Enhanced catalytic activity and thermal stability of 2,4-dichlorophenol hydroxylase by using microwave irradiation and imidazolium ionic liquid for 2,4-dichlorophenol removal

Abstract

Enzymatic removal of 2,4-dichlorophenol (2,4-DCP) has become more attractive recently due to its high efficiency, low cost and environmental benefits. A highly active 2,4-DCP hydroxylase for 2,4-DCP removal was obtained and used in 2,4-DCP removal by employing microwave irradiation as a heating mode and ionic liquid (IL) as an additive. Both [EMIM][PF₆] (1-ethyl-3-methylimidazolium hexafluorophosphate) and microwave irradiation were found to increase the 2,4-DCP removal efficiency and the thermal stability of 2,4-DCP hydroxylase, and a further incremental effect of microwave irradiation and [EMIM][PF₆] on improving the 2,4-DCP removal efficiency and enzymatic thermal stability was observed. Conditions for 2,4-DCP removal were optimized and the removal of 2,4-DCP was completed in 15 min at 25 °C with 0.66 ± 0.015 U mg⁻¹ enzyme activity under the optimum conditions, much faster than the present enzymatic removal route, which took several hours for complete 2,4-DCP removal. Only 30 min were required for complete 2,4-DCP removal by using the 2,4-DCP hydroxylase at 4 °C, indicating its psychrotrophic adaptability. These results showed that the use of 2,4-DCP hydroxylase under microwave in [EMIM][PF₆] is a fast, efficient and environmentally benign method for the removal of 2,4-DCP, and this method can be used over a wide temperature range.

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1. Introduction

2,4-Dichlorophenol (2,4-DCP) is a serious global concern because of its carcinogenicity, toxicity and persistence. It is used for the production of germicides and soil sterilizers, as well as in the manufacture of methylated chlorophenols used in the production of antiseptics and disinfectants.^1,2 2,4-DCP ranks at the top of the list of priority pollutants by the United States environmental protection agency due to its toxicity, strong odor, bioaccumulation persistence in the environment and suspected carcinogenicity and mutagenicity to living organisms.³ Although its usage has been strictly restrained, a significant amount of 2,4-DCP is still contained in surface water, industrial effluents, wastewater, potable water, and soil, as well as in atmospheric emissions from the combustion of municipal solid waste, hazardous waste, coal, wood, and herbicides.⁴ Therefore, techniques for its disposal are critically needed.

In recent years, biochemical removal for the degradation of 2,4-DCP with microorganisms or enzymes has been highly focused on and proven to be more efficient, less expensive and more environmental-friendly than its physical or chemical counterparts.⁵ However, the application of the microorganism method is limited due to a long removal period, low degradation rates, and strong inhibitory effect by high concentrations of chlorophenols. In addition, bioremediation by using microorganisms is difficult to apply to practical remediation of pollutants at low concentrations.⁶

Enzymatic methods provide a good alternative to the current biotreatment processes for 2,4-DCP degradation. Generally, the storage and handling of enzymes are easier than those of microorganisms,⁷ and the tolerance of enzymes for concentrated 2,4-DCP makes it possible to pair with other physical, chemical and enzymatic processes simultaneously.⁸ Several enzymes in the class of oxidoreductases, such as 2,4-dichlorophenol hydroxylase (2,4-DCP hydroxylase, EC 1.14.13.20), peroxidase (EC 1.11.1.7), and laccase (EC 1.10.3.2), have been explored for the removal of 2,4-DCP.⁹ Among these enzymes, 2,4-DCP hydroxylase has been found to be a viable alternative in the 2,4-DCP removal process (the obtained 3,5-dichlorocatechol is less toxic and easy to be degraded for the subsequent ring-opening reaction), as can be observed from the great number of works published in this area.⁶ This enzyme is a key enzyme in the pathway for the degradation of 2,4-dichlorophenoxyacetic acid in many bacteria.¹⁰ In our previous study, a novel 2,4-dichlorophenol hydroxylase, designated as TfdB-JLU, was identified from a metagenome constructed from polychlorinated biphenyl-contaminated soil by functional screening, and it showed great potential for bioremediation of 2,4-DCP due to its broad substrate specificity and its ability to operate at wide ranges of pH.¹¹ Although this enzyme exhibited higher 2,4-DCP affinity than other enzymes, the enzyme activity and thermal stability of the 2,4-DCP hydroxylase are still not satisfactory from an economic point of view. Therefore, it is necessary to find an adequate method to increase the enzyme activity and thermal stability and decrease the energy consumption to promote the application of the enzymatic degradation of 2,4-DCP.

