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

Article abstract of DOI:10.1039/c4ra10637g

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.

Full text of DOI:10.1039/c4ra10637g

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Enhanced catalytic activity and thermal stability of
2,4-dichlorophenol hydroxylase by using
microwave irradiation and imidazolium ionic liquid
for 2,4-dichlorophenol removal†
Cite this: RSC Adv., 2014, 4, 62631
a
b
b
b
*
Hejun Ren, Yang Zhan, Xuexun Fang and Dahai Yu
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 mgꢂ1 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
Received 17th September 2014
Accepted 10th November 2014
4
ꢀC, indicating its psychrotrophic adaptability. These results showed that the use of 2,4-DCP
DOI: 10.1039/c4ra10637g
hydroxylase under microwave in [EMIM][PF6] is a fast, efficient and environmentally benign method for
www.rsc.org/advances
the removal of 2,4-DCP, and this method can be used over a wide temperature range.
atmospheric emissions from the combustion of municipal solid
waste, hazardous waste, coal, wood, and herbicides.4 Therefore,
1. Introduction
techniques for its disposal are critically needed.
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 signicant
amount of 2,4-DCP is still contained in surface water, industrial
effluents, wastewater, potable water, and soil, as well as in
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 degrada-
tion rates, and strong inhibitory effect by high concentrations of
chlorophenols. In addition, bioremediation by using microor-
ganisms is difficult to apply to practical remediation of pollut-
ants 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 concen-
trated 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-dichlor-
ophenol 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
aKey Laboratory of Ground Water Resources and Environment of the Ministry of
Education, College of Environment and Resources, Jilin University, 2519 Jiefang
Road, Changchun, 130021, P. R. China. E-mail: renhejun@126.com; Fax: +86-431-
85155240; Tel: +86-431-85155249
bKey Laboratory for Molecular Enzymology and Engineering of Ministry of Education,
College of Life Science, Jilin University, 2699 Qianjin Street, Changchun, 130012, P. R.
China. E-mail: yudahai@jlu.edu.cn; Fax: +86-431-85155240; Tel: +86-431-85155249
† Electronic supplementary information (ESI) available: Details of abbreviations
of ionic liquids; electrophoresis analysis of puried His-tagged 2,4-DCP
hydroxylase; and purication of recombinant 2,4-DCP hydroxylase were
included here. See DOI: 10.1039/c4ra10637g
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is less toxic and easy to be degraded for the subsequent ring- on the enzyme activity were screened and optimized. Further-
opening reaction), as can be observed from the great number more, the thermal stability of 2,4-DCP hydroxylase under
of works published in this area.6 This enzyme is a key enzyme in microwave irradiation in IL was also evaluated.
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
2. Material and methods
2.1 Materials
identied from
a metagenome constructed from poly-
chlorinated biphenyl-contaminated soil by functional
screening, and it showed great potential for bioremediation of
2,4-DCP due to its broad substrate specicity 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 nd 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.
All chemicals used in the present study were of analytical grade.
2,4-DCP and other chemicals were obtained from Sigma.
Recombinant Escherichia coli. DH5a containing the TfdB-JLU
gene for 2,4-DCP hydroxylase expression was from our lab.
BugBuster protein extraction reagent was from Novagen (Not-
tingham, 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.†
Microwave irradiation and ionic liquids (ILs) are two
important and rapid developing technologies in green chem-
istry, and these two methods have been employed to accelerate
enzymatic reactions and increase enzyme stability.12 Microwave
2.2 Methods
2.2.1 Protein expression and purication. The recombi-
irradiation, in conjunction with other techniques, has been nant E. coli was cultivated in LB medium containing 30 mg
proved to increase the decomposition efficiency and reduce the kanamycin per ml and 34 mg chloramphenicol per ml at 37 ꢀC.
