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Fluorometric determination of extracellular enzyme activities in peat using a BMG LABTECH microplate reader

SAF Bonnett, R. Leah, E. Maltby Institute for Sustainable Water, University of Liverpool 09/2008

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).


Conclusion

 

The extracellular enzymes β-glucosidase, cellobio-hydrolase 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 cellobio-hydrolase 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 a BMG LABTECH microplate reader.

Peatland ecosystems represent a significant global store of carbon (C) due to the historic accumulation of organic matter resulting from suppressed microbial de-composition. Decomposition is limited by extracellular enzymes that are produced by the plant and microbial communities. The storage of carbon in peatland ecosystems is therefore regulated in part by the environmental factors that determine extracellular enzyme activities.

 

Here, we present the extracellular enzyme activities in peat measured on a BMG LABTECH microplate reader. The assay is based on methylumbelliferyl (MUF) substrates that fluoresce upon enzymatic cleavage allowing the amount of product to be measured.

 

The extracellular enzymes β-glucosidase, cellobio-hydrolase and N-acetylglucosaminidase reached substrate saturation at 200 μM, 200 μM and 300 μM of MUF-substrate, respectively.

 

The results show that extracellular enzyme activities can be determined in peat at low cost and within a short period of time using a BMG LABTECH microplate reader.

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