Degradation of acenaphthylene and anthracene by chemically modified laccase from Trametes versicolor

Article abstract of DOI:10.1039/c4ra02807d

We are studying the chemically modified laccase from Trametes versicolor for use in the in vitro oxidation of two polycyclic aromatic hydrocarbons (PAHs), acenaphthylene and anthracene, in combination with 2,2′-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a redox mediator. The results indicate that the maleic anhydride modified laccase (MA-Lac) improved the stability of laccase to temperature, pH and storage time compared with the free enzyme. After incubation for 72 h, the MA-Lac-ABTS system oxidized acenaphthylene and anthracene to more than 70% from the reaction mixture. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra02807d

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Degradation of acenaphthylene and anthracene
by chemically modified laccase from Trametes
versicolor†
Cite this: RSC Adv., 2014, 4, 31120
Yulong Liu and Xiufu Hua*b
a
We are studying the chemically modified laccase from Trametes versicolor for use in the in vitro oxidation of
two polycyclic aromatic hydrocarbons (PAHs), acenaphthylene and anthracene, in combination with
Received 30th March 2014
Accepted 23rd June 2014
0
2
,2 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a redox mediator. The results indicate
that the maleic anhydride modified laccase (MA-Lac) improved the stability of laccase to temperature,
pH and storage time compared with the free enzyme. After incubation for 72 h, the MA-Lac–ABTS
system oxidized acenaphthylene and anthracene to more than 70% from the reaction mixture.
DOI: 10.1039/c4ra02807d
www.rsc.org/advances
Polycyclic aromatic hydrocarbons (PAHs) are highly toxic and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
1
3
organic pollutants found at contaminated industrial sites (ABTS).
around the world. Due to their toxic effects, PAHs pose a
1,2
However, the practical application of laccase, mainly as an
serious health risk to microorganisms, plants and animals, agent for environmental clean-up purposes, is still prevented by
including humans. Many compounds, such as acenaphthylene several limitations. In general, low stability and the potential for
and anthracene, are admitted to be highly mutagenic and drastic reductions in enzymatic activity have always been
carcinogenic.
considered as hindrances to the practical application of enzy-
14,15
Many research efforts have been expended to nd suitable matic systems.
Chemical modication is a rapid and inex-
16
methods for the remediation of soil and water environments pensive method for stabilizing the enzyme. In this paper,
contaminated with PAHs. In recent years, many studies on the chemical modication of laccase with maleic anhydride was
biodegradation of PAHs became the focus of scientic performed to increase its stability. The oxidative potential of
3,4
research. For example, the use of various white rot fungi for maleic anhydride-modied laccase mediator system for in vitro
5,6
the biodegradation of PAHs has been extensively studied.
reaction with PAHs has not been studied. The objective of this
Unfortunately, these approaches could be very slow and may study is to evaluate the potential of maleic anhydride modied
present some undesirable limitations and incomplete removal laccase (MA-Lac) to oxidize acenaphthylene and anthracene in
of pollutants. One of the strategies to overcome this limitation the presence of ABTS.
7
is through enzymatic treatment.
Ion exchange chromatography of free laccase and MA-Lac
Laccase (E.C. 1.10.3.2) is a copper-containing oxidoreductive was performed on HiTrap DEAE FF, as described in the exper-
enzyme that catalyzes one-electron oxidation of a broad range of imental section. The elution curves at 215 nm and enzyme
8
polyphenols and aromatic substrates. Using isolated laccase in activity are shown in Fig. 1(a) and (b), respectively. Three peaks
its soluble form, the detoxifying effect of laccase in reaction appeared in the elution curve at 215 nm, as shown in Fig. 1(a)
9–11
with xenobiotics has been widely studied.
Radical mediator and only one peak of each enzyme activity is observed in
compounds acting as “electron shuttles” between the enzyme
and the substrate extended the substrate spectrum of laccase;
therefore, they could accelerate the laccase catalysis of a
12
substrate. The degradation of PAHs by a laccase mediator
system was reported to be signicantly effective in the presence
of mediator compounds such as 1-hydroxybenzotriazole (HBT)
a
Department of Basic Teaching, Yancheng Institute of Technology, Yancheng, 224051,
China
b
Department of Scientic Research and Development, Tsinghua University, Beijing,
1
†
1
00084, China. E-mail: hua_xiufu@163.com
Fig. 1 Anion-exchange chromatography of free and modified laccase
on HiTrap DEAE FF (1 ml columns); (a) protein content of the elution;
(b) laccase activity of the elution.
Electronic supplementary information (ESI) available. See DOI:
0.1039/c4ra02807d
31120 | RSC Adv., 2014, 4, 31120–31122
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ꢀ
Fig. 3 Thermal deactivation of free and modified laccases at 55 C; (a)
deactivation plot; (b) fluorescence emission spectra.
Fig. 2 (a) CD spectra; (b) fluorescence spectra.
Table 1 The enzymatic reaction parameters
ꢁ1
ꢁ1
ꢁ1
ꢁ1
Enzyme
K
m
(mmol l
)
kcat (s
)
kcat/K
m
(s mmol l
)
6
6
6
Free laccase
MA-Lac
0.68
0.47
9.04 ꢂ 10
6.89 ꢂ 10
13.29 ꢂ 10
14.66 ꢂ 10
6
Fig. 1(b). The elution curve of MA-Lac in Fig. 1(a) showed that
only the third peak shied when the NaCl concentration Fig. 4 Deactivation of free and modified laccases under acidic
conditions (pH 3.5); (a) deactivation plot; (b) fluorescence emission
spectra.
increased from 0.0466 mol to 0.0524 mol, and this shi also
reappeared in the enzyme activity of the elution (Fig. 1(b)).
These results indicate that the structure of laccase had changed
aer the modication and conrm the success of the chemical
modication with maleic anhydride.
The stabilization of laccase under acidic conditions will
extend its application spectrum and thus is constantly pursued.
The secondary and tertiary structures of the free and modi- Here, the stability of free and chemically modied laccases was
ed laccases were analyzed by circular dichroism (CD) and compared at pH 3.5 using 20 mM disodium hydrogen phos-
uorescence emission spectroscopy to probe the structural phate–citric acid as the buffer and during the experiment, the


