Characterization of a robust cold-adapted and thermostable laccase from Pycnoporus sp. SYBC-L10 with a strong ability for the degradation of tetracycline and oxytetracycline by laccase-mediated oxidation

Article abstract of DOI:10.1016/j.jhazmat.2019.121084

A native laccase (Lac-Q) with robust cold-adapted and thermostable characteristics from the white-rot fungus Pycnoporus sp. SYBC-L10 was purified, characterized, and used in antibiotic treatments. Degradation experiments revealed that Lac-Q at 10.0 U mL^?1 coupled with 1.0 mmol L^?1 ABTS could degrade 100% of the tetracycline or oxytetracycline (50 mg L^?1) within 5 min with a static incubation at 0 °C (pH 6.0). The presence of the Mn²⁺ ion inhibited the removal rate of tetracycline and oxytetracycline by the Lac-Q–ABTS system, and the presence of Al³⁺, Cu²⁺, and Fe³⁺ accelerated the removal rate of tetracycline and oxytetracycline by the Lac-Q–ABTS system. Furthermore, seven transformation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible degradation pathway including deamination, demethylation, and dehydration was proposed. Furthermore, the growth inhibition of Bacillus altitudinis SYBC hb4 and E. coli by tetracycline antibiotics revealed that the antimicrobial activity was significantly reduced after treatment with the Lac-Q–ABTS system. Finally, seven transformation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible degradation pathway including deamination, demethylation, and dehydration was proposed. These results suggest that the Lac-Q–ABTS system shows a great potential for the treatment of antibiotic wastewater containing different metal ions at various temperatures.

Full text of DOI:10.1016/j.jhazmat.2019.121084

Journal of Hazardous Materials 382 (2020) 121084
Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Characterization of a robust cold-adapted and thermostable laccase from
Pycnoporus sp. SYBC-L10 with a strong ability for the degradation of
tetracycline and oxytetracycline by laccase-mediated oxidation
a,⁎
a
a
a,c
a
a
b
Qiaopeng Tian , Xin Dou , Lin Huang , Lei Wang , Di Meng , Lixin Zhai , Yu Shen ,
b
a
a,⁎
Cuiping You , Zhengbing Guan , Xiangru Liao
a
b
c
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, PR China
School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, PR China
School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia, 014010, PR China
G R A P H I C A L A B S T R A C T
A R T I C L E I N F O
Editor: R. Debora
A B S T R A C T
A native laccase (Lac-Q) with robust cold-adapted and thermostable characteristics from the white-rot
fungus Pycnoporus sp. SYBC-L10 was purified, characterized, and used in antibiotic treatments.
Keywords:
−
−
1
−1
Laccase
Degradation experiments revealed that Lac-Q at 10.0 U mL
coupled with 1.0 mmol L
1
ABTS could
Circular dichroism
Emerging contaminants
Antimicrobial activity
Transformation products
degrade 100% of the tetracycline or oxytetracycline (50 mg L ) within 5 min with a static incubation at
0 °C (pH 6.0). The presence of the Mn ion inhibited the removal rate of tetracycline and oxytetracycline
by the Lac-Q–ABTS system, and the presence of Al , Cu , and Fe accelerated the removal rate of
2
+
3+
2+
3+
tetracycline and oxytetracycline by the Lac-Q–ABTS system. Furthermore, seven transformation products
of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were identified
during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible degradation pathway
including deamination, demethylation, and dehydration was proposed. Furthermore, the growth inhibi-
tion of Bacillus altitudinis SYBC hb4 and E. coli by tetracycline antibiotics revealed that the antimicrobial
activity was significantly reduced after treatment with the Lac-Q–ABTS system. Finally, seven transfor-
mation products of oxytetracycline (namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351)
were identified during the Lac-Q-mediated oxidation process by using UPLC–MS/MS. A possible de-
gradation pathway including deamination, demethylation, and dehydration was proposed. These results
⁎
Corresponding authors.
E-mail addresses: qiaopengt@126.com (Q. Tian), liaoxiangru@163.com (X. Liao).
https://doi.org/10.1016/j.jhazmat.2019.121084
Received 6 March 2019; Received in revised form 21 August 2019; Accepted 21 August 2019
Available online 22 August 2019
0304-3894/ © 2019 Elsevier B.V. All rights reserved.

