title: 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.
abstract: 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.
authors: Laboratoire Chirosciences, UMR 6263 CNRS, Institut des Sciences Moléculaires de Marseille (ISM2), Ecole Centrale Marseille, Université Aix-Marseille, Avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
authors: Lilia D
authors: LD
authors: Mendoza
authors: Jorge A
authors: JA
authors: Rodriguez
authors: Julien
authors: J
authors: Leclaire
authors: Gerard
authors: G
authors: Buono
authors: Frédéric
authors: F
authors: Fotiadu
authors: Frédéric
authors: F
authors: Carrière
authors: Abdelkarim
authors: A
authors: Abousalham
mesh_terms: D002175:Candida / Q000201:enzymology; D006801:Humans; D008049:Lipase / Q000378:metabolism*; D010938:Plant Oils / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism*; D025062:Yarrowia
chemical_list: C571173:1,3-O-dioleoyl-2-O-alpha-eleostearoylglycerol; D010938:Plant Oils; D014280:Triglycerides; C009612:tung oil; D008049:Lipase; C518354:PNLIP protein, human