LacMeta shows the best decolorization activity in a slightly acidic medium (pH 5-6.5) and at relatively high temperatures (50-60 °C)
We performed enzymatic characterization of LacMeta in the decolorization of MG. In terms of pH, the highest percentage of decolorization (72.62%dc) (Fig. 1A) was observed at pH 5 in sodium acetate buffer, but no significant differences in the percentages of decolorization at pH 5.0-6.5 were noted. Ten temperatures were tested from 20 to 80 °C, and the best temperature was 50 °C, followed by 60 °C, with 91.7%dc and 85%dc, respectively (Fig. 1B). Once the temperature tests were performed at the optimum pH, the result obtained at a temperature of 50 °C was the optimized parameter, and this result was achieved after 1 h of treatment, which was the stipulated time for the reactions. After 1 h of treatment, LacMeta decolorized more than 50%dc of MG at temperatures of 35 °C, 37 °C, and 40 °C.
Thermostability was determined by incubating the enzyme for 60 min at the established temperatures and then measuring the decolorization capacity at each incubation time. LacMeta was thermostable at temperatures of 50 °C and 70 °C (Fig. S1). At 50 °C, the heat inactivation half-life was reached only after 240 min of incubation. At 70 °C, the percentage of decolorization abruptly decreased after incubation for 60 min but then rebounded and remained > 60%dc throughout the test.
The enzymatic decolorization process by LacMeta is resistant to solvents, detergents, ions, and NaCl
Five compounds were tested, including detergents and solvents, of which only two inhibited severe decolorization: SDS and DTT. At all concentrations tested (1 and 2%), the SDS detergent substantially decreased the decolorization, allowing 35.48%dc decolorization at 1%, 0%dc decolorization at 2%, and no decolorization at 10% (Table 1). When DTT was added to the test, the concentrations of 5 mmol L-1 and 10 mmol L-1 resulted in 39.80%dc and 30.50%dc decolorization, respectively. The other compounds were not as impactful. For propanone, the lowest percentage values were more harmful, while the maximum concentration (10 mmol L-1) resulted in a decolorization of 91%dc, which was similar to the value obtained under the control conditions.
When copper was added at higher concentrations, the decolorization capacity of LacMeta was not intensified, and the highest decolorization percentage was approximately 89%dc (Table S1). However, when 10 mmol L-1 cobalt was added to the reaction, approximately 92%dc decolorization was obtained; although it was only 77.41dc at 1 mmol L-1, the decolorization activity was restored starting at 5 mmol L-1. These results were the opposite of those obtained by the authors of (Du et al. 2011), who reported that Co2+ strongly inhibited MG decolorization; however, in similarity to a previous study, Mg2+ and Mn2+ had no inhibitory effects on decolorization. In addition to Co2+, other ions maintained a decolorization capacity of 91%, including Ca2+ at 10 mmol L-1 and Mn2+ at 5 mmol L-1 and 10 mmol L-1, and none of the tested concentrations of both ions resulted in less than 80% decolorization. Co2+ (3 mmol L-1) and other ions negatively affected MG decolorization, with levels of 16%, 3.42%, and 3.52% with Li2+ (1 mmol L-1) and Fe3+ (5 mmol L-1 and 10 mmol L-1), respectively.
The percentage of decolorization was maintained at an equal control until 50 mmol L-1 NaCl, and then it decreased only at higher concentrations (Fig. 2). Even then, the only concentration that resulted in less than 50% decolorization was 2 mol L-1, thus demonstrating that the enzyme tolerates high salt concentrations.
To characterize the decolorization of MG in the presence of some of these compounds, a test was performed at the optimum pH and temperature by adding each of the tested ions (Fe3+, Ca2+, Co2+, Zn2+, Li2+, Mg2+, Mn2+, and Cu2+), including NaCl (Na+) at 1 mmol L-1, combined with methanol and acetone at 1%. After 1 h of incubation, the decolorization rate was 87%dc (Fig. 3A), very similar to that obtained under optimal conditions (Fig. 1B); in addition, it was higher than the values obtained with only Li2+, Co2+, or Mn2+ evaluated at the same concentration. A serious problem with effluents that have excess dyes is their excessive coloration, which prevents the photorespiration of aquatic organisms.
