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.
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...
Published in: | Journal of Lipid Research |
---|---|
Main Authors: | , , , , , , |
Other Authors: | , , , , , , , |
Format: | Article in Journal/Newspaper |
Language: | English |
Published: |
HAL CCSD
2012
|
Subjects: | |
Online Access: | https://hal.science/hal-00917267 https://doi.org/10.1194/jlr.D019489 |
id |
ftinsalyonhal:oai:HAL:hal-00917267v1 |
---|---|
record_format |
openpolar |
spelling |
ftinsalyonhal:oai:HAL:hal-00917267v1 2024-05-12T07:55:38+00:00 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. Mendoza, Lilia D Rodriguez, Jorge A Leclaire, Julien Buono, Gerard Fotiadu, Frédéric Carrière, Frédéric Abousalham, Abdelkarim 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) 2012-01 https://hal.science/hal-00917267 https://doi.org/10.1194/jlr.D019489 en eng HAL CCSD American Society for Biochemistry and Molecular Biology info:eu-repo/semantics/altIdentifier/doi/10.1194/jlr.D019489 info:eu-repo/semantics/altIdentifier/pmid/22114038 hal-00917267 https://hal.science/hal-00917267 doi:10.1194/jlr.D019489 PUBMED: 22114038 PUBMEDCENTRAL: PMC3243475 ISSN: 0022-2275 Journal of Lipid Research https://hal.science/hal-00917267 Journal of Lipid Research, 2012, 53 (1), pp.185-94. ⟨10.1194/jlr.D019489⟩ 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] info:eu-repo/semantics/article Journal articles 2012 ftinsalyonhal https://doi.org/10.1194/jlr.D019489 2024-04-16T02:46:33Z 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. Article in Journal/Newspaper Antarc* Antarctica INSA Lyon HAL (Institut National des Sciences Appliquées) Journal of Lipid Research 53 1 185 194 |
institution |
Open Polar |
collection |
INSA Lyon HAL (Institut National des Sciences Appliquées) |
op_collection_id |
ftinsalyonhal |
language |
English |
topic |
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] |
spellingShingle |
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] Mendoza, Lilia D Rodriguez, Jorge A Leclaire, Julien Buono, Gerard Fotiadu, Frédéric Carrière, Frédéric Abousalham, Abdelkarim 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. |
topic_facet |
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] |
description |
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. |
author2 |
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 |
author |
Mendoza, Lilia D Rodriguez, Jorge A Leclaire, Julien Buono, Gerard Fotiadu, Frédéric Carrière, Frédéric Abousalham, Abdelkarim |
author_facet |
Mendoza, Lilia D Rodriguez, Jorge A Leclaire, Julien Buono, Gerard Fotiadu, Frédéric Carrière, Frédéric Abousalham, Abdelkarim |
author_sort |
Mendoza, Lilia D |
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. |
title_short |
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. |
title_full |
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. |
title_fullStr |
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. |
title_full_unstemmed |
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. |
title_sort |
ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-o-dioleoyl-2-o-α-eleostearoyl-sn-glycerol as substrate. |
publisher |
HAL CCSD |
publishDate |
2012 |
url |
https://hal.science/hal-00917267 https://doi.org/10.1194/jlr.D019489 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
ISSN: 0022-2275 Journal of Lipid Research https://hal.science/hal-00917267 Journal of Lipid Research, 2012, 53 (1), pp.185-94. ⟨10.1194/jlr.D019489⟩ |
op_relation |
info:eu-repo/semantics/altIdentifier/doi/10.1194/jlr.D019489 info:eu-repo/semantics/altIdentifier/pmid/22114038 hal-00917267 https://hal.science/hal-00917267 doi:10.1194/jlr.D019489 PUBMED: 22114038 PUBMEDCENTRAL: PMC3243475 |
op_doi |
https://doi.org/10.1194/jlr.D019489 |
container_title |
Journal of Lipid Research |
container_volume |
53 |
container_issue |
1 |
container_start_page |
185 |
op_container_end_page |
194 |
_version_ |
1798835442604310528 |