Microwave irradiation and ionic liquids (ILs) are two important and rapid developing technologies in green chemistry, and these two methods have been employed to accelerate enzymatic reactions and increase enzyme stability.¹² Microwave irradiation, in conjunction with other techniques, has been proved to increase the decomposition efficiency and reduce the time required for removing pollutants.¹³ On the other hand, dramatically improved enzyme stability and activity in enzymatic degradation were observed by choosing suitable ILs.¹⁴ ILs can be regarded as clean and green solvents for separation, chemical production, and catalysis because of their high vapor pressure, non-flammability and excellent solubility for both organic and inorganic compounds.¹⁴ Notably, the excellent microwave-absorbing ability of ILs makes them good solvents for microwave-assisted catalysis. The incremental effects of microwave irradiation and IL on improving enzyme activity in biodiesel production and in the isomerization of xylose to xylulose have recently been verified by our lab.^15,16 However, investigation of microwave irradiation and ILs in biodegradation has not been reported to our knowledge.

The aim of this study was to develop an efficient enzymatic 2,4-DCP removal process (convert to 3,5-dichlorocatechol). The expression and purification process for 2,4-DCP hydroxylase was optimized to improve the enzyme activity, and enzymatic removal of 2,4-DCP by using 2,4-DCP hydroxylase was conducted through a new combined method (microwave/IL) (Scheme 1). The effects of the reaction conditions (microwave power, ILs, temperature, 2,4-DCP and enzyme concentration) on the enzyme activity were screened and optimized. Furthermore, the thermal stability of 2,4-DCP hydroxylase under microwave irradiation in IL was also evaluated.

Scheme 1 2,4-DCP treatment by using 2,4-DCP hydroxylase under microwave irradiation in ionic liquid.

2. Material and methods

2.1 Materials

All chemicals used in the present study were of analytical grade. 2,4-DCP and other chemicals were obtained from Sigma. Recombinant Escherichia coli. DH5α containing the TfdB-JLU gene for 2,4-DCP hydroxylase expression was from our lab. BugBuster protein extraction reagent was from Novagen (Nottingham, UK). ILs were purchased from Cheng Jie chemical company (Shanghai, China), whose purities were claimed to be more than 99% (w/w) with a residual chloride content less than 0.01% (w/w). ILs were dried in vacuum oven for at least one day to remove water before the reaction. The abbreviated names and full names of the used ILs are shown in ESI.†

2.2 Methods

2.2.1 Protein expression and purification. The recombinant E. coli was cultivated in LB medium containing 30 μg kanamycin per ml and 34 μg chloramphenicol per ml at 37 °C. Protein expression was induced at 18 °C by the addition of 0.2 mM isopropyl-β-D-1-thiogalactoside (IPTG) (Fisher Scientific, Fairlawn, NJ) at an OD600 of 0.4. After 15 h incubation, the cell pellets were harvested by centrifugation at 12 000 rpm and washed with 50 mM sodium phosphate buffer, pH 8.0. For the preparation of crude extract, cells (1.11 g of E. coli cell paste from 400 ml fermentation culture) were suspended in 4 ml pH 8.0 Bugbuster Protein Extraction Reagent with 1 mM dithiothreitol (DTT) and 0.6 mM PMSF (phenylmethylsulfonyl fluoride) to yield a higher specific activity compared to ultrasonication. The protein extraction was performed for 10 min at 20 °C at 150 rpm. Then, the lysate was centrifuged at 12 000 rpm for 10 min using a Thermo Sorvall WX Ultracentrifuge (Fisher Scientific, Fairlawn, NJ, USA) at 4 °C. The supernatant was transferred onto a Hislink™ column (Promega, Madison, WI, USA), rinsed with wash buffer (10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM imidazole, pH 8.0), and eluted with elution buffer (10 mM HEPES, 1 M imidazole, pH 8.0). The protein supernatant was loaded onto a nickel-nitrilotriacetic agarose resin (Qiagen, Germany) equilibrated with the same buffer. After washing with 5 column volumes of the wash buffer (40 mM imidazole), the bound enzyme was eluted with the elution buffer (200 mM imidazole). The fractions containing 2,4-DCP hydroxylase activity were pooled and concentrated by ultrafiltration by using Amicon Ultra-15 centrifugal filter units (Millipore, USA) and then were diafiltered with 50 mM sodium phosphate buffer, pH 7.5, containing 10% (v/v) glycerol. A total of 4 ml protein solution (300 μg ml⁻¹) was obtained after 4000 rpm centrifugation. Samples were stored at −80 °C for further analysis.

2.2.2 2,4-DCP removal. The experiments on 2,4-DCP removal were performed in a 10 ml three-neck round-bottomed flask. The reaction mixture was placed into the microwave reactor (MCR-3, Shanghai JieSi Microwave Chemistry Corporation), with an immersion well placed into one of the necks of the flask and a glass connector (linking the flask inside the cavity with a reflux condenser outside the cavity) into the other. A temperature probe was placed into the immersion well. The reaction mixture was heated to the desired temperature. The conventional heating reactions were performed using a digital heating circulating water bath (Changzhou Electrical Instrument Factory, HH-42). The activities of 2,4-DCP hydroxylase were determined by monitoring the decrease in absorbance at 340 nm (ε₃₄₀ = 6220 M⁻¹ cm⁻¹) following the substrate-dependent oxidation of NADPH.¹¹ Unless otherwise indicated, standard reactions were performed by incubating purified enzyme (final concentration 12 μg ml⁻¹) with 0.1 mM 2,4-DCP and 0.2 mM NADPH in 50 mM sodium phosphate buffer (pH 7.5) containing 60 μM IL at 25 °C. After the reaction, samples were quickly moved to 100 °C hot water to deactivate the enzyme. All the removals were performed for three times and statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's test.