time required for removing pollutants.13 On the other hand, Protein expression was induced at 18 C by the addition of 0.2
ꢀ
dramatically improved enzyme stability and activity in enzy- mM isopropyl-b-D-1-thiogalactoside (IPTG) (Fisher Scientic,
matic degradation were observed by choosing suitable ILs.14 ILs Fairlawn, NJ) at an OD600 of 0.4. Aer 15 h incubation, the cell
can be regarded as clean and green solvents for separation, pellets were harvested by centrifugation at 12 000 rpm and
chemical production, and catalysis because of their high vapor washed with 50 mM sodium phosphate buffer, pH 8.0. For the
pressure, non-ammability and excellent solubility for both preparation of crude extract, cells (1.11 g of E. coli cell paste
organic and inorganic compounds.14 Notably, the excellent from 400 ml fermentation culture) were suspended in 4 ml pH
microwave-absorbing ability of ILs makes them good solvents 8.0 Bugbuster Protein Extraction Reagent with 1 mM dithio-
for microwave-assisted catalysis. The incremental effects of threitol (DTT) and 0.6 mM PMSF (phenylmethylsulfonyl uo-
microwave irradiation and IL on improving enzyme activity in ride) to yield
a higher specic activity compared to
biodiesel production and in the isomerization of xylose to ultrasonication. The protein extraction was performed for 10
xylulose have recently been veried by our lab.15,16 However, min at 20 ꢀC at 150 rpm. Then, the lysate was centrifuged at
investigation of microwave irradiation and ILs in biodegrada- 12 000 rpm for 10 min using a Thermo Sorvall WX Ultracen-
tion has not been reported to our knowledge.
trifuge (Fisher Scientic, Fairlawn, NJ, USA) at 4 ꢀC. The
The aim of this study was to develop an efficient enzymatic supernatant was transferred onto a Hislink™ column (Prom-
2,4-DCP removal process (convert to 3,5-dichlorocatechol). The ega, Madison, WI, USA), rinsed with wash buffer (10 mM HEPES
expression and purication process for 2,4-DCP hydroxylase (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM
was optimized to improve the enzyme activity, and enzymatic imidazole, pH 8.0), and eluted with elution buffer (10 mM
removal of 2,4-DCP by using 2,4-DCP hydroxylase was con- HEPES, 1 M imidazole, pH 8.0). The protein supernatant was
ducted through a new combined method (microwave/IL) loaded onto a nickel-nitrilotriacetic agarose resin (Qiagen,
(Scheme 1). The effects of the reaction conditions (microwave Germany) equilibrated with the same buffer. Aer washing with
power, ILs, temperature, 2,4-DCP and enzyme concentration) 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 ultraltration by using
Amicon Ultra-15 centrifugal lter units (Millipore, USA) and
then were dialtered with 50 mM sodium phosphate buffer, pH
7.5, containing 10% (v/v) glycerol. A total of 4 ml protein solu-
tion (300 mg mlꢂ1) was obtained aer 4000 rpm centrifugation.
ꢀ
Samples were stored at ꢂ80 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
Scheme 1 2,4-DCP treatment by using 2,4-DCP hydroxylase under
microwave irradiation in ionic liquid.
ask. The reaction mixture was placed into the microwave
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reactor (MCR-3, Shanghai JieSi Microwave Chemistry Corpora-
In this study, lowering the temperature and IPTG concen-
tion), with an immersion well placed into one of the necks of the tration for protein induction and the use of BugBuster® protein
ask and a glass connector (linking the ask inside the cavity extraction reagent, which was an effective alternative to
with a reux condenser outside the cavity) into the other. A mechanical methods for releasing expressed target protein,
temperature probe was placed into the immersion well. The were performed to improve the recombinant protein soluble
reaction mixture was heated to the desired temperature. The expression and enzyme activity. As shown in ESI Table SI-1,† the
conventional heating reactions were performed using a digital target protein was highly overexpressed, and the enzyme
heating circulating water bath (Changzhou Electrical Instru- accounted for an estimated 23% of the total protein of the crude
ment Factory, HH-42). The activities of 2,4-DCP hydroxylase cell free extract in this study. Consistent with literature reports,
were determined by monitoring the decrease in absorbance at we observed that induction at 18 ꢀC for 15 h with 0.2 mM IPTG
340 nm (3340 ¼ 6220 Mꢂ1 cmꢂ1) following the substrate- (at OD ¼ 0.4) increased the puried protein yield and specic
dependent oxidation of NADPH.11 Unless otherwise indicated, activity compared with our previous results. The specic activity
standard reactions were performed by incubating puried of the puried enzyme towards 2,4-DCP was 0.66 ꢁ 0.015 U
enzyme (nal concentration 12 mg mlꢂ1) with 0.1 mM 2,4-DCP mgꢂ1 at pH 7.5 and 25 ꢀC, a 4.1-fold increase in specic activity
and 0.2 mM NADPH in 50 mM sodium phosphate buffer compared to our previous result, which was 0.16 U mgꢂ1. The
(pH 7.5) containing 60 mM IL at 25 ꢀC. Aer the reaction, total puried 2,4-DCP hydroxylase yield of the recombinant
samples were quickly moved to 100 C hot water to deactivate enzymes aer the purication procedure was 10.44 mg gꢂ1
ꢀ
the enzyme. All the removals were performed for three times protein per cell, a 3.6-fold increase in yield compared to our
and statistical signicance was determined by one-way analysis previous result, which was 2.9 mg gꢂ1 protein per cell, and this
of variance (ANOVA) followed by Dunnett's test.
