w(0) 15 showed the highest conversion yield, 38% in 2 h, while the system with w(0) 10 showed the highest initial velocity, 0.43 mu M/min. This last system had a Rg of 19.3 angstrom, similar to that of the TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w(0) 10, 55% in 2 h. However, the initial rate was much lower than that of TLL, suggesting less affinity for the substrates, which is expected since Lecitase Ultra is a phospholipase. In summary, we here used several spectroscopic and scattering techniques to reveal the shape and stability of TTL and Lecitase Ultra encapsulated systems, which allowed the selection of w(0) values to provide optimized enzymatic activity.