2.2.3 Thermal stability. The thermal stability assays were performed by incubating 2,4-DCP hydroxylase at 35 °C under conventional heating without IL, under microwave irradiation without IL, in the presence of pH 7.5 PBS buffer containing 60 μM [EMIM][PF₆] under conventional heating and under microwave irradiation. After incubation for different times, samples were removed and assayed for residual activity. All the thermal stability tests were performed for three times and statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's test.

2.2.4 Enzyme assay and characterization. The enzyme assay and kinetic parameters (K_m: Michaelis–Menten constant and V_max values: the maximum reaction velocity) for 2,4-DCP during removal were determined as described previously.¹¹ One unit of activity was defined as the amount of enzyme required to consume 1 μmol NADPH per min at 25 °C. Protein concentrations were determined by the BCA method (Novagen® BCA Protein Assay Kit) using bovine serum albumin as the standard.

3. Results and discussion

3.1 Protein expression and purification

In previous works, we reported the cloning and characterization of a novel 2,4-DCP hydroxylase. However, the activity of this enzyme was lower than those reported in the literature.^17–19 The heterologous expression of enzyme is usually limited by the loss of activity at high temperatures or by low soluble recombinant protein yield.²⁰ Additionally, the lysis and extraction of cells using sonication was regarded to decrease the solubility and activity of enzymes.²¹ The overexpression of proteins in E. coli at low temperatures²² and low IPTG concentration²³ usually improves both protein solubility and activity.²⁴ In order to increase active enzyme yield, the expression conditions and cell disruption method were modified, and the purification process was shortened on the premise of not affecting the purity of the enzyme.

In this study, lowering the temperature and IPTG concentration for protein induction and the use of BugBuster® protein extraction reagent, which was an effective alternative to mechanical methods for releasing expressed target protein, were performed to improve the recombinant protein soluble expression and enzyme activity. As shown in ESI Table SI-1,† the target protein was highly overexpressed, and the enzyme accounted for an estimated 23% of the total protein of the crude cell free extract in this study. Consistent with literature reports, we observed that induction at 18 °C for 15 h with 0.2 mM IPTG (at OD = 0.4) increased the purified protein yield and specific activity compared with our previous results. The specific activity of the purified enzyme towards 2,4-DCP was 0.66 ± 0.015 U mg⁻¹ at pH 7.5 and 25 °C, a 4.1-fold increase in specific activity compared to our previous result, which was 0.16 U mg⁻¹. The total purified 2,4-DCP hydroxylase yield of the recombinant enzymes after the purification procedure was 10.44 mg g⁻¹ protein per cell, a 3.6-fold increase in yield compared to our previous result, which was 2.9 mg g⁻¹ protein per cell, and this enzyme yield result was superior to the literature. The one step purification in this study did not have a significant effect on the protein yield and purity, an electrophoretically homogeneous enzyme, as shown in ESI Fig. SI-1,† was obtained with a yield of 96% after the purification process. Thus, the protein expression and purification process were proved more amenable and efficient in this study.

3.2 Optimization of reaction conditions for improving activity of 2,4-DCP hydroxylase

In order to study the 2,4-DCP removal ability of the obtained enzyme, 2,4-DCP removal using the obtained 2,4-DCP hydroxylase was conducted and the conditions improving 2,4-DCP hydroxylase were first optimized. The activity of 2,4-DCP hydroxylase in 2,4-DCP removal under microwave irradiation in IL was evaluated by changing the microwave power, ILs, temperature, 2,4-DCP and enzyme concentration of the reaction in a 5 ml removal system.

3.2.1 Effect of microwave power. To obtain the enzymatic activity at the cost of the possible minimum energy consumption, the microwave power was set in the range of 160 W to 800 W to investigate its effect on 2,4-DCP hydroxylase activity. 0 W could not be used as control since there was no heating source at that power. Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM [EMIM][PF₆], 0.1 mM 2,4-DCP and 0.2 mM NADPH at 25 °C under microwave irradiation in 5 min. As shown in Fig. 1, the enzyme activity increased as the microwave power increased from 160 W to 400 W, and then it was followed by a decrease at higher microwave power. The differences between these enzyme activities were statistical significant (P < 0.03). Generally, microwave irradiation induces molecular rotation arising from dipole alignment with the external oscillating electric field.²⁵ As a result, microwave irradiation significantly impacted molecules with high dipole moments. Because the enzyme, substrate and solvent used in the present study have significant dipole moments, the higher the microwave power from 160 W to 400 W was used, the faster the dipole reoriented under the microwave irradiation, which might allow the functional groups to obtain much higher reactivity.²⁶ The decrease of the enzymatic activity at high microwave power (>400 W) might be due to the deactivation of enzyme caused by the rapid change of the temperature during the reaction.²⁷ It is noteworthy that in each experiment under a preset microwave power, the microwave power was not constant during the heating process. The microwave power was first operated at the preset value for the temperature to quickly approach approximate 25 °C. And then the power slowly decreased with the further increase of temperature until it arrived at 25 °C. For the rest of the heating process, the power was kept at a relatively small value to keep the temperature constant at 25 °C. The variation of microwave power was achieved by the feedback temperature control in the microwave reactor to adjust the power according to the change of the temperature. Based on our results, 400 W was chosen as the optimum microwave power.