enzyme yield result was superior to the literature. The one step
2.2.3 Thermal stability. The thermal stability assays were purication in this study did not have a signicant effect on the
performed by incubating 2,4-DCP hydroxylase at 35 ꢀC under protein yield and purity, an electrophoretically homogeneous
conventional heating without IL, under microwave irradiation enzyme, as shown in ESI Fig. SI-1,† was obtained with a yield of
without IL, in the presence of pH 7.5 PBS buffer containing 96% aer the purication process. Thus, the protein expression
60 mM [EMIM][PF6] under conventional heating and under and purication process were proved more amenable and effi-
microwave irradiation. Aer incubation for different times, cient in this study.
samples were removed and assayed for residual activity. All the
thermal stability tests were performed for three times and
statistical signicance was determined by one-way analysis of
variance (ANOVA) followed by Dunnett's test.
3.2 Optimization of reaction conditions for improving
activity of 2,4-DCP hydroxylase
2.2.4 Enzyme assay and characterization. The enzyme In order to study the 2,4-DCP removal ability of the obtained
assay and kinetic parameters (Km: Michaelis–Menten constant enzyme, 2,4-DCP removal using the obtained 2,4-DCP
and Vmax values: the maximum reaction velocity) for 2,4-DCP hydroxylase was conducted and the conditions improving
during removal were determined as described previously.11 One 2,4-DCP hydroxylase were rst optimized. The activity of
unit of activity was dened as the amount of enzyme required to 2,4-DCP hydroxylase in 2,4-DCP removal under microwave
ꢀ
consume 1 mmol NADPH per min at 25 C. Protein concentra- irradiation in IL was evaluated by changing the microwave
tions were determined by the BCA method (Novagen® BCA power, ILs, temperature, 2,4-DCP and enzyme concentration of
Protein Assay Kit) using bovine serum albumin as the standard. 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 consump-
tion, 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
3. Results and discussion
3.1 Protein expression and purication
In previous works, we reported the cloning and characterization at that power. Reactions were carried out in 50 mM sodium
of a novel 2,4-DCP hydroxylase. However, the activity of this phosphate buffer (pH 7.5), 12 mg mlꢂ1 puried enzyme, 0.1 mM
enzyme was lower than those reported in the literature.17–19 The [EMIM][PF6], 0.1 mM 2,4-DCP and 0.2 mM NADPH at 25 ꢀC
heterologous expression of enzyme is usually limited by the loss under microwave irradiation in 5 min. As shown in Fig. 1, the
of activity at high temperatures or by low soluble recombinant enzyme activity increased as the microwave power increased
protein yield.20 Additionally, the lysis and extraction of cells from 160 W to 400 W, and then it was followed by a decrease at
using sonication was regarded to decrease the solubility and higher microwave power. The differences between these enzyme
activity of enzymes.21 The overexpression of proteins in E. coli at activities were statistical signicant (P < 0.03). Generally,
low temperatures22 and low IPTG concentration23 usually microwave irradiation induces molecular rotation arising from
improves both protein solubility and activity.24 In order to dipole alignment with the external oscillating electric eld.25 As
increase active enzyme yield, the expression conditions and cell a result, microwave irradiation signicantly impacted mole-
disruption method were modied, and the purication process cules with high dipole moments. Because the enzyme, substrate
was shortened on the premise of not affecting the purity of the and solvent used in the present study have signicant dipole
enzyme.
