Determination of lipid degradation by marine lipase-producing bacteria: critical evaluation of lipase activity assays.
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.
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@univmed.fr
Marie
M
Duflos
Madeleine
M
Goutx
France
F
Van Wambeke
D020591:Alteromonas / Q000201:enzymology* / Q000254:growth & development; D001681:Biological Assay; D007700:Kinetics; D008049:Lipase / Q000032:analysis / Q000378:metabolism*; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D019308:Palmitic Acid / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*; D013050:Spectrometry, Fluorescence
D019308:Palmitic Acid; D008049:Lipase