Page 1 - Caruso_Zaccone_2000

Journal of Applied Microbiology 2000, 89, 951ÿ959

Estimates of leucine aminopeptidase activity in different marine

and brackish environments

G. Caruso and R. Zaccone
Istituto Sperimentale Talassografico CNR, Messina, Italy
342/4/00: received 25 April 2000, revised 28 July 2000 and accepted 10 August 2000
G. CARUSO AND R. ZACCONE. 2000.
Aim: Leucine aminopeptidase (LAP), an enzyme involved in the decomposition of natural
peptides, was measured in different marine and brackish ecosystems, together with some
environmental and microbiological parameters.
Methods and Results: The ¯uorogenic compound L-leucine-7-amido-4-methyl coumarin
was speci®cally used for the determination of this in situ activity. The enzyme data obtained
from this comparative study highlighted the strong spatial and temporal variability of the
distribution of LAP in aquatic ecosystems, which was sometimes related to the course of
environmental variables such as salinity and organic carbon content.
Conclusions: LAP assay has proved to be a rapid method providing useful information on
the microbial metabolic processes involved in the mineralization of organic matter.
Signi®cance and Impact of the Study: The determination of the potential rates of
extracellular enzyme activity is of great ecological importance to extend knowledge on the
role played by bacteria in aquatic biogeochemical cycles.

INTRODUCTION has been proposed as an analogue for natural dissolved
peptides and therefore, as an indicator of the potential
Through their metabolic activities, micro-organisms play a microbial peptidase activity existing in the ecosystems. The
key role in the functioning of natural marine environments; aim of this paper has been the study of the distribution of
the recent hypothesis of the `microbial loop' has high- this enzyme in different Italian marine and brackish areas,
lighted their involvement in the carbon, nitrogen and phos- in relation to the main environmental and microbiological
phorous ¯uxes throughout the ecosystem (Azam et al. variables. Very few data on leucine aminopeptidase activity
1994). Microbial enzymes are of great importance in in the Mediterranean Sea are now available (Misic and
organic matter mineralization, a fundamental step in nutri- Fabiano 1996; Zaccone et al. 1999).
ent cycling. By means of extracellular enzymes, hetero-
trophic bacteria are able to hydrolyse particulate or
dissolved compounds into low molecular weight monomers, MATERIALS AND METHODS
which can be easily metabolized. The development of reli-
According to Hoppe's method (1983), the biochemical
able methods to measure bacterial activity on organic mat-
assay involves the incubation at the in situ temperature of
ter represents one of the goals of microbial ecology
three replicate subsamples (10 ml) with increasing amounts
research. Different ¯uorogenic compounds, such as the ÿ1
of a 05 mmol l stock solution of the substrate L-leucine-
methylumbelliferyl substrates, have been introduced as a
7-amido-4-methyl-coumarin hydrochloride (Leu-MCA,
useful and effective tool for the estimation of the extracel- Sigma), in order to obtain ®nal concentrations ranging
lular enzyme activity (EEA) (Hoppe 1983; Hoppe et al. from 10 to 200 mmol l . One boiled-water blank was pre-
ÿ1
1988; Chrost 1990). By this method, it has been recognized pared from the same sample. The aminopeptidase converts,
that many extracellular enzymes are associated with bacter- by hydrolysis, the Leu-MCA into a ¯uorescent product, 7-
ial cells (Hoppe 1986). In particular, the substrate L-leu-
amino-4-methylcoumarin (MCA), which was detected with
cine-7-amido-4-methyl coumarin (Leu-MCA), hydrolysed
a Hitachi F-2000 spectro¯uorimeter (Hitachi, Osaka,
speci®cally by the enzyme leucine aminopeptidase (LAP),
Japan) at 380 nm and 440 nm excitation and emission wave-
lengths, respectively. The increase in ¯uorescence after 3 h
of incubation is a function of the amount of enzyme pre-
Correspondence to: Dr G. Caruso, Istituto Sperimentale, Talassogra®co CNR,
Messina, Italy (e-mail: caruso@talas.ist.me.cnr.it). sent in the sample, and provides a measure of the `poten-
= 2000 The Society for Applied Microbiology