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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.
MedLine Citation:
PMID:  22114038     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Lilia D Mendoza; Jorge A Rodriguez; Julien Leclaire; Gerard Buono; Frédéric Fotiadu; Frédéric Carrière; Abdelkarim Abousalham
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Publication Detail:
Type:  Journal Article; Validation Studies     Date:  2011-11-23
Journal Detail:
Title:  Journal of lipid research     Volume:  53     ISSN:  0022-2275     ISO Abbreviation:  J. Lipid Res.     Publication Date:  2012 Jan 
Date Detail:
Created Date:  2011-12-16     Completed Date:  2012-04-09     Revised Date:  2013-06-27    
Medline Journal Info:
Nlm Unique ID:  0376606     Medline TA:  J Lipid Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  185-94     Citation Subset:  IM    
Affiliation:
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.
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MeSH Terms
Descriptor/Qualifier:
Candida / enzymology
Humans
Lipase / metabolism*
Plant Oils / metabolism
Spectrophotometry, Ultraviolet / methods*
Stereoisomerism
Substrate Specificity
Triglycerides / metabolism*
Yarrowia
Chemical
Reg. No./Substance:
0/1,3-O-dioleoyl-2-O-alpha-eleostearoylglycerol; 0/Plant Oils; 0/Triglycerides; 68038-50-6/tung oil; EC 3.1.1.3/Lipase; EC 3.1.1.3/PNLIP protein, human
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