moments, the higher the microwave power from 160 W to 400 W
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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 mg mlꢂ1 purified enzyme, 0.1 mM [EMIM][PF6], 0.1
mM 2,4-DCP and 0.2 mM NADPH at 25 ꢀC under microwave irradia-
tion in 5 min. The microwave power varied from 160 W to 800 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 deacti-
vation 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
rst 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
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
arrived at 25 C. For the rest of the heating process, the power sodium phosphate buffer (pH 7.5), 12 mg mlꢂ1 purified enzyme, 0.1 mM
ꢀ
2,4-DCP and 0.2 mM NADPH at 25 ꢀC under 400 W microwave irra-
was kept at a relatively small value to keep the temperature
diation 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 concen-
ꢀ
constant at 25 C. The variation of microwave power was ach-
ieved 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.
tration was 60 mM in (a) and in the range of 0 to 500 mM in (b).
3.2.2 Effect of ILs and organic solvents. Enzyme perfor-
mance is signicantly 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 mg mlꢂ1 puried enzyme, 0.1 mM 2,4-DCP and 0.2
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
ꢀ
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.
Signicantly 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
Enzyme properties are salt-concentration dependent,31 so
enzyme activity might be greatly inuenced by the IL concen-
tration in the reaction mixture. The inuence of the IL
concentration on 2,4-DCP removal was investigated using
different [EMIM][PF6] concentrations in the range of 0 to
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500 mM. As shown in Fig. 2b, the enzyme activity decreased with collision between the enzyme and substrate molecules
the increase of the [EMIM][PF6] concentration from 0 to 20 mM, increased, which might help form enzyme–substrate complexes
then it increased with the increase of the [EMIM][PF6] and lead to an increase in enzyme activity. As for the decrease of
concentration, and the maximum activity was achieved at an the enzyme activity with further increase of the temperature
[EMIM][PF6] concentration of 60 mM. Further increasing the above 25 ꢀC, it was likely due to the denaturation (alteration) of
[EMIM][PF6] concentration resulted in a decrease of the enzyme the protein structure resulting from a heat-induced destruction
activity. The differences between these enzyme activities were of the non-covalent interactions. The results also showed that
statistical signicant (P < 0.05). The explanation of the microwave irradiation did not change the optimum tempera-
enhanced enzyme activity with the increase of the [EMIM][PF6] ture (25 ꢀC) for the degradation compared to that of conven-
concentration might be that the higher the [EMIM][PF6] tional heating.
concentration, the higher the solubility of substrate and
3.2.4 Effect of the enzyme concentration. The high cost of
product, which might indirectly improve the enzyme activity. enzymes is one of the main problems for the application of
The decrease of enzyme activity with higher [EMIM][PF6] enzymes in industrial degradation processes. The effect of the
concentration (>60 mM) might be due to the increase of viscosity enzyme concentration on 2,4-DCP hydroxylase activity was
of IL due to the increasing mass transfer limitations and studied. Reactions were carried out in 50 mM sodium phos-
decreasing interaction between substrates and enzyme. phate buffer (pH 7.5), 0.1 mM [EMIM][PF6], 0.1 mM 2,4-DCP
ꢀ
Another possibility is that the high ionic strength of the and 0.2 mM NADPH at 25 C under 400 W microwave irradia-
reaction media at a high concentration of IL might denature the tion in 5 min. The enzyme concentrations were kept constant
enzyme.
while the enzyme concentration was changed from 0 to 96 mg
3.2.3 Effect of temperature. Theoretically, an elevated mlꢂ1. As the concentration of enzyme increased from 0 to 12 mg
temperature could help the substrate molecules obtain mlꢂ1, the activity of 2,4-DCP hydroxylase also increased (Fig. 4).
adequate energy to pass over the energy barrier and enhance the The enzyme activity then decreased with a further increase of
reaction rate. In contrast, enzymes are very sensitive to the enzyme concentration. The differences between these
temperature and are easily deactivated at high temperature. The enzyme activities were statistical signicant (P < 0.05). The
effect of temperature on the activity of 2,4-DCP hydroxylase was decrease of enzyme activity might be due to protein aggregation
examined in the range of 15 ꢀC (reactions under lower at high concentrations, which might cause the external mass
temperatures could not be performed under microwave condi- transfer resistance and limit the degradation. A concentration
tion) to 50 ꢀC. Reactions were carried out in 50 mM sodium of 12 mg mlꢂ1 of 2,4-DCP hydroxylase was proven to be the most
phosphate buffer (pH 7.5), 12 mg mlꢂ1 puried enzyme, 0.1 mM efficient concentration and was adopted in the removal
[EMIM][PF6], 0.1 mM 2,4-DCP and 0.2 mM NADPH under experiments.