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
suggest that the Lac-Q–ABTS system shows a great potential for the treatment of antibiotic wastewater
containing different metal ions at various temperatures.
1
. Introduction
Antibiotics have been recently cited as a group of emerging con-
cold-adapted enzyme for the removal of TCs. This gap in the research
prompted the search for an enzyme that can effectively degrade environ-
mental pollutants at low temperatures. Moreover, the utilization of laccase
from the genus Pycnoporus in TCs treatment remains unexplored. Among
the various white-rot fungi, the genus Pycnoporus has the ability to over-
produce high redox potential laccase as the predominant ligninolytic en-
zyme (Lomascolo et al., 2011), which could save purification steps for direct
use in biotechnological processes (Lomascolo et al., 2002).
taminants in the environment since they have been widely used in
human and veterinary medicine (Chen et al., 2016). As a result, a
variety of antibiotic residues was detected in different environmental
areas (Qiao et al., 2018; Carvalho and Santos, 2016; Luo et al., 2011;
Sim et al., 2011). For example, a study published in 2013 showed that
the total consumption of 36 antibiotics was 92,700 tons in China, and
approximately 58% of the antibiotics were eventually released into the
receiving environment following various wastewater treatments (Zhang
et al., 2015). These phenomena indicate that using conventional
treatment methods is ineffective for removing antibiotic residues from
wastewater (Zhang et al., 2015; Liang et al., 2019; Guo et al., 2017).
Antibiotic residues in the environment directly affect the community
structure of microbes and serve as a selective pressure for antibiotic
resistant bacteria (ARB) and antibiotic resistance genes (ARGs) (Jia
et al., 2017; Christou et al., 2017).
In this study, an extracellular fungal laccase (Lac-Q) with strong
cold-adapted and thermostable characteristics from Pycnoporus sp.
SYBC-L10 was purified and characterized. Batch experiments to in-
vestigate the impact of different conditions (e.g., mediators, pH, tem-
perature, enzyme-substrate ratio, mediator concentrations, and metal
ions) on the degradation rates of TC and OTC using Lac-Q coupled with
mediators were performed. The transformation products of OTC were
identified by using UPLC–MS/MS, and a possible degradation pathway
of OTC by Lac-Q-mediated oxidation was proposed. Growth inhibition
tests were performed using gram-positive Bacillus altitudinis SYBC hb4
(B. altitudinis) and gram-negative Escherichia coli BL21 (E. coli). Finally,
the transformation products of OTC were identified by using UPLC–MS/
MS, and a possible degradation pathway of OTC by Lac-Q-mediated
oxidation was proposed. The findings of this study demonstrate new
evidence and novel areas of application for a laccase-mediator system
to remove antibiotics at low temperatures.
Tetracycline antibiotics (TCs), such as tetracycline (TC), oxytetracycline
(
OTC), chlortetracycline (CTC), and doxycycline (DC), are broad-spectrum
antimicrobial agents that often have unrestricted use in animal husbandry
and aquaculture (Suda et al., 2012). Therefore, the removal of TCs in nat-
ural environments has garnered increasing attention (He et al., 2019;
Taheran et al., 2017; de Cazes et al., 2014; Gomez-Pacheco et al., 2011).
White-rot fungi and their extracellular ligninolytic enzymes are promising in
the degradation of various environmental pollutants (Mir-Tutusaus et al.,
2
018; Wesenberg et al., 2003). As described by Wen et al. in 2009, crude
2. Experimental
lignin peroxidase from Phanerochaete chrysosporium could effectively de-
grade TC and OTC in the presence of hydrogen peroxide and veratryl al-
cohol (Wen et al., 2009). In contrast to peroxidases, laccase only needs
oxygen as the final electron acceptor for the oxidation reaction to occur,
which offers an alternative green method for the biodegradation of various
environmental pollutants (Wong, 2009). Moreover, the oxidation of re-
calcitrant substrates, such as non-phenolic compounds, with redox poten-
tials greater than those of laccases usually requires the help of the mediators
that constitute the laccase-mediator systems (LMSs) (Canas and Camarero,
2.1. Chemicals, strain, and culture conditions
Tetracycline (TC, 95%, CAS: 60-54-8) and oxytetracycline (OTC,
98%, CAS: 79-57-2) were purchased from Shanghai Yuanye Biological
Technology Co., Ltd. (Shanghai, China). 2,2′-azino-bis (3-ethylben-
zothiazoline-6-sulfonic acid) diammonium salt (ABTS, 98%, CAS:
30931-67-0), 2,6-dimethoxyphenol (2,6-DMP, 99%, CAS: 91-10-1),
syringaldehyde (SA, 98%, CAS: 134-96-3), and 1-hydroxybenzotriazole
(HBT, 99%, CAS: 123333-53-9) were purchased from Sigma-Aldrich
Chemical Company (St. Louis, MO, USA). 3-Hydroxyanthranilic acid (3-
HAA, 98%, CAS: 548-93-6) was purchased from TCI Chemical Company
(Shanghai, China). Protein molecular mass markers (C610013) were
purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China).
A Major Science MC-0203 cooling dry bath incubator (made in Taipei,
China) was used for controlling the temperature (−10 °C – 100 °C) in
the characterization of Lac-Q and the antibiotics degradation experi-
ments. Additionally, methanol and acetonitrile (HPLC grade) were
purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai,
China). All other chemicals and solvents were reagent grade.
2010; Morozova et al., 2007). The more-studied mediators are 2,2′-azino-bis
(
3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), syr-
ingaldehyde (SA), 1-hydroxybenzotriazole (HBT), and 3-hydroxyanthranilic
acid (3-HAA) (Canas and Camarero, 2010; Naghdi et al., 2018). ABTS
oxidizes the substrate via an electron transfer (ET) route, whereas HBT, SA,
and 3-HAA follow a hydrogen atom transfer (HAT) mechanism (Canas and
Camarero, 2010; Yang et al., 2017).
Previous studies clearly exemplified that the combination of laccase and
redox mediators can efficiently transform antibiotics (Suda et al., 2012;
Weng et al., 2012). For example, in 2012, Suda et al. reported that 10 nkat
−1
−1
mL
laccase from Trametes versicolor coupled with 0.2 mmol L
HBT
could degrade 100% of the CTC and DC after 15 min and degrade 100% of
the TC and OTC after 60 min of incubation at 30 °C (pH 4.5) with shaking at
Pycnoporus sp. SYBC-L10 was a stock culture in the Biocatalysis and
Transformation Biology lab at Jiangnan University (Tian et al., 2018);
the identification of the fungus is shown in Fig. S1 (Supplemental
Material). Pycnoporus sp. SYBC-L10 was first cultured on a potato
dextrose agar (PDA) plate at 30 °C for 5 d and subsequently inoculated
into a potato dextrose broth (PDB) medium to gain a seed culture at
−1
1
50 r min (Suda et al., 2012). Recently, laccase from Trametes versicolor
and other fungi was extensively and successfully used in the degradation of
TCs (de Cazes et al., 2014; Shao et al., 2019; Sun et al., 2017a; Ding et al.,
2
016; Llorca et al., 2015; de Cazes et al., 2015; Abejon et al., 2015a; Abejon
−1
et al., 2015b). However, the degradation rates found in the literature are
still slower than the rates of some other methods, such as ozonation
30 °C and 200 r min . After 2 d of incubation, a modified basal
medium (Saparrat et al., 2002) with pH 4.0 was inoculated with the
seed culture at a concentration of 10% (v/v) for laccase production
(50 mL in 250 mL Erlenmeyer flasks), and incubated at 30 °C with
(Gomez-Pacheco et al., 2011), ferrate(VI) oxidation and adsorption (Ma
et al., 2012), photocatalysis (He et al., 2019), and lignin peroxidase oxi-
dation (Wen et al., 2009), and there is a scarcity of research addressing the
removal of TCs by fungal laccase at low temperatures. In winter, the
ambient temperature is generally very low, which indicates the need for a
−1
shaking at 200 r min . The modified basal medium contained the
−1
−1
−1
following: glucose, 15 g L ; soybean meal, 5 g L ; diammonium hy-
−
1
−1
drogen citrate, 0.5 g L ; CuSO
4
·5H
2
O, 0.125 g L ; KCl, 0.5 g L
;
−
1
−1
−1
KH
2
PO
4
, 1 g L ; MgSO
4
·7H O, 0.5 g L ; yeast extract, 1 g L ; and
2
2

