Conditions Optimizing and Application of Laccase-mediator System (LMS) for the Laccase-catalyzed Pesticide Degradation

A high capacity of laccase from Trametes versicolor capable of degrading pesticides has been revealed. The conditions for degrading of five selected pesticides including chlorpyrifos, chlorothalonil, pyrimethanil, atrazine and isoproturon with the purified laccases from Trametes versicolor were optimized. The results showed that the optimum conditions for the highest activity were pH at 5.0 and temperature at 25 °C. The best mediators were violuric acid for pyrimethanil and isoproturon, vanillin for chlorpyrifos, and acetosyringone and HBT for chlorothalonil and atrazine, respectively. The laccase was found to be stable at a pH range from 5.0 to 7.0 and temperature from 25 to 30 °C. It was observed that each pesticide required a different laccase mediator concentration typically between 4.0–6.0 mmol/L. In the experiment, the degradation rates of pyrimethanil and isoproturon were significantly faster than those of chlorpyrifos, chlorothalonil and atrazine. For example, it was observed that pyrimethanil and isoproturon degraded up to nearly 100% after 24 hours while the other three pesticides just reached up 90% of degradation after 8 days of incubation.

Scientific RepoRts | 6:35787 | DOI: 10.1038/srep35787 In order to better understand the pesticide degradation by laccases, this research group optimized the conditions of laccase from white-rot fungus Trametes versicolor for degrading five selected pesticide, including chlorpyrifos, chlorothalonil, pyrimethanil, atrazine, and isoproturon, with the presence of different mediators at different pH, temperature and mediator concentrations.

Results
Effects of pH and temperature on the laccase activity. The results of relative activity are given in Tables 1 and 2. Overall, the relative activities of laccases decrease as the incubation day increases. As can be seen, the effects are different with incubation temperature (Table 1) and pH ( Table 2). In Table 1, the relative laccase activities on day 8 were 70.4%, 74.3%, 71.6%, 40.5%, and 11.5% for the incubation temperature of 25,30,20,35, and 40 °C, respectively. Thus, the optimum temperature is 25 °C (with no significant change at 20-30 °C, 8 days incubation). In Table 2, the relative laccase activities on day 8 were 71.6%, 65.7%, 60.3%, 27.7%, 2.7%, and 0% for the pH of 5.0, 6.0, 7.0, 4.0, 3.0, and 2.0, respectively. It should be noted that, after 24 hours incubation, the relative laccase activities decreased to 6.8% and 14.2% when pH at 2.0 and 3.0, respectively (Table 2). From 24 hours to day 8, in Table 2, the relative laccase activities were observed to be relatively stable, i.e., no significant changes within the experimental error. In contrast, in Table 1, the relative laccase activities seemed to have clear decreasing trends with incubation days. For example, at 40 °C, the relative activity of laccase was 100%, 71%, 42%, 29%, 16% and 14% on days 0, 1, 2, 4, 6, and 8, respectively.
Roles of the mediators in the process of pesticide degradation. White-rot fungi with an appropriate mediator are able to degrade the pesticides. The results are presented in Fig. 1. Data showed that the laccase extracted from Trametes versicotor with an appropriate mediator promoted the degradations of five selected pesticides including atrazine, chlorothalonil, isoproturon, pyrimethanil and chlorpyrifos. Comparing to the treatments of CK and without a mediator, the optimizing mediators were HBT for pesticide atrazine. Its degradation rate was up to 75.0% with HBT as a mediator. But with other mediators, the rates were found to be ranging from 21.7% to 38.9%. For isoproturon and pyrimethanil, the best mediators were found to be acetosyringone, ABTS, HBT and violuric acid. Syringaldehyde and vanillin were the best mediators for chlorpyrifos only. As can be seen in Fig. 1, the mediators play important roles in the degradation of the pesticides by laccase catalysis except for chlorothalonil. The degradation of isoproturon and pyrimethanil reached above 60% in the presence of a proper mediator 6 and 10 h incubation, respectively. However, it took 2 days for other selected pesticides to reach the similar degradation (60%). The data demonstrated that the degradation rates of isoproturon and pyrimethanil were higher than other three pesticidee. With violuric acid as a mediator, the decline rates of isoproturon and pyrimethanil were approximately 98% within 24 h. The degradation of atrazine, chlorothalonil, and chlorpyrifos ranged from 70.4% to 91.6% (8 days) with an appropriate mediator. However, without any mediator, the degradation rate of chlorothalonil was 78.6%. Similarly, for other four pesticides, the rates were from 6.1% to 38.9%.
Optimization of pH, temperature and mediator concentration for pesticide degradation. In order to obtain the optimum conditions for pesticide residues in the biodegradation, pH of culture solution, incubation temperature and mediator concentration were studies. The pH values for laccase were determined by measuring the pesticide degradation rates in a citric acid-dibasic sodium phosphate buffer at varying pHs (pH 3-7). As can be seen in Fig. 2a, comparing to CK treatments, the pH optima were at 4.0 for chlorothalonil, isoproturon and pyrimethanil while for atrazine and chlorpyrifos, their optimum pHs were found to be pH 5.0. The best temperatures for the degradations of chlorothalonil, atrazine, chlorpyrifos and isoproturon, pyrimethanil demonstrated at 30 and 35 °C, respectively (Fig. 2b). The rates of pesticide degradation were found to be greater than 79% with the mediator concentrations ranging from 2.0 to 10.0 mmol/L. For isoproturon and pyrimethanil, the rates reached to nearly 100% with 4.0 mmol/L of mediator after 1 day treatment. The results suggested that the pesticide degradation rates increased with increasing the fortification of the mediator concentration until it reached the maximum, i.e., approximately 100% at 8.0 mmol/L (Fig. 2c).

