Fluorometric determination of extracellular enzyme
activities in peat using the FLUOstar OPTIMA
Bonnett, S.A.F., Leah, R., and Maltby, E.
Institute for Sustainable Water, Integrated Management and Ecosystem Research, University of Liverpool, Merseyside, UK
Application Note 179
Rev. 09/2008
Fluorescence method to measure extracellular enzyme
activities in peat
Methylumbelliferone (MUF) and derivatives used as substrates
Fast measurement in 96-well format using the FLUOstar
OPTIMA microplate reader
Introduction
Peatland ecosystems represent a significant global store of carbon
(C) due to the historic accumulation of organic matter resulting
from suppressed microbial decomposition.1 Decomposition is
limited by extracellular enzymes that are produced by the plant
and microbial communities and function independently of the
microbial community. The storage of carbon in peatland ecosystems is therefore regulated in part by the environmental factors
that determine extracellular enzyme activities.2 Understanding the
environmental controls and processes that regulate extracellular
enzyme activities is therefore critical in determining the fate of
carbon in peatland ecosystems.
Here we present the results from a method developed to measure
potential extracellular enzyme activities in peat using the BMG
LABTECH FLUOstar OPTIMA. The assay is based on the use of
fluorogenic methylumbelliferyl (MUF) substrates that fluoresce upon
enzymic cleavage allowing the amount of product to be measured.
Materials and Methods
Black 96-well microplates, F-bottom, Greiner Bio-One, UK
MUF and MUF substrates, Sigma-Aldrich, UK
FLUOstar OPTIMA, BMG LABTECH, Offenburg, Germany (Figure 1)
pipette tip, 0.75 mL of peat slurry was placed into a 1.7 mL centrifuge
vial. To the vial, 0.75 mL of MUF substrate was added and samples
were incubated at field temperature for one hour. Samples were
centrifuged for 5 minutes at 12,000 rpm and 300 µL of
supernatant transferred to a well in a plate. Fluorescence was
determined on a BMG LABTECH FLUOstar OPTIMA fluorometer at
330 nm excitation and 450 nm emission wavelength. Enzyme activities
were determined from the fluorescence units using a standard
calibration curve of methylumbelliferone (MUF) and expressed as
rates of MUF production (µmol MUF per g-1 dry peat weight per
min-1). Fluorescence quenching is a potentially interfering process
which decreases the intensity of the fluorescence emission and occurs
in water containing peat-derived compounds. The standard calibration
curve accounted for quenching by dissolving the MUF standard in
150 µL of centrifuged peat slurry for each peat replicate.
Table 1: Enzymes and enzyme MUF substrates
Enzyme
MUF substrate
Reaction
Cellobiohydrolase
MUF-β-D-cellobioside
Cellulose polymers
to dimers
β-glucosidase
MUF-β-Dglucopyranoside
Cellulose dimers
to monomers
Chitinase
MUF-N-acetyl-β-Dglucosaminide
Chitin to
acetylglucosamine
The assay above was performed with substrate concentrations
varying from 0 to 500 µM in order to determine the optimal substrate
concentration. Using the optimal substrate concentrations, samples
were then incubated for varying lengths of time to determine the
optimal assay incubation length.
Results and Discussion
Figure 2 shows the effect of increasing substrate concentration
on the activity of cellobiohydrolase. Activity followed MichaelisMenten kinetics and reached saturation at approximately 200 µM of
substrate.
Fig. 1: BMG LABTECH’s FLUOstar OPTIMA microplate reader
Peat samples were collected from Langdon moor, Durham, UK in
order to determine the concentration of methylumbelliferyl (MUF)
substrates to be added to peat solutions to achieve enzyme ‘active-site’
saturation and the optimal assay incubation length.
The hydrolytic enzymes β-glucosidase, cellobiohydrolase, and
N-acetylglucosaminidase (or chitinase) were determined using MUF
artificial substrates 3 (see Table 1). For each peat replicate sample, ten
cm3 of peat was placed in a 50 mL centrifuge vial and deionised water
added up to the 50 mL mark. The vial was shaken by hand for 30 seconds
and mixed using a vortex for a further 30 seconds. Using a sawn-off
Activity (molg-1 dry weight min-1)
8
7
6
5
4
3
2
1
0
0
100
200
300
400
500
MUF-cellobioside (M)
Fig. 2: Effect of substrate concentration on cellobiohydrolase activity (n = 3).
Figure 3 shows the effect of increasing substrate concentration on the
activity of β-glucosidase. Activity followed Michaelis-Menten kinetics
and reached saturation at approximately 200 µM of substrate. One of
the replicates exhibited substrate inhibition at 100 µM of substrate
resulting in a relative standard deviation of 91 % at 100 µM of
substrate.
30
Activity (molg-1 dry weight min-1)
25
Figure 5 shows the significant quenching effect of peat on
fluorescence.
70000
Standard
Standard in peat
60000
50000
Fluorescence units
One of the replicates exhibited substrate inhibition at 100 µM of
substrate resulting in a relative standard deviation of 85 % at 100 µM
of substrate. This is mainly due to spatial variation of enzyme activity
within the block of peat being analysed, NOT analytical variation.
40000
30000
20000
20
10000
15
10
0
0
5
20
40
50
80
100
MUF-concentration (M)
0
Fig. 5: The quenching effect of peat on fluorescence.
0
100
200
300
400
500
Time course incubations performed using optimal substrate
concentrations for cellobiohydrolase, β-glucosidase and chitinase
showed that one hour was the maximum amount of time that reaction
rates remained linear (data not shown).
MUF-glucoside (M)
Fig. 3: Effect of substrate concentration on β-glucosidase activity (n = 3).
Figure 4 shows the effect of increasing substrate concentration on the
activity of chitinase. Activity followed Michealis-Menten kinetics and
reached saturation at approximately 300 µM of substrate.
3.5
Activity (molg-1 dry weight min-1)
3
2.5
Conclusion
The extracellular enzymes β-glucosidase, cellobiohydrolase and
N-acetylglucosaminidase reached substrate saturation at 200 µM,
200 µM and 300 µM of MUF respectively. Measured enzyme activities
were linear for up to 120 minutes although 60 minutes duration of
incubation was chosen for future applications to minimise variation
between replicates. One replicate exhibited substrate inhibition for the
β-glucosidase and cellobiohydrolase assays.
The results show that potential extracellular enzyme activities can be
determined in peat at low cost and within a short period of time using
the BMG LABTECH FLUOstar OPTIMA microplate reader.
2
1.5
References
1
0.5
0
0
100
200
300
400
MUF-N-acetyl--glucosaminide (M)
Fig. 4: Effect of substrate concentration on chitinase activity (n = 3).
500
1) Gorham, E. (1991) Northern peatlands: role in the carbon cycle and
probable responses to climatic warming. Ecol. App. 1, 182-195.
2) Freeman, C., Ostle, N. and Kang, H. (2001) An enzymic ‘latch’ on a
global carbon store. Nature 409, 149.
3) Freeman, C., Liska, G., Ostle, N.J., Jones, S.E. and Lock, M.A. (1995)
The use of flurogenic substrates for measuring enzyme activity in
peatlands. Plant and Soil 175, 147-152.
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