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
O, 0.01 g L−1. After 7 d of incubation, the cultures were
HBT, or 3-HAA; they were statically incubated in 0.1 mol L buffers at
−1
MnSO4·H
2
harvested for laccase purification.
each of the experimental conditions for 5 min before the laccase and
metal ions were added. Control tests, including those with heat-in-
activated purified Lac-Q, were performed under identical conditions. A
2
.2. Laccase purification, gel electrophoresis, N-terminal sequence, and
−1
mass spectrometry
control with the individual antibiotic (50 mg L ) in buffers was used to
rule out any possible physicochemical process during the degradation
of TCs. At different time intervals of the reaction, a 0.25 mL sample
from each reaction mixture was collected and immediately added to
0.5 mL methanol to stop the reaction (the effect of methyl alcohol on
the Lac-Q activity is shown in Fig. S2 in the supplemental material); this
sample was then stored in a vial at -20 °C for the determination of the
TCs’ concentration by HPLC (Sun et al., 2017a; Ding et al., 2016). The
removal efficiency (R) of the TCs in the degradation experiments was
calculated according to the formulas in a previous study (Ding et al.,
2016).
Pycnoporus sp. SYBC-L10 laccase was purified using ammonium
sulfate precipitation (40–70%), a Sephadex G-25 size-exclusion chro-
matography column (GE Healthcare, Little Chalfont, UK), and a Hi Trap
Q HP anion exchange chromatography column (1.6 cm × 30 cm, 5 mL,
GE Healthcare, Little Chalfont, UK). The details of this purification
process are shown in the supplemental material. Then, the purified Lac-
Q was kept at -20 °C for further use.
Laccase isozymes in the supernatant were analyzed by Native-PAGE.
The Native-PAGE of crude laccase was performed as described by (Zeng
−
1
et al. (2011)), laccase activity was stained with DMP in a 0.1 mol L
citrate-phosphate buffer (pH 3.0). The SDS-PAGE of the purified Lac-Q
was performed as described by (Liu et al. (2016)).
2
.6. Test of growth inhibition
The N-terminal amino acid sequence of Lac-Q was determined by
automated Edman degradation in a PPSQ-33A Protein Sequencing
System (Sangon Biotech (Shanghai) Co., Ltd.). The protein mass spec-
trometry of Lac-Q was performed according to the method discussed in
a previous study (Wang et al., 2010) via the matrix-assisted laser des-
orption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).
Growth inhibition tests were performed using gram-positive B. al-
titudinis (Zhang et al., 2016) and gram-negative E. coli during the Lac-
Q–ABTS-mediated TC or OTC reactions, as described previously, with
minor modifications (Suda et al., 2012; Yang et al., 2017). B. altitudinis
and E. coli were cultivated in a Luria-Bertani (LB) medium at 37 °C with
−1
shaking at 200 r min . After 24 h of incubation, the percentage of the
bacterial growth inhibition (%) was calculated using the same method
utilized by (Yang et al. (2017)). The 24-h IC50 values (50% effective
concentration) of the TC or OTC against B. altitudinis and E. coli were
determined. Furthermore, the Lac-Q–ABTS mediated TC or OTC reac-
tion solution was added into a fresh LB medium that included the
bacterial suspension and incubated at 37 °C with shaking at 200 r
2.3. Laccase activity assay
Laccase activity was determined using the methods given in a pre-
vious study (Wang et al., 2010).
2.4. Properties and CD spectra analysis of purified Lac-Q
−1
min
for 24 h. The initial concentration of TC or OTC before the en-
−1 −1
zymatic treatment corresponded to 0.5 mg L and 1.67 mg L for the
The effects of various conditions (e.g., pH, temperature, metal ions,
tests using B. altitudinis and E. coli, respectively.
and organic solvents) on the purified Lac-Q activity and the stability, as
well as the effect of inhibitors on the purified Lac-Q activity, were
studied. The methods were performed using the approach found in a
previous study (Wang et al., 2010), with some modifications. The de-
tails are shown in the supplemental material.
2.7. Analytical methods
2
.7.1. Quantification of TCs by HPLC
The concentrations of TCs in the reaction mixtures during the Lac-Q-
The evaluation of the kinetic parameters of the purified Lac-Q were
performed using the approach found in an earlier study (Zeng et al.,
mediated reactions were determined by high-performance liquid
chromatography (HPLC), and each sample was replicated twice. The
HPLC system (Chromaster, Hitachi High-Technologies, Tokyo, Japan)
was equipped with the XSelect® HSS T3 column (length × I.D.:
2
012), with some modifications. The details are shown in the supple-
mental material.
UV-CD spectrum of the purified Lac-Q affected by various condi-
2
5 cm × 4.6 mm; 5-μm particles, Waters, Milford, Massachusetts, USA)
tions (e.g., pH, temperature, CuSO
4
, Al
2
(SO
4
)
3
, NaF, and NaN ) were
3
and a diode array detector (DAD-Chromaster5430, Hitachi). The details
of the analytical methods used in our work are described elsewhere
studied. The methods were performed using the approach from a pre-
vious study (Liu et al., 2016), with some modifications. The details are
shown in the supplemental material.
(
Sun et al., 2017a). TC and OTC were detected at 268 nm. Quantifica-
tion was achieved by a 5-point external calibration curve ranging from
5
to 25 mg L−1
.
2.5. Degradation experiments
The effect of the pH and temperature on the TC and OTC physico-
2.7.2. Identification of transformation products
chemical degradation experiments were investigated; the details are
shown in the supplemental material. The effects of mediators on the TCs
The transformation products of OTC were identified during the Lac-
Q-mediated oxidation process using ultra-performance liquid chroma-
tography tandem mass spectrometry with a triple quadrupole mass
spectrometer (UPLC–MS/MS). The details of the analytical methods are
described elsewhere (Pan et al., 2018), and UPLC–MS/MS analyses
were conducted in the positive ionization mode. The reaction mixture
was as described previously, with minor modifications (Sun et al.,
−
1
−1
(
50 mg L ) degradation were investigated. Individual TCs (50 mg L
)
−
1
in the presence or absence of 0.5 mmol L
ABTS, SA, HBT, or 3-HAA
−
1
−1
were statically incubated with 5.0 U mL Lac-Q in 0.1 mol L citrate-
phosphate buffer (pH 6.0) at 25 °C or 0 °C for 3 min. Furthermore, the
effects of the pH values (3.0–7.0), temperature (0 °C – 70 °C), laccase
−
1
−1
−1
activity (1.0–12.0 U mL ), ABTS concentrations (0–4.0 mmol L ),
2017a); the reaction solution initially contained 50 mg L OTC, 0.25 U
−
1
−1
−1
−1
and metal ions (in the presence or absence of 1 mmol L ) on the TCs’
mL
Lac-Q, and 0.5 mmol L
ABTS in a 4.5 mL 0.1 mol L
citrate-
−
1
(
50 mg L ) degradation were investigated. Finally, the degradation
phosphate buffer (pH 6.0) at 25 °C. Control samples that were Lac-Q-
free were created for comparison in UPLC–MS/MS. After a 40-min in-
cubation, the reaction solution was subjected to solid-phase extraction
(SPE) on the C18 column (6 mL, 500 mg) as described previously (Sun
et al., 2017a), and then the elution was collected and constituted to
0.25 mL for UPLC–MS/MS analysis.
curves of the TCs in the Lac-Q–ABTS system at 25 °C or 0 °C and de-
gradation curves of the TCs in the Lac-Q–SA system, Lac-Q–HBT system,
and Lac-Q–3-HAA system at 25 °C were investigated. All the degrada-
tion experiments were placed in 1.5-mL autoclave-sterilized tubes and
individual TCs (50 mg L− ) in the presence or absence of ABTS, SA,
1
3

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
2
.8. Data analysis
original activity after a 3 h incubation at 40 °C, 50 °C, 60 °C, and 70 °C,
respectively, and lost 96.3% of its original activity after a 20-min in-
cubation at 80 °C (Fig. 2D). These results clearly indicate that Lac-Q
exhibited a better thermostability and pH stability than found in most
of the previous studies (Lomascolo et al., 2011; Wang et al., 2010;
Ramirez-Cavazos et al., 2014).
All the experiments were performed in triplicate, and the error bar
represents the standard deviations (n = 3). The software Origin 9.1 was
used to calculate the mean value/error bar and to plot figures of the
collected data.
2
+
2+
As shown in Fig. 2E, Mg and Ni showed slight activation effect
−
1
2+
on the Lac-Q activity. Lac-Q was slightly activated by 1 mmol L Cu
3
. Results and discussion
−1
−1
2+
and slightly inactivated by 5 mmol L
and 10 mmol L
Cu . This
+
result is in agreement with previous study (Wang et al., 2010). Na
,
showed a slight inhibitory effect on
exhibited a strong inhibitory effect
3.1. Properties of purified Lac-Q
+
2+
2+
2+
3+
3+
K
, Ca , Mn , Zn , and Cr
2+
the Lac-Q activity. Fe
and Fe
Laccase secreted by Pycnoporus sp. SYBC-L10 was purified as de-
on the Lac-Q activity. A similar phenomenon was explained in earlier
scribed in Table S1 and shown in Fig. S3 (Supplemental material). After
2+
studies (Liu et al., 2016; Luo et al., 2018). Fe
is competitively in-
purification, the specific activity of the laccase from Pycnoporus sp.
2+
+▪
hibited in the laccase activity because the Fe ion restored the ABTS
−1
SYBC-L10
increased
16.82-fold,
from
57.92 U mg
to
3+
to the ABTS. However, Al
showed a strong activation effect on the
−
1
1
032.27 U mg , using ABTS as a substrate with yield of 46.32%. As
Lac-Q activity. This result is opposite to the results from earlier reports
shown in Fig. 1A and B, a single band was observed according to Na-
tive-PAGE as well as SDS-PAGE, indicating only one isoenzyme in the
culture supernatant; a purified form of laccase with an apparent mo-
lecular weight of about 60 kDa was obtained (Lac-Q). The analysis of
the N-terminal amino acid sequence (Table S2, Supplemental Material)
and protein mass spectrometry (Table S3, Supplemental Material) in-
dicated that Lac-Q was a Pycnoporus protein; the details are shown in
the supplemental material.
(
Wang et al., 2010; Liu et al., 2013; Zhu et al., 2011). This result
3+
probably indicates that Lac-Q is an Al
activated protein, and the
3+
presence of Al
significantly altered the structure of Lac-Q. Interest-
−1 2+ + + 2+ 2+
ingly, the presence of 10 mmol L
Mg , Na , K , Ca , Mn
,
2+
2+
3+
3+
Zn , Ni , Fe , and Cr exhibited no obvious inactivation effect on
the Lac-Q stability (shown in Fig. 2F). However, the presence of
−1
3+
2+
1
0 mmol L Al and Cu showed a slight inactivation effect on the
Lac-Q stability (Fig. 2F). Lac-Q exhibited a strong resistance to organic
solvents, and the details are shown in Fig. S4 (Supplemental Material).
Lac-Q was slightly inhibited by L-Cysteine and Dithiothreitol and
The optimum pH of Lac-Q was 3.0 for the two substrates (Fig. 2A).
The study of the pH stability revealed that Lac-Q was stable from pH 3.0
to pH 10.0, especially at pH 6.0 (Fig. 2B). The optimum temperature of
Lac-Q was 70 °C for the two substrates (Fig. 2C). Remarkably, Lac-Q
showed about 51% of the maximal activity incubation at 0 °C, implying
that Lac-Q has a strong tolerance to cold conditions. A similar phe-
nomenon has also been observed in previous studies (Wang et al.,
strongly inhibited by NaCl, NaF and NaN
3
(especially NaN , see
3
Table 1).
The CD spectrum (Fig. 3) and Table S4 show that Lac-Q is 10.6% α-
helix, 36.5% β-sheet, 21.3% β-turn, and 31.6% random coils, which is
very similar with other laccases (Liu et al., 2016; Mot et al., 2012). As
2
010). Lac-Q retained 108.9%, 106.7%, 106.5%, and 68.0% of its
Fig. 1. (A) Native-PAGE of crude laccase. (B) SDS-PAGE of purified Lac-Q; lane 1: molecular mass markers; lane 2: laccase in Tubes 3 and 4 from the Hi Trap Q HP.
4