microwave irradiation in 5 min. No substrate volatilization was
3.2.5 Enzyme kinetics. A concentration of 0.1 mM 2,4-DCP
observed in this temperature range since the boiling point of was adopted in the removal experiments. The enzyme kinetics
2,4-DCP is 210 ꢀC. The results in Fig. 3 showed that the enzyme were also studied to reveal the catalytic mechanism of the
activity increased as the temperature increased from 4 to 25 ꢀC, enzyme, its role in metabolism, and how its activity was
followed by a decrease at higher temperatures. The differences controlled.33 Reactions were carried out in 50 mM sodium
between these enzyme activities were statistical signicant (P < phosphate buffer (pH 7.5), 0.1 mM [EMIM][PF6], 12 mg mlꢂ1
0.02). As the reaction temperature increased, the chance of a puried enzyme, and 0.2 mM NADPH at 25 ꢀC under 400 W
Fig.
3 Effect of temperature on 2,4-DCP hydroxylase activity. Fig. 4 Effect of enzyme concentration on 2,4-DCP hydroxylase
Conditions: reactions were carried out in 50 mM sodium phosphate activity. Conditions: reactions were carried out in 50 mM sodium
buffer (pH 7.5), 12 mg mlꢂ1 purified enzyme, 0.1 mM [EMIM][PF6], phosphate buffer (pH 7.5), 0.1 mM [EMIM][PF6], 0.1 mM 2,4-DCP and
0.1 mM 2,4-DCP and 0.2 mM NADPH under microwave irradiation in 0.2 mM NADPH at 25 ꢀC under 400 W microwave irradiation in 5 min.
5 min. The temperature varied from 4 ꢀC to 50 ꢀC.
The enzyme concentration varied from 0 to 96 mg mlꢂ1
.
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microwave irradiation in 5 min. The concentration of 2,4-DCP
varied from 0.5 to 200 mM. The Km and Vmax values were
obtained from Lineweaver–Burk Plots for 2,4-DCP. The higher
Km (6.5 mM) for 2,4-DCP compared to our previous results (5
mM)10 suggested that the substrate affinity of the 2,4-DCP
hydroxylase was slightly lower in this study. However, the high
Vmax (0.027 mM minꢂ1) in this study corresponded to a 4.1-fold
increase of the enzyme specic 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
mg mlꢂ1 puried 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 mgꢂ1) 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 mgꢂ1) 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 mgꢂ1).
A further incremental effect of microwave and IL on improving
the enzymatic activity and 2,4-DCP removal efficiency (15 min
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 (A) under microwave irradiation at 25 ꢀC. Conditions: reactions
were carried out in 50 mM sodium phosphate buffer (pH 7.5), 12
mg mlꢂ1 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
mg mlꢂ1 purified enzyme, 0.1 mM 2,4-DCP and 0.2 mM NADPH.
Statistical significance (*P < 0.01) was determined by ANOVA.
for complete removal, enzymatic activity of 0.66 ꢁ 0.015 U mgꢂ1
,
1.5-fold) was also observed. The differences between these data
were statistical signicant (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
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 mM) 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). Aer
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
ꢀ
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 exibility 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].
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stability of 2,4-DCP hydroxylase, tests were performed. To stability of the enzyme. Microwave irradiation was also found
test if the thermal stability enhancement was caused during to increase the thermal stability during the pre-treatment
the incubation step or during the degradation step, experi- process. A total of 100 ꢁ 3.0% enzyme activity remained in
ments were also performed by adding additional [EMIM][PF6] experiment 6 when assaying under microwave irradiation
in the reaction mixture aer the incubation step. For compared to 37.7 ꢁ 1.2% in experiment 6 assaying under
comparison, control reactions in [EMIM][PF6] under conventional heating. Thermal stability improvement using
conventional heating and in the solvent-free system under [EMIM][PF6] was also observed under conventional heating
both microwave irradiation and conventional heating were (88.2 ꢁ 3.3% for experiment 1 compared to 63.6 ꢁ 2.4% for
conducted under identical conditions.
experiment 2).