differences caused by the chemical modication. The identical enzyme solutions were incubated at room temperature. The
adsorption curves, as shown in Fig. 2, indicate that the maleic changes in the residual activity are shown in Fig. 4(a), in which
anhydride modied laccase retained its secondary and tertiary MA-Lac exhibits improved stability with an increase in the half-
structure.
The determination of the reacted amino lysine groups was
performed according to the method described in the experi- MA-Lac at a high temperature of 55 C or under acidic condi-
life of enzyme activity from 6.45 h to 11.36 h.
The changes in the tertiary structure of the free laccase and
ꢀ
mental session. The modication ratio was determined to be tions (pH 3.5) were monitored by uorescence intensity. As
7
3.77% for maleic anhydride.
The biocatalytic activity of native and modied laccase was position of the uorescence spectra was observed from free
examined using ABTS as the substrate. Michaelis–Menten laccase, as compared to that of the MA-Lac, which indicates that
parameters, K and kcat, interpreted from the Lineweaver–Burk the enzyme structure was more stable due to the chemical
plots are listed in Table 1. Here, the chemical modication modication with maleic anhydride.
shown in Fig. 3(b) and 4(b), a more signicant shi in the peak
m
leads to a reduction in Km while an increase in kcat/K , indi-
In the experiments, two compounds (acenaphthylene and
m
cating that the modied laccase has an enhanced affinity and anthracene) were chosen as representatives of PAHs. The
activity due to ABTS.
degradation by free laccase and MA-Lac was conducted at pH
The thermal stability of the free laccase and MA-Lac was 4.5, using 20 mM disodium hydrogen phosphate–citric acid
ꢀ
ꢀ
compared at 55 C and at pH 7.0. During the experiment, the buffer at a temperature of 30 C. The metabolites were studied
enzyme solution incubated without ABTS was sampled at given at different time points within the entire incubation time of
time intervals and subjected to activity assay using ABTS as the 72 h. HPLC analysis indicated that the main products detected
substrate. To magnify the difference in thermal stability, free aer the incubation of acenaphthylene were 1,8-naphthalic acid
laccase solution incubated at zero degree was used as a control. anhydride and 1,2-acenaphthenedione and that of anthracene
The residual activities are shown in Fig. 3, which indicate that was 9,10-anthraquinone.
MA-Lac has the highest stability. The half-lives of enzyme
The degradation ratios of acenaphthylene and anthracene
activity interpreted from the curves in Fig. 3(a) are 138.6 and are listed in Fig. 5. From the results, the presence of ABTS as an
693 min for the free laccase and MA-Lac, respectively, i.e. a electron shuttle intensies the oxidation of PAHs by both free
5-fold increase in thermal stability is achieved by MA-Lac.
and the chemically modied laccases in vitro within 72 h of
incubation. The oxidation of acenaphthylene and anthracene
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Conclusions
Although the oxidation of PAHs by free laccase in the presence of an
immobilized laccase-mediator system has been reported, this is the
rst report that showed how laccase modied with maleic anhy-
dride oxidized acenaphthylene and anthracene in combination
with ABTS. The results showed that the chemical modication of
laccase with maleic anhydride could improve stability under acidic
conditions and thermal stability compared with the free enzyme
and also increase the oxidation of acenaphthylene and anthracene.
In the presence of the ABTS mediator, both the free and chemically
modied laccases increased the oxidation of anthracene, but had
no signicant inuence on acenaphthylene. The results indicate a
new opportunity in the practical application of laccase for biore-
mediation of environmental pollution from PAHs.
Fig. 5 Oxidation of PAHs; (a) acenaphthylene; (b) anthracene.
signicantly increased when 1 mM of ABTS is added to the
reaction mixture.
In the case of MA-Lac, the oxidation ratios of acenaphthylene
and anthracene increased from 50% and 36% without ABTS to
Notes and references
8
3% and 94% in the presence of the mediator, respectively. The
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oxidation by free laccase increased from 35% and 7% without
ABTS to 70% and 97% in its presence, respectively. Aer treat-
2
3
ꢀ
ment for 72 h at 30 C, the activities of the free and modied
laccases were reduced by 82.8% and 43.3%, respectively.
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of acenaphthylene and anthracene. The modication of laccase
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1
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20
electron transfer.
31122 | RSC Adv., 2014, 4, 31120–31122
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