Nanoencapsulated Lecitase Ultra and Thermomyces lanuginosus Lipase, a Comparative Structural Study.
Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase A1 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 (Rg) changed from 16 to 38 Å, as the water content (w0) 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 w0 15 showed the highest conversion yield, 38% in 2 h, while the system with w0 10 showed the highest initial velocity, 0.43 μM/min. This last system had a Rg of 19.3 Å, similar to that of the TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w0 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 w0 values to provide optimized enzymatic activity.
Faculty of Pharmacy, Federal University of Rio de Janeiro , Rio de Janeiro 21941-902, Brazil.
Karen M
KM
Gonçalves
Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro , Rio de Janeiro 21941-909, Brazil.|Department of Biochemical Engineering, School of Chemistry, Federal University of Rio de Janeiro , Rio de Janeiro 21941-909, Brazil.
Ivaldo I
II
Junior
Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation , Athens 116 35, Greece.
Vassiliki
V
Papadimitriou
Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation , Athens 116 35, Greece.
Maria
M
Zoumpanioti
Faculty of Pharmacy, Federal University of Rio de Janeiro , Rio de Janeiro 21941-902, Brazil.
Ivana C R
IC
Leal
Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro , Rio de Janeiro 21941-909, Brazil.
Rodrigo O M A
RO
de Souza
Faculty of Pharmacy, Federal University of Rio de Janeiro , Rio de Janeiro 21941-902, Brazil.
Yraima
Y
Cordeiro
Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation , Athens 116 35, Greece.
Aristotelis
A
Xenakis
D001203:Ascomycota / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D008823:Micelles; D054466:Phospholipases A1 / Q000737:chemistry*; D000072417:Protein Domains; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D014961:X-Ray Diffraction
D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D008823:Micelles; D054466:Phospholipases A1