The results in Table 1 showed that without microwave or
Enhancement of the thermal stability of 2,4-DCP hydroxy-
[EMIM][PF6] protection, the remaining activity of 2,4-DCP lase was also achieved using microwave pre-treatment.
hydroxylase was low aer pretreatment (15.5 ꢁ 0.5% and NA Without [EMIM][PF6] incubation, higher enzyme activity
for experiment 5 under conventional heating and microwave remained assaying under conventional heating conditions
assay conditions, respectively) as shown in Table 1, which (69.3 ꢁ 2.7% for experiment 8 compared to 15.5 ꢁ 0.5% for
indicated its low thermal stability at 35 ꢀC. This deactivation experiment 5) or assaying under microwave conditions (45.3
could not be recovered by additional adding of [EMIM][PF6] ꢁ 1.6% for experiment 8 compared to NA for experiment 5).
in the assay system (11.2 ꢁ 0.4% and 0.6 ꢁ 0.02% for exper- The microwave eld might change the interactions between
iment 4 under conventional heating and microwave assay the enzyme and its microenvironment, and thus prevent
conditions, respectively). Increased thermal stability of 2,4- enzyme thermodenaturation. The highest enzyme activity
DCP hydroxylase was achieved by [EMIM][PF6] pre-treatment. (100 ꢁ 3.0%) was obtained by incubation of the enzyme in
Without microwave incubation, [EMIM][PF6] pre-treatment [EMIM][PF6] with microwave pre-treatment and assaying in
could increase the remaining enzyme activity of 2,4-DCP [EMIM][PF6] under microwave irradiation, which suggested a
hydroxylase under conventional heating incubation, either further incremental effect of microwave irradiation and
assaying under conventional heating conditions (29.9 ꢁ 1.0% [EMIM][PF6] on improving thermal stability. The highest
for experiment 3 compared to 11.2 ꢁ 0.4% for experiment 4) enzyme activity was even higher than those of the experi-
or assaying under microwave conditions (89.8 ꢁ 2.9% for ments (experiments 1 and 2) without thermal deactivation, as
experiment 3 compared to 0.6 ꢁ 0.02% for experiment 4). The shown in Table 1. The differences between these data were
reason for the improvement of the thermal stability of statistical signicant (P < 0.05). The enhancement of the
enzymes in [EMIM][PF6] might be related to its ionic thermal stability in [EMIM][PF6] under microwave irradiation
nature. During the pre-treatment process, the presence of might be due to the microwave irradiation acting directly on
[EMIM][PF6] might increase the hydrophobic interactions [EMIM][PF6] because of its excellent microwave-absorbing
between water and the enzyme, which increased the rigidity ability, which created a compatible system for maintaining
of enzyme and thus maintained its catalytic conformation. its rigidity. Moreover, microwave irradiation might eliminate
One interesting result was that, although microwave did not the water from the system and prevent the occurrence of a
have a signicant effect on the enzyme activity without pre- number of detrimental chemical reactions that would lead to
treatment, as shown in experiment 1 and 2, microwave irra- protein inactivation in aqueous solutions. This result indi-
diation signicantly increased the activity of the denatured cated that, aer microwave irradiation and [EMIM][PF6]
enzyme incubated in [EMIM][PF6] (89.8 ꢁ 2.9% compared to preꢀtreatment, the removal process would also be effective at
29.9 ꢁ 1.0% for experiment 3) and thus increased the thermal 35 C.37
Table 1 Thermal stability experiments
Relative enzyme activity
Relative enzyme activity
Exp. no.
(%) remained under conventional heating
(%) remained under microwave
1
2
3
4
5
6
7
8
88.2 ꢁ 3.3
63.6 ꢁ 2.4
29.9 ꢁ 1.0
11.2 ꢁ 0.4
15.5 ꢁ 0.5
37.7 ꢁ 1.2
77.1 ꢁ 2.8
69.3 ꢁ 2.7
95.9 ꢁ 3.4
78.1 ꢁ 2.6
89.8 ꢁ 2.9
0.6 ꢁ 0.02
NAa
100 ꢁ 3.0
84.7 ꢁ 3.1
45.3 ꢁ 1.6
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.
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62638 | RSC Adv., 2014, 4, 62631–62638
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