Fig. 1 Effect of microwave power on 2,4-DCP hydroxylase activity. Conditions: reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM [EMIM][PF₆], 0.1 mM 2,4-DCP and 0.2 mM NADPH at 25 °C under microwave irradiation in 5 min. The microwave power varied from 160 W to 800 W.

3.2.2 Effect of ILs and organic solvents. Enzyme performance is significantly affected by using different additives and reaction media.²⁸ The effects of different types of ILs on the 2,4-DCP hydroxylase activity were investigated by using 10 types of imidazolium ILs with different cations and anions. For comparison, reactions in some organic solvents were also studied, and a solvent free system was used as the control. Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM 2,4-DCP and 0.2 mM NADPH at 25 °C under 400 W microwave irradiation in 5 min. As shown in Fig. 2a, the enzyme activity was greatly dependent on the properties of the ILs and the organic solvent. Significantly different reactivity patterns were observed in different ILs. The activity of 2,4-DCP hydroxylase increased with the increase of the alkyl chain length on the cation or the cation's hydrophobicity ([BMIM] < [EMIM]) when the anion was the same in the ILs. The activity of 2,4-DCP hydroxylase in ILs with the same cation [BMIM] followed a decreasing order of [Br] > [Cl] > [BF₄] > [N(CN)₂] > [NTf₂] > [CF₃SO₃] and [PF₆] > [OTf] > [Ac] > [BF₄] with [EMIM] cation. The effect of cations on enzyme activity might be due to the ion kosmotropicity (Hofmeister series), which was important in interpreting the enzyme's behaviors in aqueous IL solutions (kosmotropic anions and chaotropic cations stabilize the enzyme, whereas chaotropic anions and kosmotropic cations destabilize it).²⁹ It might be that chaotropic cations stabilized the enzyme to cause the high activity in [EMIM] IL, whereas kosmotropic cations destabilized it to cause the low activity in [BMIM] IL, as illustrated above. The effect of anions on the enzyme activity was complex.³⁰ The highest enzyme activities was observed in IL [EMIM][PF₆] compared with the other media. The enhancement of enzyme activity in [EMIM][PF₆] might be due to the high capability for dissolving substrates, which enabled the reaction to occur in homogeneous systems.^30,31 [EMIM][PF₆] was chosen as the best reaction medium for optimal reaction conditions because of its highest enzyme activity improvement ability, its lowest cost and its toxicity compared with other ILs.³²

Fig. 2 Effect of ILs (a) and [EMIM][PF₆] concentration (b) on 2,4-DCP hydroxylase activity. Conditions: reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM 2,4-DCP and 0.2 mM NADPH at 25 °C under 400 W microwave irradiation in 5 min. Statistical significance for difference between control and solvent groups (*P < 0.05) was determined by one-way analysis of variance (ANOVA) followed by Dunnett's test. The solvent concentration was 60 μM in (a) and in the range of 0 to 500 μM in (b).

Enzyme properties are salt-concentration dependent,³¹ so enzyme activity might be greatly influenced by the IL concentration in the reaction mixture. The influence of the IL concentration on 2,4-DCP removal was investigated using different [EMIM][PF₆] concentrations in the range of 0 to 500 μM. As shown in Fig. 2b, the enzyme activity decreased with the increase of the [EMIM][PF₆] concentration from 0 to 20 μM, then it increased with the increase of the [EMIM][PF₆] concentration, and the maximum activity was achieved at an [EMIM][PF₆] concentration of 60 μM. Further increasing the [EMIM][PF₆] concentration resulted in a decrease of the enzyme activity. The differences between these enzyme activities were statistical significant (P < 0.05). The explanation of the enhanced enzyme activity with the increase of the [EMIM][PF₆] concentration might be that the higher the [EMIM][PF₆] concentration, the higher the solubility of substrate and product, which might indirectly improve the enzyme activity. The decrease of enzyme activity with higher [EMIM][PF₆] concentration (>60 μM) might be due to the increase of viscosity of IL due to the increasing mass transfer limitations and decreasing interaction between substrates and enzyme. Another possibility is that the high ionic strength of the reaction media at a high concentration of IL might denature the enzyme.

