3. RESULTS AND DISCUSSION
3.5.3. Influence of humic acid in laccase decolorization of methyl orange
3.5.3.1. Laccase alone
The study of the influence of HA in laccase activity was followed using MO as a different substrate. Even in the absence of mediators, laccase can oxidize MO but at a slow rate as it was seen in previous experiments (Section 3.3). Laccase-catalyzed MO decolorization was assayed in the presence of HA to determine how it is affected by the presence of HA. These assays were carried with greater concentrations of laccase (300 µg/mL) to achieve substantial decolorization rates and to reduce experimental errors. MO decolorization assays by laccase were performed using the method described in Section 2.6.4. In the assays containing the highest HA concentrations (100 mg/L), it was registered pH values of 4.95 ± 0.02, indicating that no significant pH changes occurred during the assays.
Similar to what was observed with ABTS, the presence of HA shows an inhibitory effect on MO decolorization by laccase (Figure 3.27). Experimental results indicate that the presence of concentrations of HA as small as 5 mg/L are sufficient to reduce the rate of MO decolorization significantly (11%). It was also possible to observe that, in the presence of 20 mg/L of HA there is an inhibition of MO decolorization of 30% and plateaus for higher concentrations of HA. The apparent saturation behavior of the inhibition caused by HA observed for ABTS oxidation and MO decolorization assays suggests that the inhibition mechanism of HA might not be dependent on interactions between HA and the substrate. Instead, since a plateauing of the inhibition on laccase activity is observed with both substrates (ABTS and MO), it is possible that the inhibitory effect caused by HA could be due to a direct interaction with laccase.
The inhibition of MO decolorization by high concentrations of HA was further investigated in additional assays as shown in Figure 3.28. These assays confirmed that the laccase catalyzed reaction can be further inhibited, but not even 200 mg/L of HA was able to completely inhibit the enzymatic activity.
Figure 3.27 – Effect of humic acid on methyl orange decolorization by laccase. Decolorization assays were carried with 300 µg/mL laccase and an initial dye concentration of 10 mg/L (30.6 µM), in 100 mM acetate buffer pH 5.0.
Dye decolorization was followed at 477 nm at 25 ºC in the absence and in the presence of different humic acid concentrations from 0.5 to 100 mg/L. The decolorization rates in the presence of humic acid were normalized to the rate observed in the corresponding controls carried using equivalent volumes of Blank solution (buffer without humic acid), which were taken as 0% inhibition. The control decolorization rates in ΔAbs/Δt (min-1) in absence of humic acid were -0.003 ± 0.001 min-1. The data plotted is the Mean ± SE from three independent experiments.
Figure 3.28 – Representative assays of the effect of humic acid on methyl orange decolorization by laccase. The absorbance of methyl orange, initial concentration 10 mg/L (30.6 µM), in 100 mM acetate buffer pH 5.0, was followed at 477 nm, at 25 ºC. The concentration of the enzyme was 900 µg/mL. In the humic acid assay shown, volumes of humic acid stock solution were added to reach the final concentrations indicated, whereas in the Control assay equivalent volumes of blank solution (buffer without humic acid) were added to the spectrophotometer cuvette. The Control assay trace is displaced vertically and horizontally for better visualization.
3.5.3.2. Laccase-ABTS system
It became clear from the experiments performed that HA inhibited laccase activity in both ABTS oxidation and MO decolorization, however, it is still important to investigate how HA affects the activity of the laccase-ABTS mediator system that is being studied in the present work. The inhibitory effect of HA in laccase activity mediated by ABTS was studied using two different concentrations of ABTS (5 and 50 µM).
The results from these assays show that the activity of the laccase-ABTS system is inhibited by the presence of HA. At the resemblance of the results obtained using ABTS as substrate (Section 3.5.2.1), HA concentrations smaller than 5 mg/L did not have a significant effect on the activity of the laccase-ABTS system. HA above 25 mg/L produced a significant inhibitory effect on the activity of laccase-ABTS systems, which is stronger at higher concentrations of HA, reaching around 27%
inhibition on the presence of 100 mg/L. However, the inhibitory effect of HA is not directly proportional to the concentration of HA, as inhibition percentages seem to reach a maximum of inhibition at the highest concentrations studied. In these assays, it is verified that the percentages of laccase-ABTS system inhibition are very similar for systems with 5 µM and 50 µM ABTS, suggesting that the inhibitory effect that HA generates on laccase-ABTS systems is independent of the ABTS concentration.
Figure 3.29 – Effect of humic acid on methyl orange decolorization by laccase-ABTS system in the presence of ABTS. Decolorization assays were carried with 100 µg/mL laccase, an initial dye concentration of 10 mg/L (30.6 µM) and ABTS 5 µM or 50 µM, in 100 mM acetate buffer pH 5.0. Dye decolorization was followed at 477 nm, at 25 ºC, in the absence and in the presence of different humic acid concentrations from 0.5 to 100 mg/L.
The decolorization rates in the presence of humic acid were normalized to the rate observed in the corresponding controls carried using equivalent volumes of Blank solution (buffer without humic acid), which was taken as 0% inhibition. The control decolorization rates in ΔAbs/Δt (min-1) in absence of humic acid were -0.009 ± 0.002 min-1 for assays with 5 µM ABTS and -0.029 ± 0.005 min-1 for assays with 50 µM ABTS. The data plotted is the Mean ± SE are representative of at least triplicate assays.