Optimization of a β-glucosidase enzyme assay in peat using the FLUOstar OPTIMA

Samuel Bonnett, Rick Leah* & Ed Maltby
Institute for Sustainable Water, Integrated Management and Ecosystem Research, University of Liverpool.
*died on the 1st February 2009

Introduction
Peatland ecosystems represent a significant global store of carbon (C) due to the historic accumulation of organic matter resulting from suppressed microbial decomposition (Gorham, 1991). 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 (Freeman et al, 2001). 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 further optimization of a method developed to measure potential β-glucosidase activities in peat using the BMG Labtech FLUOstar OPTIMA. The assay is based on the use of fluorogenic methylumbellifone (MUF) substrates that fluoresce upon enzymic cleavage, allowing the amount of product to be measured. This work was part of a United Utilities funded project investigating the impact of moorland re-wetting on peat degradation processes in the Goyt Moors (Bonnett et al. 2008a) and builds on previous assay work (Bonnett et al. 2008b).

Materials and methods
Peat samples were collected from four sites (sites 1-4) from blanket bog on the Goyt valley estate, UK. MUF substrate of varying concentration was added to peat solutions to determine the enzyme ‘active-site’ saturation (Vmax) and the optimal assay incubation length. Sites 1 and 2 were located close to blocked grips with high water table levels whilst sites 3 and 4 had unblocked grips with low water table levels.

The hydrolytic enzyme β-glucosidase was determined using MUF artificial substrates (Freeman et al., 1995). Enzyme activities were determined by placing 5 cm3 of peat in a 50 ml vial with deionised water added to the 50 ml mark. Each sample was thoroughly homogenised using a spatula and vortexer for 1 minute before filtering the suspension through a 2 mm sieve. For three replicate suspensions from each site, 1 ml of suspension was placed in a 2 ml microcentrifuge vial and 1 ml of MUF glucopyranoside substrate added in increasing concentrations from 0 to 500 µmol/l. Samples were incubated in a cold room for 1 hour followed by centrifugation at 12 000 rpm for 5 minutes. 300 µl of extract were pipetted into a well on a 96-well plate and the fluorescence was determined on a BMG LABTECH FLUOstar OPTIMA fluorometer at 450 nm emission and 330 nm excitation wavelength. Enzyme activities were determined from the fluorescence units using a standard calibration curve of MUF and expressed as rates of MUF production (µmol MUF per g dry peat weight per min). 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 peat replicates from site 1 and 3 which were representative of sites 1/2 and 3/4.

Results and discussion

Sites 1/2 and 3/4 differed in both Vmax (maximum rate of reaction) and Km (inverse of the affinity of the enzyme for substrate) that may be explained by the differences in water table level between sites. However, neither site reached full enzyme saturation at a high concentration of substrate (>500 µmol/l). This was an issue for routine determinations of Vmax as were the differences in Km, leading to speculation regarding the optimization of the assay. Therefore the methods were evaluated and developed further. Figure 2 shows the quenching (interference) effect of peat from sites 1 and 3 on enzyme MUF concentration. Quenching is a potentially complex phenomenon that reduces fluorescence although it did not differ between sites suggesting that quenching cannot explain differences in Vmax and Km between the sites.

Figure 2 Standard curves for methylumbelliferone standard in peat extract and water.

The background fluorescence of the MUF glucopyranoside substrate was measured in deionised water and peat extracts to check for interference. Normally, the enzyme-specific substrate is chemically attached to the fluorescent MUF moiety causing the suppression of the fluorescent property of the chemical. It is only upon enzymic hydrolysis of the bond between MUF and the attached compound that the MUF is liberated and its fluorescence measured and related to enzyme activity (also called cleavage or unquenching). However, figure 3 shows that significant background fluorescence of MUF substrate can occur although estimated to have a magnitude of less than 1 % of the concentration of MUF substrate. Whilst the amount of MUF that fluoresces is low relative to the concentration of total MUF substrate in the assay, it is similar in magnitude to the amount of substrate cleaved by enzymes in peat and therefore greatly reduces the sensitivity of enzyme activity estimations within this study. The correction for the presence of unreacted MUF substrate was best predicted by a sigmoidal function. Based on this equation the background interference for each substrate could be estimated from the concentration of MUF substrate used in the assay. The fluorescence of MUF glucoside substrate in peat is probably lower due to quenching.