3.2.3 Effect of temperature. Theoretically, an elevated temperature could help the substrate molecules obtain adequate energy to pass over the energy barrier and enhance the reaction rate. In contrast, enzymes are very sensitive to temperature and are easily deactivated at high temperature. The effect of temperature on the activity of 2,4-DCP hydroxylase was examined in the range of 15 °C (reactions under lower temperatures could not be performed under microwave condition) to 50 °C. Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM [EMIM][PF₆], 0.1 mM 2,4-DCP and 0.2 mM NADPH under microwave irradiation in 5 min. No substrate volatilization was observed in this temperature range since the boiling point of 2,4-DCP is 210 °C. The results in Fig. 3 showed that the enzyme activity increased as the temperature increased from 4 to 25 °C, followed by a decrease at higher temperatures. The differences between these enzyme activities were statistical significant (P < 0.02). As the reaction temperature increased, the chance of a collision between the enzyme and substrate molecules increased, which might help form enzyme–substrate complexes and lead to an increase in enzyme activity. As for the decrease of the enzyme activity with further increase of the temperature above 25 °C, it was likely due to the denaturation (alteration) of the protein structure resulting from a heat-induced destruction of the non-covalent interactions. The results also showed that microwave irradiation did not change the optimum temperature (25 °C) for the degradation compared to that of conventional heating.

Fig. 3 Effect of temperature on 2,4-DCP hydroxylase activity. Conditions: reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM [EMIM][PF₆], 0.1 mM 2,4-DCP and 0.2 mM NADPH under microwave irradiation in 5 min. The temperature varied from 4 °C to 50 °C.

3.2.4 Effect of the enzyme concentration. The high cost of enzymes is one of the main problems for the application of enzymes in industrial degradation processes. The effect of the enzyme concentration on 2,4-DCP hydroxylase activity was studied. Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 0.1 mM [EMIM][PF₆], 0.1 mM 2,4-DCP and 0.2 mM NADPH at 25 °C under 400 W microwave irradiation in 5 min. The enzyme concentrations were kept constant while the enzyme concentration was changed from 0 to 96 μg ml⁻¹. As the concentration of enzyme increased from 0 to 12 μg ml⁻¹, the activity of 2,4-DCP hydroxylase also increased (Fig. 4). The enzyme activity then decreased with a further increase of the enzyme concentration. The differences between these enzyme activities were statistical significant (P < 0.05). The decrease of enzyme activity might be due to protein aggregation at high concentrations, which might cause the external mass transfer resistance and limit the degradation. A concentration of 12 μg ml⁻¹ of 2,4-DCP hydroxylase was proven to be the most efficient concentration and was adopted in the removal experiments.

Fig. 4 Effect of enzyme concentration on 2,4-DCP hydroxylase activity. Conditions: reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 0.1 mM [EMIM][PF₆], 0.1 mM 2,4-DCP and 0.2 mM NADPH at 25 °C under 400 W microwave irradiation in 5 min. The enzyme concentration varied from 0 to 96 μg ml⁻¹.

3.2.5 Enzyme kinetics. A concentration of 0.1 mM 2,4-DCP was adopted in the removal experiments. The enzyme kinetics were also studied to reveal the catalytic mechanism of the enzyme, its role in metabolism, and how its activity was controlled.³³ Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 0.1 mM [EMIM][PF₆], 12 μg ml⁻¹ purified enzyme, and 0.2 mM NADPH at 25 °C under 400 W microwave irradiation in 5 min. The concentration of 2,4-DCP varied from 0.5 to 200 μM. The K_m and V_max values were obtained from Lineweaver–Burk Plots for 2,4-DCP. The higher K_m (6.5 μM) for 2,4-DCP compared to our previous results (5 μM)¹⁰ suggested that the substrate affinity of the 2,4-DCP hydroxylase was slightly lower in this study. However, the high V_max (0.027 μM min⁻¹) in this study corresponded to a 4.1-fold increase of the enzyme specific activity compared to our previous result.