Discussion
Temperature, pH and mediator play important roles in laccase activity and its catalyzed pesticide degradation.
In the literature, most of the fungal laccase optimum pH is 5.0 (form 4.0 to 6.0). For example, the pH optimum was at 5.0 for the laccase from the edible wild mushrooms, including Albatrella dispansus 20 , Cantharellus cibarius 21 . Coriolus hirsutus 22 , Lentinula edodes 23 and Tricholoma giganteum 24 and pH 4.0 for Hericium erinaceum 25 and Panus tigrinus 26 . However, in terms of maximal activities, these reports indicated that the most of laccases required higher incubation temperatures in the previous work than the that for selected Trametes versicolor in this study. The laccases from C. cibarius and H. erinaceum which had an optimum temperature at 50 °C, while C. hirsutus and R. lignosus were at 45 °C and 40 °C, respectively. Laccase requires a temperature of 40 °C to exhibit its maximal activity. The high activity observed at 35-45 °C for Rigidoporus lignosus (38). As shown in Table 2, an optimum temperature of 25 °C was required for the laccase from Trametes versicolor to reach its maximal activity. When the temperature was at 20 and 30 °C, it also showed a good stability of laccase activity in this study.
The results demonstrated that the laccase from Trametes versicolor with an appropriate mediator could accelerate the degradations of five selected pesticides compared to the treatments of without a mediator. A number of studies reported the capacity of removal of pesticides in contaminated agricultural environment by LMS. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator of 2,2′ -azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) indicated complete and rapid oxidative degradations  28 . The results of twelve halogenated organic pesticide analyses in the presence of nine different mediators indicated that the acetosyringone and syringaldehyde appeared to be the best mediator 29 . The degradation rates of herbicide glyphosate 24 hours incubation were 40.9%, 62.8% and 90.1% by three kinds of mixture of laccase and ABTS, Mn 2+ , Tween 80 with laccase, respectively 3 . Fungicide cyprodinil did not show transformation when incubated alone with a laccase from Trametes villosa. But it was transformed to a significant extent, when a mediator was present 30 . In the literatures, the laccase-catalyzed pesticide degradations depended on selected pesticide physicochemical properties and different optimum of LMS. The pH optima for fungal laccases rely generally on the acidic region. The pH optima were reported that had a wide range showing from 2.7 to 7.5, and typically in the range of 3.5 to 6.0, depending on the different substrate 31,32 . Amitai et al. reported that the optimal pH for degradation of VX and RVX was 7.4 as compared to DiPr-Amiton that is degraded more rapidly at pH 8.0 27 . Stepanova et al. showed that the pH optimum of catechol oxidation by Pleurotus oastreatus 0432 laccase was 6.0 33 . The results demonstrated that the LMS with pH optima for chlorothalonil and chlorpyrifos were at 5.0, for atrazine, isoproturon and pyrimethanil were at 4.0, respectively.
Mediator concentration is also a factor in laccase-catalyzed pesticide degradation. The optimal molar ratio of ABTS/OP for VX and RVX degradation was 1:20, whereas, the rate of diPr-amiton degradation rate reached its peak at a molar ratio of 1:10 27 . The natural mediator syringaldehyde showed to be an efficient mediator and the highest pesticide transformation rates of recalcitrant halogenated pesticides were obtained with a mediatorsubstrate proportion of 5:1 29 . For all the selected pesticides in this study, the maximum activity was reached at a mediator concentration of 4.0 mmol/L, which was slightly higher than the values in the literature. But the initial concentrations of the selected pesticides in this study (20.0 mg/L for each) were significantly higher than those above-mentioned.

