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Determination of Lipid Degradation by Marine Lipase-Producing Bacteria: Critical Evaluation of Lipase Activity Assays

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Lipids

Abstract

With the aim of obtaining a better understanding of lipids-lipases interactions in bacterioplankton communities in oceans, we used different methods for measuring lipase activities in pure cultures of the marine strain Alteromonas macleodii. The decay of tripalmitate added to cultures was followed chemically over time. In an enzymatic approach, lipase activities were measured using the fluorogenic lipid analogs MUF-palmitate and ELF-palmitate. When hydrolyzed by lipase, the non-fluorescent substrates release MUF and ELF Alcohol (ELFA) which are fluorescent. As shown by spectrofluorometry, ELF-palmitate was an efficient competitor for MUF-palmitate. However, the activities reached using these two fluorogenic substrates were different, but still much higher than the tripalmitate hydrolysis rate, measured chemically. MUF- and ELF-palmitate would not be hydrolyzed by lipase sensu stricto (defined as triacylglycerol acylesterase E.C. 3.1.1.3) but rather reflects lipolytic activities in a broad sense. ELFA is also water-insoluble and theoretically precipitates in the external membrane of bacteria causing its hydrolysis, which would allow microscopic identification of active cells. By epifluorescence microscopy, the accumulation of ELFA fluorescence over time was detected (as large, diffuse halos), but no precipitates were clearly associated with bacteria on slide preparations, neither for pure cultures of Alteromonas macleodii nor for natural samples from the Bay of Marseille, France. Among possible biases, those related to the hydrophobic/hydrophilic conditions required for precipitation are discussed.

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Abbreviations

DAPI:

4′,6-Diamidino-2-phenylindole

DMSO:

Dimethylsulfoxide

ELFA:

Enzyme-labeled fluorescence alcohol

ELF-palmitate:

Enzyme-labeled fluorescence-palmitate

FFA:

Free fatty acid

IC50:

ELF-palmitate concentration giving a 50% inhibition of MUF-palmitate hydrolysis

K m :

Michaelis–Menten constant

MUF:

4-Methylumbelliferyl

MUF-palmitate:

4-Methylumbelliferyl-palmitate

OM:

Organic matter

PYR culture:

Culture grown in medium where the single carbon source is pyruvate

PYR medium:

Medium where the single carbon source is pyruvate

TLC-FID:

Thin layer chromatography-flame ionization detection

TG:

Triglyceride

TG culture:

Culture grown in triglyceride-enriched medium

TG medium:

Triglyceride-enriched medium

V max :

Maximum hydrolysis rate (determined by Michaelis–Menten equation)

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Acknowledgments

We acknowledge the anonymous reviewers for their helpful comments. We also thank Tracy Bentley and Rose Campbell for improving the English. This study was supported by a graduate research fellowship from the Conseil Régional Provence-Alpes-Côte d’Azur. Funding was obtained from the “Agence de l’eau” de Marseille in the framework of the IBISCUS project (Indicateurs Biologiques et chImiqueS de Contaminations UrbaineS). This work is a CNRS-INSU and Université d’Aix-Marseille II research contribution.

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  1. Laboratoire de Microbiologie, Géochimie et Ecologie Marines, Centre d’Océanologie de Marseille, CNRS UMR 6117, Université de la Méditerranée, Campus de Luminy, 13288, Marseille Cedex 09, France

    Marie Duflos, Madeleine Goutx & France Van Wambeke

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  1. Marie Duflos
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  2. Madeleine Goutx
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  3. France Van Wambeke
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Correspondence to Marie Duflos.

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Duflos, M., Goutx, M. & Van Wambeke, F. Determination of Lipid Degradation by Marine Lipase-Producing Bacteria: Critical Evaluation of Lipase Activity Assays. Lipids 44, 1113–1124 (2009). https://doi.org/10.1007/s11745-009-3358-7

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