3.3 Enzymatic removal of 2,4-DCP under microwave irradiation in [EMIM][PF₆]

Enzyme activity and product yield are strongly affected by additives and the heating mode.³⁴ Herein, we demonstrate that combined use of IL and microwave irradiation was an efficient method for the enzymatic removal of 2,4-DCP. [EMIM][PF₆], in which 2,4-DCP hydroxylase exhibited the best activity, was used as the reaction medium for the enzymatic removal of 2,4-DCP under microwave heating. Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM 2,4-DCP and 0.2 mM NADPH under 400 W microwave irradiation at 25 °C. Our result in Fig. 5a showed that the enzymatic activity and 2,4-DCP removal efficiency (20 min for complete removal, enzymatic activity of 0.50 ± 0.021 U mg⁻¹) of 2,4-DCP hydroxylase increased approximately 10% under microwave heating compared to under conventional heating, and a 1.2-fold increase in enzymatic activity and 2,4-DCP removal efficiency (18 min for complete removal, enzymatic activity of 0.55 ± 0.018 U mg⁻¹) were observed in [EMIM][PF₆] compared to that in the solvent free system under conventional heating (22 min for complete removal, enzymatic activity of 0.45 ± 0.013 U mg⁻¹). A further incremental effect of microwave and IL on improving the enzymatic activity and 2,4-DCP removal efficiency (15 min for complete removal, enzymatic activity of 0.66 ± 0.015 U mg⁻¹, 1.5-fold) was also observed. The differences between these data were statistical significant (P < 0.02). These results indicated that microwave irradiation was a good heating method, and [EMIM][PF₆] was a favorable medium for 2,4-DCP removal. The time course result in Fig. 5b showed that a 100% removal percentage of 2,4-DCP was achieved in 15 min under microwave irradiation in [EMIM][PF₆]. This result was superior to the results of other enzymatic 2,4-DCP removal processes reported in the literature,³⁵ which took hours for the complete removal. The removal experiment was also performed at 4 °C under conventional heating to test the 2,4-DCP removal ability using the 2,4-DCP hydroxylase. Only 30 min was required to complete 2,4-DCP removal at 4 °C, and a 98.1% removal percentage was obtained at 15 min, which indicated that the enzymatic method used in this study has potential application in cold areas even without microwave irradiation. The high catalytic activity of this enzyme from 4 °C to 25 °C might suggest that this enzyme possessed local flexibility in the active site (which is responsible for the activity at low temperatures) and high overall rigidity (which is responsible for the thermal stability) under microwave irradiation in [EMIM][PF₆].

Fig. 5 (a) Effect of operation method on complete 2,4-DCP removal at 25 °C. (b) Time course of reaction process in [EMIM][PF₆] at (■) 4 °C and (♦) under microwave irradiation at 25 °C. Conditions: reactions were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM 2,4-DCP and 0.2 mM NADPH under 400 W microwave irradiation in 5 min at 25 °C. Reaction conditions at 4 °C was carried out in 50 mM sodium phosphate buffer (pH 7.5), 12 μg ml⁻¹ purified enzyme, 0.1 mM 2,4-DCP and 0.2 mM NADPH. Statistical significance (*P < 0.01) was determined by ANOVA.

3.4 Thermal stability

The obtained 2,4-DCP hydroxylase showed high activity and 2,4-DCP removal ability in the range of 4 °C to 25 °C, which suggested that this enzyme would be a psychrophilic (cold-adapted) enzyme. Psychrophilic enzymes usually show low stability in mesophilic conditions,³⁶ and the thermal stability is one of the key factors for its practical use. Thus, the thermal stability of the obtained 2,4-DCP hydroxylase was studied to measure the effect of microwave irradiation and ILs during the removal process. The enzyme mixtures were incubated in the presence and absence of [EMIM][PF₆] (60 μM) at 35 °C (lower than 40% enzyme activity remained at this temperature, according to the above temperature screening study) for 30 min (200 rpm rotary shaker). After incubation, samples were assayed under the optimum conditions described above for residual activity. Microwave irradiation and IL have been reported to increase the thermal stability of enzymes in certain cases. To study the effect of microwave irradiation and [EMIM][PF₆] on the thermal stability of 2,4-DCP hydroxylase, tests were performed. To test if the thermal stability enhancement was caused during the incubation step or during the degradation step, experiments were also performed by adding additional [EMIM][PF₆] in the reaction mixture after the incubation step. For comparison, control reactions in [EMIM][PF₆] under conventional heating and in the solvent-free system under both microwave irradiation and conventional heating were conducted under identical conditions.

The results in Table 1 showed that without microwave or [EMIM][PF₆] protection, the remaining activity of 2,4-DCP hydroxylase was low after pretreatment (15.5 ± 0.5% and NA for experiment 5 under conventional heating and microwave assay conditions, respectively) as shown in Table 1, which indicated its low thermal stability at 35 °C. This deactivation could not be recovered by additional adding of [EMIM][PF₆] in the assay system (11.2 ± 0.4% and 0.6 ± 0.02% for experiment 4 under conventional heating and microwave assay conditions, respectively). Increased thermal stability of 2,4-DCP hydroxylase was achieved by [EMIM][PF₆] pre-treatment. Without microwave incubation, [EMIM][PF₆] pre-treatment could increase the remaining enzyme activity of 2,4-DCP hydroxylase under conventional heating incubation, either assaying under conventional heating conditions (29.9 ± 1.0% for experiment 3 compared to 11.2 ± 0.4% for experiment 4) or assaying under microwave conditions (89.8 ± 2.9% for experiment 3 compared to 0.6 ± 0.02% for experiment 4). The reason for the improvement of the thermal stability of enzymes in [EMIM][PF₆] might be related to its ionic nature. During the pre-treatment process, the presence of [EMIM][PF₆] might increase the hydrophobic interactions between water and the enzyme, which increased the rigidity of enzyme and thus maintained its catalytic conformation. One interesting result was that, although microwave did not have a significant effect on the enzyme activity without pre-treatment, as shown in experiment 1 and 2, microwave irradiation significantly increased the activity of the denatured enzyme incubated in [EMIM][PF₆] (89.8 ± 2.9% compared to 29.9 ± 1.0% for experiment 3) and thus increased the thermal stability of the enzyme. Microwave irradiation was also found to increase the thermal stability during the pre-treatment process. A total of 100 ± 3.0% enzyme activity remained in experiment 6 when assaying under microwave irradiation compared to 37.7 ± 1.2% in experiment 6 assaying under conventional heating. Thermal stability improvement using [EMIM][PF₆] was also observed under conventional heating (88.2 ± 3.3% for experiment 1 compared to 63.6 ± 2.4% for experiment 2).

