New lipase assay using Pomegranate oil coating in microtiter plates.
Lipases play various roles in fat digestion, lipoprotein metabolism, and in the mobilization of fat stored in lipid bodies in animals, plants and microorganisms. In association with these physiological functions, there is an important field of research for discovering lipase inhibitors and developing new treatments of diseases such as obesity, atherosclerosis, diabetes and tuberculosis. In this context, the development of convenient, specific and sensitive analytical methods for the detection and assay of lipases and/or lipase inhibitors is of major importance. It is shown here that purified triacylglycerols (TAGs) from Punica granatum (Pomegranate) seed oil coated on microtiter plates can be used for the continuous assay of lipase activity by recording the variations with time of the UV absorption spectra at 275 nm. UV absorption is due the release of punicic acid (9Z,11E,13Z-octadeca-9,11,13-trienoic acid), a conjugated triene contained in Pomegranate oil. This new microtiter plate assay allows to accurately measure the activity of a wider range of lipases compared to the similar assay previously developed with Tung oil containing α-eleostearic acid (9Z,11E,13E-octadeca-9,11,13-trienoic acid), including the LipY lipase from Mycobacterium tuberculosis. Although punicic acid is a diastereoisomer of α-eleostearic acid, the Δ(13)cis double bound found in punicic acid gives a different structure to the acyl chain that probably favours the interaction of Pomegranate TAGs with the lipase active site. The microplate lipase assay using Pomegranate TAGs shows high sensitivity, reproducibility and remarkable relevance for the high-speed screening of lipases and/or lipase inhibitors directly from raw culture media without any purification step.
CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Serdar
S
Ülker
CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Camille
C
Placidi
CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Vanessa
V
Point
ITERG, Parc Industriel Bersol 2, 11 Rue Gaspard Monge, 33600 Pessac, France.
Benoît
B
Gadenne
CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Carole
C
Serveau-Avesque
CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Stéphane
S
Canaan
CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Frédéric
F
Carrière
CNRS - Aix-Marseille Université - Enzymologie Interfaciale et Physiologie de la Lipolyse - UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Electronic address: jfcavalier@imm.cnrs.fr.
Jean-François
JF
Cavalier
D001426:Bacterial Proteins / Q000737:chemistry*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D008049:Lipase / Q000737:chemistry*; D029561:Lythraceae / Q000737:chemistry*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010938:Plant Oils / Q000737:chemistry*; D037521:Virulence Factors / Q000737:chemistry*
D001426:Bacterial Proteins; D010938:Plant Oils; D037521:Virulence Factors; D002265:Carboxylic Ester Hydrolases; C000594149:LipY protein, Mycobacterium tuberculosis; D008049:Lipase
High-throughput screening; LipY lipase from Mycobacterium tuberculosis; Orlistat; Pancreatic lipase; Punicic acid