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
−
1
Fig. 2. Properties of purified Lac-Q. (A) Effect of pH on Lac-Q activity. (B) Effect of pH on the stability of Lac-Q. Lac-Q was incubated in 0.1 mol L
buffers at
different pH values (3–10) at room temperature for 24 h. (C) Effect of temperature on Lac-Q activity. (D) Effect of temperature on the stability of Lac-Q. Lac-Q was
−
1
incubated at different temperatures (40 °C – 80 °C) in a 0.1 mol L citrate-phosphate buffer (pH 6.0) for 0–3 h. (E) Effect of different metal ions on Lac-Q activity. (F)
−1
−1
Effect of different metal ions on the stability of Lac-Q. Lac-Q was incubated with various metal ions (final concentration: 10 mmol L ) in a 0.1 mol L
phosphate buffer (pH 6.0) at room temperature for 24 h.
citrate-
shown in Fig. 3A, no significant changes in the CD spectra were ob-
served from pH 2.5 to pH 6.5. A similar phenomenon was also observed
in other studies (Liu et al., 2016). As shown in Fig. 3B and Table S4,
slight changes in the CD spectra and a slow decrease of the β-sheet in
the secondary structure were observed as the temperature increased
altered the CD spectra (Fig. 3D), suggesting a dramatic decrease of the
β-sheet content in laccase (Table S4). As shown in Fig. 3E–F and Table
S4, the presence of NaF and NaN slightly changed in the CD spectra
3
and a slow increase of the β-sheet in the secondary structure was ob-
−
1
−1
served. The mechanisms of the F and N
3
inhibition of the laccases’
−
1
−1
2+
−1
−1
from 30 to 60 °C. Interestingly, 1 mmol L or 5 and 10 mmol L Cu
activity are due to the F
and N
3
anions binding to the type 2
exhibited different changes in the CD spectra (Fig. 3C), and this result is
copper ion at the active site of the laccases and interrupting the internal
−1
consistent with the fact that Lac-Q was slightly activated by 1 mmol L
2+
electron transfer pathway (Baminger et al., 1999).
2
+
−1
−1
Cu
and slightly inactivated by 5 mmol L
and 10 mmol L
Cu
.
The purified Lac-Q exhibited different activities with DMP and
3
+
−1
−1
The presence of Al
(ranging from 1 to 50 mmol L ) significantly
ABTS, with K
m
values of 0.0712 and 0.0191 mmol L , respectively
5

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Table 1
The effect of inhibitors on purified Lac-Q activity.
−
1
Concentration (mmol L−1
Concentration (mmol L
)
Relative activity (%)
)
Relative activity (%)
None
–
100
L-Cysteine
0.1
109.77 ± 2.35
93.74 ± 0.60
76.11 ± 2.35
100.83 ± 1.46
82.14 ± 4.07
71.55 ± 2.20
91.68 ± 3.59
57.37 ± 2.66
42.51 ± 0.54
NaF
0.1
0.5
1
13.45 ± 1.53
2.93 ± 0.07
1.37 ± 0.15
20.02 ± 1.60
4.01 ± 0.10
1.79 ± 0.17
0
1
.5
Dithiothreitol
NaCl
0.1
NaN
3
0.01
0.05
0.1
0
1
1
5
1
.5
0
Laccase activity was measured using 1 mmol L−1 ABTS as the substrate at 30 °C (pH 3.0).
(
Table 2). In addition, the catalytic efficiencies (kcat/K
m
) varied from
et al., 2016). Remarkably, the Lac-Q–ABTS system shows an efficient
removal of TCs at pH 4.0, ranging from 0 °C to 70 °C, suggesting definite
potential for practical applications (Fig. 5C). The removal of TC and
OTC at pH 6.0 increased from 0 °C to 40 °C, and then remained stable
from 40 °C to 70 °C (Fig. 5D). This result is in substantial agreement
with previous studies that found TCs degradation rates are mostly in-
creased by increasing temperatures (de Cazes et al., 2014; Ding et al.,
2016). As described by Wen et al., the degradation rates of TC and OTC
by crude manganese peroxidase quickly increased with the increase of
−
1
−1
−1
−1
3
012.92 L s mmol
for DMP up to 60,536.13 L s mmol
for
ABTS, indicating that ABTS was a more effective substrate than DMP.
3
.2. Removal of TC and OTC by the Lac-Q-mediator systems under various
conditions
Previous studies found that mediators considerably impact the de-
gradation rates of antibiotics in laccase-mediated oxidation (Shao et al.,
−1
2
019; Ding et al., 2016; Weng et al., 2013). Margot et al. proposed a
the enzyme-substrate ratio from 0 to 2.0 U mg , and remained at
−1
laccase-mediator reaction model, indicating mediators can be oxidized
by laccases to reactive radicals and the reactive radical oxidation of the
target pollutants (shown in Fig. 4A). The mechanisms of the laccase-
mediated oxidation pollutants are frequently described in previous
studies (Canas and Camarero, 2010; Naghdi et al., 2018; Margot et al.,
about 92% after a threshold ratio (2.0 U mg ) was achieved (Wen
−1
et al., 2010). In the present study, the threshold ratio was 100 U mg
−
1
for the Lac-Q removal of TCs at 25 °C (Fig. 5E) and 200 U mg for the
Lac-Q removal of TCs at 0 °C (Fig. 5F), indicating the removal of TCs at
0 °C is more difficult than at 25 °C. This result is probably due to the
temperature effect of the Lac-Q activity and effect of the catalytic re-
action process during the incubation at 0 °C. Therefore, it is re-
2
015). As shown in Fig. 4B, the Lac-Q–ABTS system showed the highest
degradation rates, with a removal ratio of 96% of TC and a removal
ratio of 90% of OTC after a 3-min static incubation at 25 °C (pH 6.0).
The Lac-Q–SA system had the second highest removal treatment, with a
removal ratio of 49% of TC and a removal ratio of 45% of OTC after a 3-
min static incubation at 25 °C (pH 6.0). In comparison to the Lac-
Q–ABTS system, the Lac-Q–HBT system and the Lac-Q–3-HAA system
had a far lower rate. Furthermore, the Lac-Q–ABTS system showed a
greater advantage when the degradation experiments were incubated at
−1
−1
commended that the 100 U mg
and 200 U mg
enzyme-substrate
ratio be applied to obtain a cost-effective and efficient degradation at
25 °C and at 0 °C, respectively. As shown in Fig. 5G, the removal of TC
and OTC increases with the increase of the ABTS concentration from 0
−1
to 0.125 mmol L , and then the increasing trend slowed with the in-
−
1
crease of the ABTS concentration from 0.125 to 0.5 mmol L . A similar
phenomenon was also observed in Fig. 5H and in other studies (Shao
et al., 2019; Ding et al., 2016). However, with the increase of the ABTS
0
°C (pH 6.0), indicating the ABTS was a better mediator for mediated
−1
TCs oxidation (compare Figs. 4C with B). In 2014, Shi et al. found that a
higher affinity of laccase from white-rot fungus Echinodontium taxodii to
syringic acid than to syringaldehyde and acetosyringone resulted in a
higher catalytic efficiency of sulfonamide antibiotics (SAs) (Shi et al.,
concentration from 0.5 to 2.0 mmol L , the removal rate of TC slightly
increased, whereas the removal rate of OTC decreased (Fig. 5G).
However, this is unlike the removal of TC and OTC at 0 °C (both slightly
increased with the increase of the ABTS concentration from 0.5 to
−1
2
014). However, TC and OTC showed no significant reductions with
2.0 mmol L ), indicating the relatively higher mediator concentration
is beneficial to the removal of TCs at 0 °C (Fig. 5H). Therefore,
treatment with Lac-Q without any mediator when compared to the
control treatment (Fig. 4B and C), indicated that TCs could not be ef-
ficiently removed by laccase without any mediator. Similarly, laccase
from the Trametes versicolor removal of TCs without any mediator
showed no significant difference from the control treatment (Suda
et al., 2012; Ding et al., 2016; Becker et al., 2016). As shown in Fig. S5,
TC and OTC were comparatively stable at low temperatures and around
pH 6.0 – pH 7.0. Therefore, an enzyme that can effectively remove TCs
at low temperatures and around pH 6.0 – pH 7.0 has an important
significance for practical applications.
−1
0.5 mmol L ABTS is recommended for removing TCs in contaminated
−
1
water at 25 °C. Similarly, 1.0 mmol L
ABTS is recommended for re-
moving TCs in contaminated water at 0 °C.
The presence of metal cations may be an important factor that in-
fluences the TCs transformation during laccase-mediated oxidation
(Sun et al., 2017b). To further investigate the effects of metal ions on TC
and OTC removal, different metal ions were used in the Lac-Q–ABTS
2
+
system. As shown in Fig. 5I and J, the presence of the Mn
ion in-
hibited the removal rate of TC and OTC by the Lac-Q–ABTS system and
3
+
2+
3+
In consideration of the superiority of ABTS in the degradation rate
and solubility, removal of TCs by the Lac-Q–ABTS system required
further investigation. Therefore, the effects of pH, temperature, en-
zyme-substrate ratio, and ABTS concentration in the Lac-Q–ABTS
system were investigated. As shown in Fig. 5A, the TC and OTC de-
gradation rates decreased when the pH started increasing after the pH
increased to 6. The optimal pH was around 3.0–5.0 for removal of TCs
at 25 °C (Fig. 5A) and 4.0 for removal of TCs at 0 °C (Fig. 5B). This result
was opposite to the results found in previous studies, indicating that
TCs were transformed by laccase–SA or laccase–HBT systems and
showing the optimal pH was around 6.0–7.0 (Sun et al., 2017a; Ding
the presence of Al , Cu , and Fe
accelerated the removal rate of
2+
TC and OTC by the Lac-Q–ABTS system. The effect of the Mn ion was
3
+
2+
3+
inhibited and the effect of the Al , Cu , and Fe
ions were ac-
celerated, which shows a greater significance for the incubation at 0 °C
(shown in Fig. 5I and J). However, the presence of other metal ions did
not significantly affect the TC and OTC removal during a 3-min static
2
+
incubation at 25 °C. In view of the slight effect of the Mn ion on the
2
+
Lac-Q activity (Fig. 2E), this is probably due to the Mn ion inhibiting
the interaction between Lac-Q, TCs, and redox mediators (Mot et al.,
3
+
2012; Sun et al., 2017b). There are two possible causes for the Al
,
2
+
3+
Cu , and Fe
ions accelerating the removal rate of TC and OTC by
6