Table 1 Thermal stability experiments

Exp. no.	Relative enzyme activity (%) remained under conventional heating	Relative enzyme activity (%) remained under microwave
a NA stands for not detected. IL used in this experiment was [EMIM][PF6]. The incubation was performed at 35 °C for 30 min. Experiment 1: no pre-treatment, assay in IL; experiment 2: no pre-treatment, assay without IL; experiment 3: incubation (conventional heating) in IL, assay in IL; experiment 4: incubation (conventional heating), assay in IL; experiment 5: incubation (conventional heating), assay without IL; experiment 6: incubation (microwave) in IL, assay in IL; experiment 7: incubation (microwave), assay in IL; experiment 8: incubation (microwave), assay without IL.
1	88.2 ± 3.3	95.9 ± 3.4
2	63.6 ± 2.4	78.1 ± 2.6
3	29.9 ± 1.0	89.8 ± 2.9
4	11.2 ± 0.4	0.6 ± 0.02
5	15.5 ± 0.5	NA^a
6	37.7 ± 1.2	100 ± 3.0
7	77.1 ± 2.8	84.7 ± 3.1
8	69.3 ± 2.7	45.3 ± 1.6

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Enhancement of the thermal stability of 2,4-DCP hydroxylase was also achieved using microwave pre-treatment. Without [EMIM][PF₆] incubation, higher enzyme activity remained assaying under conventional heating conditions (69.3 ± 2.7% for experiment 8 compared to 15.5 ± 0.5% for experiment 5) or assaying under microwave conditions (45.3 ± 1.6% for experiment 8 compared to NA for experiment 5). The microwave field might change the interactions between the enzyme and its microenvironment, and thus prevent enzyme thermodenaturation. The highest enzyme activity (100 ± 3.0%) was obtained by incubation of the enzyme in [EMIM][PF₆] with microwave pre-treatment and assaying in [EMIM][PF₆] under microwave irradiation, which suggested a further incremental effect of microwave irradiation and [EMIM][PF₆] on improving thermal stability. The highest enzyme activity was even higher than those of the experiments (experiments 1 and 2) without thermal deactivation, as shown in Table 1. The differences between these data were statistical significant (P < 0.05). The enhancement of the thermal stability in [EMIM][PF₆] under microwave irradiation might be due to the microwave irradiation acting directly on [EMIM][PF₆] because of its excellent microwave-absorbing ability, which created a compatible system for maintaining its rigidity. Moreover, microwave irradiation might eliminate the water from the system and prevent the occurrence of a number of detrimental chemical reactions that would lead to protein inactivation in aqueous solutions. This result indicated that, after microwave irradiation and [EMIM][PF₆] pretreatment, the removal process would also be effective at 35 °C.³⁷

4. Conclusions

In the present study, a fast and efficient enzymatic method for 2,4-DCP treatment was developed. A 4.1-fold improvement of 2,4-DCP hydroxylase specific activity for 2,4-DCP degradation was achieved through modification and optimization of the expression and purification conditions. Microwave irradiation and [EMIM][PF₆] were found not only to increase the activity of 2,4-DCP hydroxylase and thus increase the 2,4-DCP removal efficiency but also to significantly increase the thermal stability of 2,4-DCP hydroxylase. One interesting result was that the thermal deactivation of enzyme incubated in [EMIM][PF₆] could be partially recovered by microwave irradiation. The applicability over a wide range of temperature from 4 °C to 35 °C of this enzyme under microwave irradiation in [EMIM][PF₆] might make the scope and economy of the removal process more attractive.

Acknowledgements

The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (no. 31100574) and Fund from Science and Technology Department of Jilin Province (no. 20130206066YY).