Figure 3 Background fluorescence of MUF glucopyranoside substrate in water and peat extract.

Fig. 4 shows the concentration of background fluorescent MUF glucopyranoside substrate in water and peat extract after quench correction using the standard curve. In this test the MUF glucopyranoside was added to peat suspension and centrifuged immediately to form a peat extract. The greater concentration of background fluorescent MUF at high substrate concentration was due to some substrate breakdown by enzymes in the peat despite the short-incubation time. This suggests that background fluorescence cannot be accurately determined in peat due to pre-steady state enzyme kinetics. Pre-steady state kinetics (or burst phase kinetics) occur within nanoseconds of substrate addition. The negative concentration on the y-axis at low MUF substrate concentration in peat was not corrected for as this suggests that quenching has important effects that are not fully understood at present. However, the background interference caused by the MUF substrate is susceptible to quenching by the peat.

Figure 4 Concentration of MUF glucopyranoside substrate that fluoresces in water without enzymic cleavage and in peat extract due to interference and pre-steady state enzyme kinetics.

Figure 5 shows the activity of β-glucosidase corrected for background interference by subtracting the appropriate MUF equivalent. This graph suggests substrate inhibition of β-glucosidase activity in which a high concentration of substrate blocks the interaction between the enzyme active site and the substrate molecule. The concentration of enzymes in peat would be expected to be low and thus be saturated at low substrate concentration.

Figure 5 β-glucosidase activity corrected for background interference of MUF glucopyranoside substrate.

Figure 6 shows glucosidase kinetics at each site at lower substrate concentration which suggests that the enzymes were saturated at different concentrations and that Vmax was higher at site 1 compared to site 3. The differences between the two figures may be due to natural variability of enzyme activities in peat rather than differences in Vmax and Km. In particular this may have been due to burst phase kinetics of the background substrate fluorescence correction.

Figure 6 β-glucosidase activity corrected for background interference of MUF glucopyranoside substrate. Note the lower substrate concentration.

In figure 6, the concentration of substrate that achieved Vmax differed between site 1 (150 µmol/l) and site 3 (75 µmol/l). Also, it appears that Km may differ between sites, although presently there is no replication to prove this. Unfortunately it is not possible to determine Km automaically as the optical density of the peat-substrate incubations masks the fluorescence. Also, it would not be appropriate to measure Vmax routinely at different substrate concentrations for each site as some replicates may not be fully saturated by substrate. Therefore, the final method for β-glucosidase was determined as 100 µmol/l of substrate incubated for 30 minutes.

Conclusion
Estimates of Vmax and Km were susceptible to interference by unexpected fluorescence of the unreacted MUF substrate compounds. By accounting for background fluorescence and burst phase kinetics, results showed significant substrate inhibition of enzyme in peat suspensions and suggested it would be necessary to use low substrate concentration (100 µmol/l) and a short-incubation period for the determination of β-glucosidase activity.

Acknowledgements
This work was funded by United Utilities PLC. We acknowledge the support of Martin McGrath at United Utilities. Sadly, Rick Leah died towards the completion of this project. He was central to the completion of this work and will be greatly missed.

References
Bonnett, SA.F., Leah, R. & Maltby, E. (2008a) An investigation of the impact of moorland re-wetting on peat degradation processes in the Goyt moors. Report for United Utilities. University of Liverpool.

Bonnett, S.A.F., Leah, R. & Maltby, E. (2008b) Fluorometric determination of extracellular enzyme activities in peat using the FLUOstar OPTIMA. BMG Labtech Application Note 179, Rev. 09/2008.

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.

Freeman, C., Ostle, N. and Kang, H. (2001) An enzymic ‘latch’ on a global carbon store. Nature 409, 149.

Gorham, E. (1991) Northern peatlands: role in the carbon cycle and probable responses to climatic warming. Ecol. App. 1, 182-195.