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Fig. 3. (A) UV-CD spectrum of purified Lac-Q affected by varying pH at room temperature. (B) UV-CD spectrum of Lac-Q affected by varying the temperature in a
−
1
0
.1 mol L citrate-phosphate buffer (pH 6.0). (C–F) UV-CD spectrum of Lac-Q affected by varying concentrations of CuSO
4
(C), Al
2
(SO
4
)
3
(D), NaF (E), or NaN
3
(F)
−
1
in a 0.1 mol L
citrate-phosphate buffer (pH 6.0) at room temperature.
ion. It is also noteworthy that heavy metals, like Zn2+, Ni , and Cr
2+
3+
,
Table 2
Kinetic parameters of purified Lac-Q from Pycnoporus sp. SYBC-L10.
did not have a significant effect on the removal of TC and OTC by Lac-Q
combined with ABTS (also shown in Fig. 5I and J). These results clearly
indicate that Lac-Q coupled with ABTS has the definite potential for the
removal of TC and OTC in practical applications.
−
1
−1
−1
mmol−1
)
Substrates
K
m
(mmol L
)
k
cat (s
)
kcat/K
m
(L s
ABTS
DMP
0.0191
0.0712
1156.24
214.52
60,536.13
3012.92
As shown in Table 3, considerable research efforts have focused on
different treatments for the degradation of TCs. However, previous
studies mostly used commercial laccase for the degradation of TCs at
the Lac-Q–ABTS system: (1) It could be due to the adsorption of TC and
OTC by metal ions or effect of the stabilization of TC and OTC by metal
2
0–30 °C. The sources of laccase are very abundant (Bilal et al., 2019;
3
+
2+
3+
Forootanfar and Faramarzi, 2015; Baldrian, 2006), but the utilization of
laccase from other sources in the TCs treatment is rarely found in the
literature. The degradation rates found in the literature are still slower
ions, such as Al , Cu , and Fe
ions, or (2) it could be due to the
3+
effect of the metal ions on the enzyme activity, such as that of the Al
7

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Fig. 4. (A) Proposition of a laccase-mediator reaction model (adapted from the literature (Margot et al., 2015)). Different treatments for the degradation of TCs at
−
1
−1
−1
2
5 °C (B) or 0 °C (C). Reaction conditions: 50 mg L TC or OTC, 5.0 U mL Lac-Q, in the presence or absence of 0.5 mmol L ABTS, SA, HBT, or 3-HAA, pH 6.0,
and static incubation.
than the rates of some other methods, such as those from ozonation
degraded 100% of the TC and OTC after 4 min and 5 min, respectively,
with a static incubation at 25 °C (pH 6.0) (Fig. 6A and B). Remarkably,
(
Gomez-Pacheco et al., 2011) and photocatalysis (He et al., 2019), and
−1
−1
there is a scarcity of research addressing the removal of TCs by fungal
laccase at low temperatures. This gap in the research prompted the
search for an enzyme that can effectively degrade environmental pol-
lutants at low temperatures. In the present study, a native laccase from
Pycnoporus sp. SYBC-L10 was introduced to degrade the TCs. The results
Lac-Q at 10.0 U mL coupled with 1.0 mmol L ABTS degraded 100%
of the TC and OTC after 5 min with a static incubation at 0 °C (pH 6.0)
(Fig. 6C and D). The present report is the first time TCs have been ef-
ficiently degraded at 0 °C (pH 6.0). Furthermore, the degradation
curves of the TC and OTC treatment in the Lac-Q–SA system, Lac-
Q–HBT system, and Lac-Q–3-HAA system at 25 °C are shown in Fig.
−
1
−1
showed that Lac-Q at 5.0 U mL
coupled with 0.5 mmol L
ABTS
8

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Fig. 5. (A–B) Effect of pH on TCs’ degradation in the Lac-
−
1
Q–ABTS system. Reaction conditions: 50 mg L
TC or
−1
−1
OTC, 3.0 U mL
Lac-Q, 0.5 mmol L
ABTS, pH 3.0–7.0,
and static incubation at 25 °C (A) and 0 °C (B). (C–D) Effect
of temperature on TCs’ degradation in the Lac-Q–ABTS
−1
system. Reaction conditions: 50 mg L
TC or OTC, 3.0 U
−1
−1
mL
D), and static incubation at 0–70 °C. (E–F) Effect of en-
zyme-substrate ratio on TCs’ degradation in the Lac-
Q–ABTS system. Reaction conditions: 50 mg L TC or
OTC, 1.0–6.0 U mL Lac-Q (E) and 1.0–12.0 U mL Lac-
Q (F), 0.5 mmol L ABTS, pH 6.0, and static incubation at
5 °C (E) and 0 °C (F). (G–H) Effect of ABTS concentration
Lac-Q, 0.5 mmol L
ABTS, pH 4.0 (C) and pH 6.0
(
−
1
−1
−1
−1
2
on TCs’ degradation in the Lac-Q–ABTS system. Reaction
−
1
−1
conditions: 50 mg L
TC or OTC, 3.0 U mL
Lac-Q,
ABTS (H),
−
1
−1
0
–2.0 mmol L
ABTS (G) or 0–4.0 mmol L
pH 6.0, and static incubation at 25 °C (G) and 0 °C (H). (I–J)
Effect of metal ions on TCs’ degradation in the Lac-Q–ABTS
−1
system. Reaction conditions: 50 mg L
TC or OTC,
−1
−1
3
.0 U mL Lac-Q, 0.5 mmol L ABTS, in the presence or
−1
absence of 1 mmol L
metal ions, pH 6.0, and static in-
cubation at 25 °C (I) and 0 °C (J).
S6A, B, and C (Supplemental Material), respectively. The removal of TC
and OTC was efficient in the Lac-Q–SA system and Lac-Q–HBT system.
However, TC and OTC were a portion of the reduction treatment with
the Lac-Q–3-HAA system during the period of the reaction initiation
(Fig. S6C, Supplemental Material). This result is in agreement with the
facts that 3-HAA oxidized and reduced forms are unstable, and laccase
rapidly oxidizes 3-HAA to form CA (Eggert et al., 1995). This result
demonstrated that the laccase–3-HAA system from Pycnoporus can be
9

