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
| Autor: | Abdelkarim Abousalham, Gérard Buono, Lilia D. Mendoza, Jorge A. Rodriguez, Frédéric Fotiadu, Julien Leclaire, Frédéric Carrière |
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| Přispěvatelé: | Institut des Sciences Moléculaires de Marseille (ISM2), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Institut de Chimie du CNRS (INC), 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-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-École Supérieure Chimie Physique Électronique de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), 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 du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2012 |
| Předmět: |
Yarrowia
01 natural sciences Biochemistry Substrate Specificity MESH: Plant Oils MESH: Lipase chemistry.chemical_compound Endocrinology MESH: Spectrophotometry Ultraviolet tung oil Methods Candida 0303 health sciences biology Stereoisomerism Candida antarctica A lipase MESH: Triglycerides Aleurites fordii QD415-436 stereoselectivity 03 medical and health sciences Hydrolysis MESH: Candida Glycerol Humans Plant Oils Lipolysis [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry Molecular Biology/Biochemistry [q-bio.BM] Lipase Triglycerides 030304 developmental biology MESH: Humans Chromatography 010405 organic chemistry Substrate (chemistry) Cell Biology biology.organism_classification MESH: Stereoisomerism 0104 chemical sciences Oleic acid chemistry β-cyclodextrin sn-2 specificity biology.protein Spectrophotometry Ultraviolet MESH: Substrate Specificity MESH: Yarrowia Candida antarctica |
| Zdroj: | Journal of Lipid Research Journal of Lipid Research, American Society for Biochemistry and Molecular Biology, 2012, 53 (1), pp.185-94. ⟨10.1194/jlr.D019489⟩ Journal of Lipid Research, Vol 53, Iss 1, Pp 185-194 (2012) Journal of Lipid Research, 2012, 53 (1), pp.185-94. ⟨10.1194/jlr.D019489⟩ |
| ISSN: | 0022-2275 |
| DOI: | 10.1194/jlr.d019489 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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