An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate.

• Published in: Journal of Lipid Research
• Main Authors: Mendoza, Lilia D, Rodriguez, Jorge A, Leclaire, Julien, Buono, Gerard, Fotiadu, Frédéric, Carrière, Frédéric, Abousalham, Abdelkarim
• Other Authors: Institut des Sciences Moléculaires de Marseille (ISM2), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Institut de Chimie - CNRS Chimie (INC-CNRS)-Centre National de la Recherche Scientifique (CNRS), Enzymologie interfaciale et de physiologie de la lipolyse (EIPL), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie - CNRS Chimie (INC-CNRS)-Centre National de la Recherche Scientifique (CNRS)
• Format: Article in Journal/Newspaper
• Language: English
• Published: HAL CCSD 2012
• Subjects: MESH: Candida; MESH: Humans; MESH: Lipase; MESH: Plant Oils; MESH: Spectrophotometry; Ultraviolet; MESH: Stereoisomerism; MESH: Substrate Specificity; MESH: Triglycerides; MESH: Yarrowia; [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry; Molecular Biology/Biochemistry [q-bio.BM]; Antarc*; Antarctica
• Online Access: https://hal.science/hal-00917267; https://doi.org/10.1194/jlr.D019489

International audience In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.