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
10

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Fig. 6. Degradation curve of TCs and antimicrobial activity of TCs and transformation products during Lac-Q–ABTS-mediated reactions. Degradation of TC (A) and
OTC (B) at 25 °C. Reaction conditions: 50 mg L TC (A) and OTC (B), 5.0 U mL Lac-Q, 0.5 mmol L ABTS, pH 6.0, and static incubation. Degradation of TC (C)
and OTC (D) at 0 °C. Reaction conditions: 50 mg L OTC (C) and OTC (D), 10.0 U mL Lac-Q, 1.0 mmol L ABTS, pH 6.0, and static incubation.
−
1
−1
−1
−
1
−1
−1
involved in the degradation of antibiotics (Gao et al., 2018; Li et al.,
3.4. Transformation products of OTC and the proposed degradation
pathway
2
016; Dhawan et al., 2005).
Previous studies have demonstrated that TC was first oxidized to
OTC as the major transformation product (de Cazes et al., 2014; Sun
et al., 2017a). In this study, some major transformation products of OTC
were identified; these products are listed in Table 4. The degradation
curve of OTC during the Lac-Q–ABTS-mediated reactions in this section
is shown in Fig. S8 (Supplemental Material) and the mass spectra data
for all derivatives are shown in Fig. S9 (Supplemental Material). As
shown in Table 4, there are seven transformation products: TP 445, TP
431, TP 413, TP 399, TP 381, TP 367, and TP 351. Similar transfor-
mation products have also been identified as described in some pre-
vious studies (de Cazes et al., 2014; Yang et al., 2017; Shao et al., 2019;
Sun et al., 2017a; Llorca et al., 2015). As shown in Fig. 7, a possible
degradation pathway including deamination, demethylation, and de-
hydration was proposed based on the seven identified products. This
degradation pathway was similar, but not exactly the same, to the re-
sults found in previous studies where TCs were transformed by lacca-
se–SA or laccase–HBT systems (Shao et al., 2019; Sun et al., 2017a).
OTC could be transformed to TP 445 by deamination (de Cazes et al.,
2014; Llorca et al., 2015), and then TP 445 could be transformed to TP
381 by bi-demethylation and di-dehydration (de Cazes et al., 2014;
Yang et al., 2017; Shao et al., 2019; Sun et al., 2017a; Llorca et al.,
2015); finally, TP 381 demethylation and deamination could result in
TP 351 (Shao et al., 2019).
3
.3. Assessment of antimicrobial activity
The antimicrobial activity of TC and OTC before and after Lac-
Q–ABTS treatment was evaluated by the growth inhibition of B. alti-
tudinis and E. coli. TC and OTC displayed 24-h IC50 values of
−
1
−1
0
.211 mg L and 0.250 mg L against B. altitudinis, respectively (Fig.
S7A and B, Supplemental Material). In comparison, E. coli was more
−
1
insensitive, and the 24-h IC50 values of TC and OTC were 0.760 mg L
−
1
and 0.824 mg L
, respectively (Fig. S7C and D, Supplemental
Material). For the incubation at 25 °C, the antimicrobial activity of TCs
and the transformation products was completely removed after a 4-min
(
TC) or 5-min (OTC) treatment with the Lac-Q–ABTS system (Fig. 6A
and B). As described by Suda et al., the growth inhibition of Bacillus
subtilis (NBRC 3134) and E. coli (NBRC 14,249) by TC or OTC was
completely lost after 60 min of treatment with the laccase–HBT system
−
1
incubation at 30 °C (pH 4.5) with shaking at 150 r min
(Suda et al.,
2
012). Remarkably, the growth inhibition of the test bacteria by TC and
OTC was completely lost after 5 min for the incubation at 0 °C (Fig. 6C
and D). The present report is the first time TCs’ antimicrobial activity
has been efficiently eliminated at 0 °C (pH 6.0). The findings of this
study demonstrate novel areas of application for the use of the lacca-
se–ABTS system for removing TCs and reducing their toxicity at low
temperatures.
4
. Conclusion
In this study, an extracellular fungal laccase (Lac-Q) with strong
11

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Table 4
Accurate mass measurements and proposed molecular structures of the OTC transformation products.
Products Measured
Calculated
Relative
Proposed
Proposed structure
References
+
mass (m/z) mass (m/z) error (ppm) formula [M+H]
TP 445
TP 431
TP 413
445.1377
431.1223
413.1123
445.1373
431.1216
413.1111
0.9
1.6
2.9
C
C
C
22
21
21
H23NO
H21NO
H19NO
9
9
8
(de Cazes et al., 2014; Llorca et al., 2015)
(Yang et al., 2017; Shao et al., 2019; Sun et al., 2017a)
(de Cazes et al., 2014; Sun et al., 2017a; Llorca et al.,
2
015)
TP 399
TP 381
TP 367
TP 351
399.0962
381.0854
367.0698
351.0511
399.0954
381.0849
367.0692
351.0505
2.0
1.3
1.6
1.7
C
C
C
C
20
20
19
19
H
H
H
H
17NO
15NO
13NO
8
7
7
(Yang et al., 2017; Shao et al., 2019; Sun et al., 2017a)
(de Cazes et al., 2014)
NF
11
O
7
(Shao et al., 2019)
NF: No relevant studies were found.
cold-adapted and thermostable characteristics from Pycnoporus sp.
SYBC-L10 was purified and characterized. In addition, the ability of a
Lac-Q-mediator system to remove TC and OTC under various conditions
was also evaluated. Furthermore, seven transformation products of OTC
time TCs’ antimicrobial activity has been efficiently eliminated at 0 °C
(pH 6.0). Finally, seven transformation products of OTC (namely TP
445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351) were
identified during the Lac-Q–mediated oxidation process. A possible
degradation pathway including deamination, demethylation, and de-
hydration was proposed based on the seven identified products. These
results clearly indicate that the Lac-Q–ABTS system shows a great po-
tential for the treatment of antibiotic wastewater containing different
metal ions at various temperatures. Nonetheless, further studies must
be conducted for the following reasons: 1) to probe the interactive
mechanisms between Lac-Q and antibiotics or mediators and 2) to
(
namely TP 445, TP 431, TP 413, TP 399, TP 381, TP 367, and TP 351)
were identified during the Lac-Q-mediated oxidation process. A pos-
sible degradation pathway including deamination, demethylation, and
dehydration was proposed based on the seven identified products.
Furthermore, the growth inhibition of B. altitudinis and E. coli by TCs
revealed that the antimicrobial activity was significantly reduced after
treatment with the Lac-Q–ABTS system. The present report is the first
12

