Résumé
Marine
microbiologists commonly assay lipase activities by using a synthetic
fluorescent analog, 4-methylumbelliferyl (MUF)-oleate. The
technique is convenient, but it is considered to be unspecific because
of the structure of this analog. This study reports the design of a new
specific and sensitive lipase assay based on the use of a radiolabeled
triglyceride, [3H]triolein. Free fatty acids (FFA)
resulting from its hydrolysis are isolated as a function of time in a
one-step liquid-liquid extraction and then radioassayed. MUF-oleate and
[3H]triolein techniques were compared by measuring
lipase activities at similar substrate concentrations along a trophic
gradient in the Southwest Lagoon of New Caledonia, near Nouméa.
Hydrolysis rates decreased from the nearshore station to the offshore
station and showed similar trends regardless of the technique used.
Rates decreased from 5.83 to 0.88 nmol of
FFA · liter−1 · h−1
and from 0.76 to 0.23 nmol of
3H-FFA · liter−1 · h−1,
respectively. These results appeared to be consistent with bacterial
production results, which also decreased similarly (from 0.59 to 0.26
μg of
C · liter−1 · h−1).
However, the ratio of MUF-oleate activities to
[3H]triolein activities, which was constant at the
offshore stations (3.8 ± 0.1), gradually increased
at the nearshore stations (from 4.1 to 7.6). This result shows that the
two assays respond in different ways to changes in environmental
conditions and validates the need to set up more specific enzymatic
assays.