Ability of LacMeta to detoxify malachite green
A phytotoxicity evaluation was performed using the metabolite generated after 24 h of MG dye treatment by LacMeta and was executed without any redox mediator, such as ABTS, simulating a less expensive treatment approach. For both cultures tested, S. bicolor (sorghum) and S. lycopersicum (tomato), the MG without treatment at a concentration of 50 mg L-1 inhibited seed development, resulting in 66.7% and 50% residual germination, respectively, indicating that the dye truly had a significant deleterious effect on germination. Surprisingly, the results obtained with the treatment product did not differ from those in the control (distilled water) for sorghum, which showed 100% germination (Fig. 4A). Good results were also obtained for tomato seed germination, which maintained 80% germination capacity and could be optimized to achieve full detoxification results. Assessing the vegetative parts also revealed that the enzymatic treatment reduced the deleterious effects of the dye. In sorghum seeds, the treatment mainly decreased the effect on the radicula, which is important since it is an important structure for establishing the seedling (Fig. 4B and C). Unlike the pure dye, the metabolic product of MG treatment with LacMeta was not toxic to the tested cultures, which indicated the efficiency of the enzyme in the degradation of the dye and the generation of a less toxic compound.
When antibiosis was evaluated against E. coli, a halo of growth inhibition was observed with the dye but was not formed after enzymatic treatment or with the control (ultrapure water) (Fig. 5A), confirming the effectiveness of the treatment. When the dye and the product of enzymatic treatment were tested against A. brasilense, no apparent inhibition halo was observed with either untreated dye (Fig. 5B).
However, the damaging effect of MG was evidenced when the treatments were compared for liquid culture medium, in which case adding 50 mg L-1 MG caused changes in the growth of cultured A. brasilense in relation to the control (Fig. 5C). As an effect of the untreated dye, the microorganism growth curve reached the decline phase (death) from 6 to 9 h, while for E. coli (Fig. 6D), the stationary phase was prolonged. However, for both microorganisms in the liquid medium assay, the difference between the growth curves of untreated dye and enzyme-treated MG was clearly evident (Fig. 5C and D) since LacMeta-metabolized MG showed growth patterns matching those of both control cultures: The efficiency of the enzyme treatment was evidenced by the treated dye and negative control (without any additive) achieving the same growth performance, reaching approximately 2.5-fold higher growth than that of the positive inhibition control (untreated dye). Moreover, the effect of the dye in the initial phase of cultivation was demonstrated to be more expressive in E. coli, causing a prolongation of the lag phase up to 6 h of cultivation. Subsequently, a short logarithmic phase lasting only 1 h was observed, followed by a stationary phase similar to that observed for the negative control and the treatment, although it reached a maximum logarithmic growth rate approximately 3-fold lower than that observed in these cases (Fig. 5D), and the performance of the metabolite generated by the treatment was consistent with that of the negative control, indicating efficient detoxification.
Interestingly, SEM results provided the magnitude of the detrimental effect of the untreated dye against each of the cultures studied. The dye noticeably altered the cell morphology of A. brasilense but was extremely harmful to E. coli, completely changing the bacterial cell shape (Fig. 6C and F). Cell membrane integrity is essential for microbial survival, and MG exerts cytotoxicity at the structural level, causing deformations, cell size changes, surface destruction, and often cell lysis and bursting (Gopinathan et al. 2015). Thus, although no halo of inhibition was observed for A. brasilense, other characteristics, such as alterations in cell growth and morphology, indicated the cytotoxicity of the dye. The metabolite generated by enzymatic treatment produced no severe deleterious effects on the bacterial strains, thus confirming that the enzyme represents an excellent bioremediation agent (Fig. 6B and E).
Potential for enzymatic decolorization/degradation of LacMeta
The UV–vis spectra of the decolorization of MG treated with LacMeta, with and without a redox mediator (Fig. 7), in ultrapure water at 37 °C indicate the effectiveness of the decolorization enzymatic treatment in relation to the spectrum of the untreated dye. The peak at 620 nm (λmax of MG) significantly decreased after 24 h and especially after 48 h for both treatments. Furthermore, although the redox mediator was used in the reaction (Fig. 7B), the spectra were not disproportionate, with the decolorization rate after 48 h of treatment being 95.5%dc and 94.8%dc with and without the mediator, respectively; that is, both treatments were effective.
H-NMR spectra
The H-NMR spectra in Fig. 8 corroborate the high efficiency of LacMeta in degrading the dye after 48 h of treatment. The MG H-NMR spectrum commonly presents aromatic signals (Fig. 8), three doublets (7.05, 7.37, 7.44 ppm), and two triplets (7.74 and 7.60 ppm), while the LCM spectrum shows three doublets (6.70, 6.91, and 7.06 ppm) and two triplets (7.13 and 7.22 ppm) (Moe et al. 2015), indicating that treating MG with LacMeta did not actually form LCM.