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Fig. 7. The proposed degradation pathway of OTC by Lac-Q-mediated oxidation.
explore the relationship between the structure of Lac-Q and its cold-
adapted and thermostable characteristics.
laccase production from Cyathus bulleri and Pycnoporus cinnabarinus. Bioresource.
Technol. 96, 1415–1418.
Ding, H.Y., Wu, Y.X., Zou, B.C., Lou, Q., Zhang, W.H., Zhong, J.Y., Lu, L., Dai, G.F., 2016.
Simultaneous removal and degradation characteristics of sulfonamide, tetracycline,
and quinolone antibiotics by laccase-mediated oxidation coupled with soil adsorp-
tion. J. Hazard. Mater. 307, 350–358.
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgements
de Cazes, M., Belleville, M.P., Petit, E., Llorca, M., Rodriguez-Mozaz, S., de Gunzburg, J.,
Barcelo, D., Sanchez-Marcano, J., 2014. Design and optimization of an enzymatic
membrane reactor for tetracycline degradation. Catal. Today 236, 146–152.
de Cazes, M., Belleville, M.P., Mougel, M., Kellner, H., Sanchez-Marcano, J., 2015.
Characterization of laccase-grafted ceramic membranes for pharmaceuticals de-
gradation. J. Membrane. Sci. 476, 384–393.
This work was financially supported by the Collaborative
Innovation Involving Production, Teaching & Research Funds of
Jiangsu Province (BY2014023-28). We thank LetPub (www.letpub.
com) for its linguistic assistance during the preparation of this manu-
script.
Eggert, C., Temp, U., Dean, J.F.D., Eriksson, K.E.L., 1995. Laccase-mediated formation of
the phenoxazinone derivative, cinnabarinic acid. FEBS Lett. 376, 202–206.
Forootanfar, H., Faramarzi, M.A., 2015. Insights into laccase producing organisms, fer-
mentation states, purification strategies, and biotechnological applications.
Biotechnol. Progr. 31, 1443–1463.
Gao, N., Liu, C.X., Xu, Q.M., Cheng, J.S., Yuan, Y.J., 2018. Simultaneous removal of ci-
profloxacin, norfloxacin, sulfamethoxazole by co-producing oxidative enzymes
system of Phanerochaete chrysosporium and Pycnoporus sanguineus. Chemosphere 195,
Appendix A. Supplementary data
1
46–155.
Gomez-Pacheco, C.V., Sanchez-Polo, M., Rivera-Utrilla, J., Lopez-Penalver, J., 2011.
Tetracycline removal from waters by integrated technologies based on ozonation and
biodegradation. Chem. Eng. J. 178, 115–121.
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121084.
Guo, N., Wang, Y.K., Yan, L., Wang, X.H., Wang, M.Y., Xu, H., Wang, S.G., 2017. Effect of
bio-electrochemical system on the fate and proliferation of chloramphenicol re-
sistance genes during the treatment of chloramphenicol wastewater. Water Res. 117,
References
9
5–101.
He, L., Dong, Y.N., Zheng, Y.N., Jia, Q.M., Shan, S.Y., Zhang, Y.Q., 2019. A novel mag-
Abejon, R., De Cazes, M., Belleville, M.P., Sanchez-Marcano, J., 2015a. Large-scale en-
zymatic membrane reactors for tetracycline degradation in WWTP effluents. Water
Res. 73, 118–131.
netic MIL-101(Fe)/TiO composite for photo degradation of tetracycline under solar
2
light. J. Hazard. Mater. 361, 85–94.
Jia, S.Y., Zhang, X.X., Miao, Y., Zhao, Y.T., Ye, L., Li, B., Zhang, T., 2017. Fate of antibiotic
resistance genes and their associations with bacterial community in livestock
breeding wastewater and its receiving river water. Water Res. 124, 259–268.
Li, X., Xu, Q.M., Cheng, J.S., Yuan, Y.J., 2016. Improving the bioremoval of sulfa-
methoxazole and alleviating cytotoxicity of its biotransformation by laccase produ-
cing system under coculture of Pycnoporus sanguineus and Alcaligenes faecalis.
Bioresource. Technol. 220, 333–340.
Abejon, R., Belleville, M.P., Sanchez-Marcano, J., 2015b. Design, economic evaluation
and optimization of enzymatic membrane reactors for antibiotics degradation in
wastewaters. Sep. Purif. Technol. 156, 183–199.
Baldrian, P., 2006. Fungal laccases - occurrence and properties. FEMS Microbiol. Rev. 30,
2
15–242.
Baminger, U., Nidetzky, B., Kulbe, K.D., Haltrich, D., 1999. A simple assay for measuring
cellobiose dehydrogenase activity in the presence of laccase. J. Microbiol. Meth. 35,
Liang, B., Ma, J.C., Cai, W.W., Li, Z.L., Liu, W.Z., Qi, M.Y., Zhao, Y.K., Ma, X.D., Deng, Y.,
Wang, A.J., Zhou, J.Z., 2019. Response of chloramphenicol-reducing biocathode re-
sistome to continuous electrical stimulation. Water Res. 148, 398–406.
Liu, J.Y., Cai, Y.J., Liao, X.R., Huang, Q.G., Hao, Z.K., Hu, M.M., Zhang, D.B., 2013.
Purification and characterisation of a novel thermal stable laccase from Pycnoporus
sp. SYBC-L3 and its use in dye decolorisation. Biol. Environ. 113B, 27–39.
Liu, J.Y., Yu, Z.H., Liao, X.R., Liu, J.H., Mao, F.J., Huang, Q.G., 2016. Scalable produc-
tion, fast purification, and spray drying of native Pycnoporus laccase and circular
dichroism characterization. J. Clean. Prod. 127, 600–609.
2
53–259.
Becker, D., Della Giustina, S.V., Rodriguez-Mozaz, S., Schoevaart, R., Barcelo, D., de
Cazes, M., Belleville, M.P., Sanchez-Marcano, J., de Gunzburg, J., Couillerot, O.,
Volker, J., Oehlmann, J., Wagner, M., 2016. Removal of antibiotics in wastewater by
enzymatic treatment with fungal laccase - Degradation of compounds does not always
eliminate toxicity. Bioresource. Technol. 219, 500–509.
Bilal, M., Adeel, M., Rasheed, T., Zhao, Y.P., Iqbal, H.M.N., 2019. Emerging contaminants
of high concern and their enzyme-assisted biodegradation - A review. Environ. Int.
1
24, 336–353.
Llorca, M., Rodriguez-Mozaz, S., Couillerot, O., Panigoni, K., de Gunzburg, J., Bayer, S.,
Czaja, R., Barcelo, D., 2015. Identification of new transformation products during
enzymatic treatment of tetracycline and erythromycin antibiotics at laboratory scale
by an on-line turbulent flow liquid-chromatography coupled to a high resolution
mass spectrometer LTQ-Orbitrap. Chemosphere 119, 90–98.
Canas, A.I., Camarero, S., 2010. Laccases and their natural mediators: biotechnological
tools for sustainable eco-friendly processes. Biotechnol. Adv. 28, 694–705.
Carvalho, I.T., Santos, L., 2016. Antibiotics in the aquatic environments: a review of the
European scenario. Environ. Int. 94, 736–757.
Chen, Y.Y., Stemple, B., Kumar, M., Wei, N., 2016. Cell surface display fungal laccase as a
renewable biocatalyst for degradation of persistent micropollutants bisphenol A and
sulfamethoxazole. Environ. Sci. Technol. 50, 8799–8808.
Lomascolo, A., Cayol, J.L., Roche, M., Guo, L., Robert, J.L., Record, E., Lesage-Meessen,
L., Ollivier, B., Sigoillot, J.C., Asther, M., 2002. Molecular clustering of Pycnoporus
strains from various geographic origins and isolation of monokaryotic strains for
laccase hyperproduction. Mycol. Res. 106, 1193–1203.
Christou, A., Aguera, A., Bayona, J.M., Cytryn, E., Fotopoulos, V., Lambropoulou, D.,
Manaia, C.M., Michael, C., Revitt, M., Schroder, P., Fatta-Kassinos, D., 2017. The
potential implications of reclaimed wastewater reuse for irrigation on the agricultural
environment: the knowns and unknowns of the fate of antibiotics and antibiotic re-
sistant bacteria and resistance genes - A review. Water Res. 123, 448–467.
Dhawan, S., Lal, R., Hanspal, M., Kuhad, R.C., 2005. Effect of antibiotics on growth and
Lomascolo, A., Uzan-Boukhris, E., Herpoel-Gimbert, I., Sigoillot, J.C., Lesage-Meessen, L.,
2011. Peculiarities of Pycnoporus species for applications in biotechnology. Appl.
Microbiol. Biot. 92, 1129–1149.
Luo, Y., Xu, L., Rysz, M., Wang, Y.Q., Zhang, H., Alvarez, P.J.J., 2011. Occurrence and
transport of tetracycline, sulfonamide, quinolone, and macrolide antibiotics in the
13

