Dimeric coumponds of comparable composition as well as (in some cases) polymeric
substrates have been shown to be competitors of MUF-Substrates. Molecule structure of
MUF-substrates and mechanisms of their enzymatic breakdown are demonstrated in
detail by Hoppe (1983).
Stock solutions were prepared by dissolving 2µM of MUF-substrate in 1 ml Methylcellusolve (ethyleneglycol monomethylether). For some of the MUF-substrates water may be
used as a solvent as well. Aliquot parts of the stock solution ( 1 to 400 µl) were added to 20
ml subsamples of the original water sample, yielding final MUF-substrate concentrations
of 0.1 - 40 µM per 1. Appropriate dilutions or more concentrated stock solutions may be
used in order to gain a more similar solvent level in the subsamples. However, the
influence of the solvent concentrations applied here on enzymatic activity proved to be
negligible. For the substrate MUF-α-D-glucopyranoside additional final concentrations
as low as 0.01 µM per 1 were applied, because saturation levels are occasionally much
lower than for the other substrates.
The fluorescing compound methylumbelliferon is liberated from the combined molecular
complex exclusively by extracellular enzymes. This has been proven by epifluorescence
microscopy, where methylumbelliferon did not penetrate the interior of bacterial cells,
while a strong blue fluorescence was observed in the surrounding medium. It is, however,
admitted that the verification of this statement needs further investigation. Fluorescence
intensity was recorded at 365 nm (excitation) and 455 nm(emission) in a Jasco spectrofluorimeter at the beginning and after 3 h of incubation. Previous time series experiments
exhibited strict linearity of enzymatic activity vs. time over several hours. The fluorescence of methylumbelliferon is pH-dependent and. hence, 2.5 ml subsamples for measuring fluorescence intensity were adjusted to pH 10.3 with a commercial buffer (Merck).
Controls were run with water samples boiled for 20 min in a water bath prior to addition
of the MUF-substrates. Incubation was performed in a water bath at 20°C or at ambient
water temperatures during the annual survey.
Extracellular enzymatic activities are expressed in terms of velocity of hydrolysis V m (µgC
1 -1 h -1 ) and hydrolyzation rate (% h -1 ).
Generally in experiments involving fractionated filtration only three MUF-substrate concentrations were used and results are presented in units of relative fluorescence.
For calibration of methylumbelliferon (which is liberated from the complexes in equimolar concentrations) a calibration curve with different small amounts of methylumbelliferon was established. Calibration was repeated before the experiments from time to time.
The standard deviation of extracellular enzyme activity measurements, as derived from
experiments in different aquatic regions, was in the range of ± 5 % from the mean value.
Methods used for determination of additional parameters: Saprophytes were grown on
ZoBell-agar with a salinity of 15 ‰. After acridine orange staining the total number of
bacteria was counted in an epifluorescence microscope according to Zimmermann and
Meyer-Reil (1974).
Heterotrophic uptake potential for 3 H-leucine was determined according to Hoppe
(1978). Inorganic chemical (NO3", NH3, PO4 3 ), physical (T, S) and planktological
(chlorophyll a, b, c. species distribution) analyses of water samples were conducted
according to standard methods (Grasshoff, 1976 ; SCOR-UNESCO recommandation,
1966).
RESULTS
Origin of extracellular enzymes
Water samples from different brackish water areas were size fractionated with Nucleo121
substrates have been shown to be competitors of MUF-Substrates. Molecule structure of
MUF-substrates and mechanisms of their enzymatic breakdown are demonstrated in
detail by Hoppe (1983).
Stock solutions were prepared by dissolving 2µM of MUF-substrate in 1 ml Methylcellusolve (ethyleneglycol monomethylether). For some of the MUF-substrates water may be
used as a solvent as well. Aliquot parts of the stock solution ( 1 to 400 µl) were added to 20
ml subsamples of the original water sample, yielding final MUF-substrate concentrations
of 0.1 - 40 µM per 1. Appropriate dilutions or more concentrated stock solutions may be
used in order to gain a more similar solvent level in the subsamples. However, the
influence of the solvent concentrations applied here on enzymatic activity proved to be
negligible. For the substrate MUF-α-D-glucopyranoside additional final concentrations
as low as 0.01 µM per 1 were applied, because saturation levels are occasionally much
lower than for the other substrates.
The fluorescing compound methylumbelliferon is liberated from the combined molecular
complex exclusively by extracellular enzymes. This has been proven by epifluorescence
microscopy, where methylumbelliferon did not penetrate the interior of bacterial cells,
while a strong blue fluorescence was observed in the surrounding medium. It is, however,
admitted that the verification of this statement needs further investigation. Fluorescence
intensity was recorded at 365 nm (excitation) and 455 nm(emission) in a Jasco spectrofluorimeter at the beginning and after 3 h of incubation. Previous time series experiments
exhibited strict linearity of enzymatic activity vs. time over several hours. The fluorescence of methylumbelliferon is pH-dependent and. hence, 2.5 ml subsamples for measuring fluorescence intensity were adjusted to pH 10.3 with a commercial buffer (Merck).
Controls were run with water samples boiled for 20 min in a water bath prior to addition
of the MUF-substrates. Incubation was performed in a water bath at 20°C or at ambient
water temperatures during the annual survey.
Extracellular enzymatic activities are expressed in terms of velocity of hydrolysis V m (µgC
1 -1 h -1 ) and hydrolyzation rate (% h -1 ).
Generally in experiments involving fractionated filtration only three MUF-substrate concentrations were used and results are presented in units of relative fluorescence.
For calibration of methylumbelliferon (which is liberated from the complexes in equimolar concentrations) a calibration curve with different small amounts of methylumbelliferon was established. Calibration was repeated before the experiments from time to time.
The standard deviation of extracellular enzyme activity measurements, as derived from
experiments in different aquatic regions, was in the range of ± 5 % from the mean value.
Methods used for determination of additional parameters: Saprophytes were grown on
ZoBell-agar with a salinity of 15 ‰. After acridine orange staining the total number of
bacteria was counted in an epifluorescence microscope according to Zimmermann and
Meyer-Reil (1974).
Heterotrophic uptake potential for 3 H-leucine was determined according to Hoppe
(1978). Inorganic chemical (NO3", NH3, PO4 3 ), physical (T, S) and planktological
(chlorophyll a, b, c. species distribution) analyses of water samples were conducted
according to standard methods (Grasshoff, 1976 ; SCOR-UNESCO recommandation,
1966).
RESULTS
Origin of extracellular enzymes
Water samples from different brackish water areas were size fractionated with Nucleo121