Methods and Materials
Instruments. Gas  Measurement of laccase activity. Laccase activity was measured at 420 nm by generation of ABTS2 + radicals from the enzymatic oxidation of ABTS at 25 °C using a spectrophotometer. The assay mixture contained 200 μ L of 0.5 mmol/L ABTS, 2700 μ L of sodium acetate buffer (pH 4.5) and 100 μ L of the enzyme-containing sample. One unit of laccase activity (U) was defined as the amount of enzyme that formed 1 μ mol ABTS per min, using the extinction coefficient ε 420 nm of 36,000 /M/cm 34 . Influence of the pH and temperature on laccase stability. Laccase was added in citric acid-dibasic sodium phosphate buffers with the pH at 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0. The incubation system were in tubes put in an incubator, and the temperature was controlled at 25 °C. Samples were collected at 1, 2, 4, 6 and 8 days intervals after laccase application for the detection of laccase activity. Laccase activity was controlled in 50 U/L of each treat at the beginning. Laccase was separately added to citric acid-dibasic sodium phosphate buffers with the pH at 5.0. The incubation system were in tubes put in the incubator, and the temperature was controlled at 20, 25, 30, 35, and 40 °C, separately. Samples were collected at 1, 2, 4, 6 and 8 days after Laccase application for the detection of laccase activity.

Degradation of pesticides by laccase.
Degradation experiment were carried out in 10 mL tubes in citric acid-dibasic sodium phosphate buffers containing 20.0 mg/L pesticides in the dark at 30 °C in the incubator. The enzyme activity was in the control of 0.05 U/mL by an ultraviolet spectrophotometer. Samples of atrazine, chlorothalonil, chlorpyrifos, and pyrimethanil, isoproturon were collected at 1, 2, 4, 6, 8 days and 0, 2, 6, 10, 16, 24 h after incubation, respectively. Then the residues were measured by HPLC or GC. All of the treatments including controls were in triplicates.
Pesticide residues analysis. For chlorothalonil, pyrimethanil, chlorpyrifos and isoproturon, 2.0 mL of the buffer solution was added into a 10 mL glass tube, aloowed by adding 2.0 mL of petroleum ether to the extract. The mixture was vortexed for 2 min and wait for the layer separation. The supernatant fluid (petroleum ether, organic phase) was transferred into a clean glass tube. Repeat the above steps 2 additional times. Then the supernatants were combined. Three (3.00) mL of the supernatant was transferred into another tube and dried under a stream of nitrogen (40 °C). The pesticide residues were dissolved in 1 mL of appropriate organic solvent* and filtered through a 0.22 μ m nylon filter into an autosampler vial. (*The residue of chlorpyrifos was dissolved in n-hexane for GC analysis, chlorothalonil and pyrimethanil were dissolved in methanol for HPLC analysis, and isoproturon were dissolved in acetonitrile for HPLC analysis. For atrazine, it was diluted using acetonitrile for HPLC analysis just after filtered using a 0.22 μ m nylon filter without additional extraction or cleanup steps).