Q. Tian, et al.
Journal of Hazardous Materials 382 (2020) 121084
Haihe River basin, China. Environ. Sci. Technol. 45, 1827–1833.
Hazard. Mater. 331, 182–188.
Luo, Q., Chen, Y., Xia, J., Wang, K.Q., Cai, Y.J., Liao, X.R., Guan, Z.B., 2018. Functional
expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-
directed mutagenesis. Enzyme. Microb. Tech. 109, 11–19.
Sun, K., Kang, F.X., Waigi, M.G., Gao, Y.Z., Huang, Q.G., 2017b. Laccase-mediated
transformation of triclosan in aqueous solution with metal cations and humic acid.
Environ. Pollut. 220, 105–111.
Ma, Y., Gao, N.Y., Li, C., 2012. Degradation and pathway of tetracycline hydrochloride in
aqueous solution by potassium ferrate. Environ. Eng. Sci. 29, 357–362.
Taheran, M., Naghdi, M., Brar, S.K., Knystautas, E.J., Verma, M., Surampalli, R.Y., 2017.
Covalent immobilization of laccase onto nanofibrous membrane for degradation of
pharmaceutical residues in water. ACS Sustain. Chem. Eng. 5, 10430–10438.
Tian, Q., Feng, Y., Huang, H., Zhang, J., Yu, Y., Guan, Z., Cai, Y., Liao, X., 2018.
Production of lactobionic acid from lactose using the cellobiose dehydrogenase-3-
HAA-laccase system from Pycnoporus sp. SYBC-L10. Lett. Appl. Microbiol. 67,
589–597.
Margot, J., Copin, P.J., von Gunten, U., Barry, D.A., Holliger, C., 2015. Sulfamethoxazole
and isoproturon degradation and detoxification by a laccase-mediator system: in-
fluence of treatment conditions and mechanistic aspects. Biochem. Eng. J. 103,
4
7–59.
Mir-Tutusaus, J.A., Baccar, R., Caminal, G., Sarra, M., 2018. Can white-rot fungi be a real
wastewater treatment alternative for organic micropollutants removal? A review.
Water Res. 138, 137–151.
Wang, Z.X., Cai, Y.J., Liao, X.R., Tao, G.J., Li, Y.Y., Zhang, F., Zhang, D.B., 2010.
Purification and characterization of two thermostable laccases with high cold
adapted characteristics from Pycnoporus sp. SYBC-L1. Process Biochem. 45,
1720–1729.
Morozova, O.V., Shumakovich, G.P., Shleev, S.V., Yaropolov, Y.I., 2007. Laccase-med-
iator systems and their applications: a review. Appl. Biochem. Micro+ 43, 523–535.
Mot, A.C., Parvu, M., Damian, G., Irimie, F.D., Darula, Z., Medzihradszky, K.F., Brem, B.,
Silaghi-Dumitrescu, R., 2012. A "yellow" laccase with "blue" spectroscopic features,
from Sclerotinia sclerotiorum. Process Biochem. 47, 968–975.
Wen, X.H., Jia, Y.N., Li, J.X., 2009. Degradation of tetracycline and oxytetracycline by
crude lignin peroxidase prepared from Phanerochaete chrysosporium - A white rot
fungus. Chemosphere 75, 1003–1007.
Naghdi, M., Taheran, M., Brar, S.K., Kermanshahi-pour, A., Verma, M., Surampalli, R.Y.,
Wen, X.H., Jia, Y.N., Li, J.X., 2010. Enzymatic degradation of tetracycline and oxyte-
tracycline by crude manganese peroxidase prepared from Phanerochaete chrysos-
porium. J. Hazard. Mater. 177, 924–928.
2
018. Removal of pharmaceutical compounds in water and wastewater using fungal
oxidoreductase enzymes. Environ. Pollut. 234, 190–213.
Pan, L.J., Li, J., Li, C.X., Tang, X.D., Yu, G.W., Wang, Y., 2018. Study of ciprofloxacin
biodegradation by a Thermus sp. Isolated from pharmaceutical sludge. J. Hazard.
Mater. 343, 59–67.
Weng, S.S., Ku, K.L., Lai, H.T., 2012. The implication of mediators for enhancement of
laccase oxidation of sulfonamide antibiotics. Bioresource. Technol. 113, 259–264.
Weng, S.S., Liu, S.M., Lai, H.T., 2013. Application parameters of laccase-mediator systems
for treatment of sulfonamide antibiotics. Bioresource. Technol. 141, 152–159.
Wesenberg, D., Kyriakides, I., Agathos, S.N., 2003. White-rot fungi and their enzymes for
the treatment of industrial dye effluents. Biotechnol. Adv. 22, 161–187.
Wong, D.W.S., 2009. Structure and action mechanism of ligninolytic enzymes. Appl.
Biochem. Biotech. 157, 174–209.
Qiao, M., Ying, G.G., Singer, A.C., Zhu, Y.G., 2018. Review of antibiotic resistance in
China and its environment. Environ. Int. 110, 160–172.
Ramirez-Cavazos, L.I., Junghanns, C., Ornelas-Soto, N., Cardenas-Chavez, D.L.,
Hernandez-Luna, C., Demarche, P., Enaud, E., Garcia-Morales, R., Agathos, S.N.,
Parra, R., 2014. Purification and characterization of two thermostable laccases from
Pycnoporus sanguineus and potential role in degradation of endocrine disrupting
chemicals. J. Mol. Catal. B-Enzym. 108, 32–42.
Yang, J., Lin, Y.H., Yang, X.D., Ng, T.B., Ye, X.Y., Lin, J., 2017. Degradation of tetra-
cycline by immobilized laccase and the proposed transformation pathway. J. Hazard.
Mater. 322, 525–531.
Saparrat, M.C.N., Cabello, M.N., Arambarri, A.M., 2002. Extracellular laccase activity in
Tetraploa aristata. Biotechnol. Lett. 24, 1375–1377.
Zeng, X.K., Cai, Y.J., Liao, X.R., Zeng, X.L., Li, W.X., Zhang, D.B., 2011. Decolorization of
synthetic dyes by crude laccase from a newly isolated Trametes trogii strain cultivated
on solid agro-industrial residue. J. Hazard. Mater. 187, 517–525.
Zeng, X.K., Cai, Y.J., Liao, X.R., Zeng, X.L., Luo, S.P., Zhang, D.B., 2012. Anthraquinone
dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase. Process
Biochem. 47, 160–163.
Shao, B.B., Liu, Z.F., Zeng, G.M., Liu, Y., Yang, X., Zhou, C.Y., Chen, M., Liu, Y.J., Jiang,
Y.L., Yan, M., 2019. Immobilization of laccase on hollow mesoporous carbon nano-
spheres: noteworthy immobilization, excellent stability and efficacious for antibiotic
contaminants removal. J. Hazard. Mater. 362, 318–326.
Shi, L., Ma, F.Y., Han, Y.L., Zhang, X.Y., Yu, H.B., 2014. Removal of sulfonamide anti-
biotics by oriented immobilized laccase on Fe
iators. J. Hazard. Mater. 279, 203–211.
O
3 4
nanoparticles with natural med-
Zhang, Q.Q., Ying, G.G., Pan, C.G., Liu, Y.S., Zhao, J.L., 2015. Comprehensive evaluation
of antibiotics emission and fate in the river basins of China: source analysis, multi-
media modeling, and linkage to bacterial resistance. Environ. Sci. Technol. 49,
6772–6782.
Sim, W.J., Lee, J.W., Lee, E.S., Shin, S.K., Hwang, S.R., Oh, J.E., 2011. Occurrence and
distribution of pharmaceuticals in wastewater from households, livestock farms,
hospitals and pharmaceutical manufactures. Chemosphere 82, 179–186.
Suda, T., Hata, T., Kawai, S., Okamura, H., Nishida, T., 2012. Treatment of tetracycline
antibiotics by laccase in the presence of 1-hydroxybenzotriazole. Bioresource.
Technol. 103, 498–501.
Zhang, Y.Z., Li, X.H., Xi, R.C., Guan, Z.B., Cai, Y.J., Liao, X.R., 2016. Characterization of
an acid-stable catalase KatB isolated from Bacillus altitudinis SYBC hb4. Ann.
Microbiol. 66, 131–141.
Zhu, Y.S., Zhang, H.B., Cao, M.L., Wei, Z.Z., Huang, F., Gao, P.J., 2011. Production of a
thermostable metal-tolerant laccase from Trametes versicolor and its application in
dye decolorization. Biotechnol. Bioprocess Eng. 16, 1027–1035.
Sun, K., Huang, Q.G., Li, S.Y., 2017a. Transformation and toxicity evaluation of tetra-
cycline in humic acid solution by laccase coupled with 1-hydroxybenzotriazole. J.
14