Notes and references

1. A. Chen, G. Zeng, G. Chen, C. Zhang, M. Yan, C. Shang, X. Hu, L. Lu, M. Chen, Z. Guo and Y. Zuo, Chemosphere, 2014, 109, 208.
2. B. Fontaine, A. Nuzzo, R. Spaccini and A. Piccolo, J. Geochem. Explor., 2013, 129, 28.
3. G. Chen, S. Guan, G. Zeng, X. Li, A. Chen, C. Shang, Y. Zhou, H. Li and J. He, Appl. Microbiol. Biotechnol., 2013, 97, 3149.
4. X. Shi, H. Leng, Y. Hu, Y. Liu, H. Duan, H. Sun and Y. Chen, Ecotoxicol. Environ. Saf., 2012, 86, 125.
5. P. K. Arora and H. Bae, Microb. Cell Fact., 2014, 13, 31.
6. M. C. Tomeia, D. M. Angeluccia and A. J. Daugulis, Environ. Technol., 2014, 35, 75.
7. Y. Wang, X. Chen, J. Liu, F. He and R. Wang, Environ. Sci. Pollut. Res. Int., 2013, 20, 6222.
8. H. Forootanfar, M. M. Movahednia, S. Yaghmaei, M. Tabatabaei-Sameni, H. Rastegar, A. Sadighi and M. A. Faramarzi, J. Hazard. Mater., 2012, 209–210, 199.
9. H. Vergara-Dominguez, M. Roca and B. Gandul-Rojas, Food Chem., 2013, 139, 786.
10. K. Oh and O. Tuovinen, J. Ind. Microbiol., 1990, 6, 275.
11. Y. Lu, Y. Yu, R. Zhou, W. Sun, C. Dai, P. Wan, L. Zhang, D. Hao and H. Ren, Biotechnol. Lett., 2011, 33, 1159.
12. R. Martinez-Palou, Ionic liquid and Microwave-Assisted Organic Synthesis: A “Green” and synergic couple, J. Mex. Chem. Soc., 2007, 51, 252.
13. J. P. Robinson, S. W. Kingman, E. H. Lester and C. Yi, Sep. Purif. Technol., 2012, 96, 12.
14. V. I. Parvulescu and C. Hardacre, Chem. Rev., 2007, 107, 2615.
15. D. Yu, C. Wang, Y. Yin, A. Zhang, G. Gui and X. Fang, Green Chem., 2011, 13, 1869.
16. D. Yu, Y. Wang, C. Wang, D. Ma and X. Fang, J. Mol. Catal. B: Enzym., 2012, 79, 8.
17. T. Ledger, D. H. Pieper and B. Gonzalez, Appl. Environ. Microbiol., 2006, 72, 2783.
18. K. Makdessi and U. Lechner, FEMS Microbiol. Lett., 1997, 157, 95.
19. T. Liu and P. J. Chapman, FEBS Lett., 1984, 173, 314.
20. T. San-Miguel, P. Pérez-Bermúdez and I. Gavidia, SpringerPlus, 2013, 2, 89.
21. V. Babu and B. Choudhury, J. Genet. Eng. Biotechnol., 2013, 11, 117.
22. A. Vera, N. González-Montalbán, A. Arís and A. Villaverde, Biotechnol. Bioeng., 2007, 96, 1101.
23. E. Winograd, M. A. Pulido and M. Wasserman, BioTechniques, 1993, 14, 886.
24. S. Sahdev, S. K. Khattar and K. S. Saini, Mol. Cell. Biochem., 2008, 307, 249.
25. D. D. Young, J. Nichols, R. M. Kelly and A. Deiters, J. Am. Chem. Soc., 2008, 130, 10048.
26. H. M. Yu, S. T. Chen, P. Suree, R. Nuansri and K. T. Wang, J. Org. Chem., 1996, 61, 9608.
27. M. Galesio, M. Mazzarino, X. de la Torre, F. Botrè and J. L. Capelo, Anal. Bioanal. Chem., 2011, 399, 861.
28. Methods in Non-Aqueous Enzymology, ed. M. N. Gupta, Birkhäuser Verlag, Basel-Boston-Berlin, Biochemistry (Moscow), 2000, vol. 67, p. 218.
29. Z. Yang, J. Biotechnol., 2009, 144, 12.
30. S. C. Lee and R. S. Mohamad, Enzyme Microb. Technol., 2006, 38, 551.
31. Z. Yang and W. Pan, Enzyme Microb. Technol., 2005, 37, 19.
32. M. Matzke, S. Stolte, K. Thiele, T. Juffernholz, J. Arning and J. Ranke, Green Chem., 2007, 9, 1198.
33. D. Yu, H. Wu, A. Zhang, L. Tian, L. Liu, C. Wang and X. Fang, Process Biochem., 2011, 46, 599.
34. A. M. Dehkordi, M. S. Tehrany and I. Safari, Ind. Eng. Chem. Res., 2009, 48, 3271.
35. C. Laane, S. Boeren, K. Vos and C. Veeger, Biotechnol. Bioeng., 1987, 30, 81.
36. M. L. Tutino, G. di Prisco, G. Marino and D. de Pascale, Protein Pept. Lett., 2009, 16, 1172.
37. B. Réjasse, S. Lamare, M. D. Legoy and T. Besson, Org. Biomol. Chem., 2004, 2, 1086.

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Footnote

† Electronic supplementary information (ESI) available: Details of abbreviations of ionic liquids; electrophoresis analysis of purified His-tagged 2,4-DCP hydroxylase; and purification of recombinant 2,4-DCP hydroxylase were included here. See DOI: 10.1039/c4ra10637g

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