Science of the Total Environment 713 (2020) 136713

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Science of the Total Environment

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The heterologous expression, characterization, and application of a novel

laccase from Bacillus velezensis
Tao Li a,b,1, Lin Huang a,1, Yanzhen Li a, Zehua Xu a, Xiuqi Ge a, Yuanfu Zhang a, Nan Wang a, Shuang Wang a,
Wei Yang b, Fuping Lu a, Yihan Liu a,⁎
a
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, The College of Biotechnology, Tianjin University of Science and
Technology, Tianjin 300457, PR China
b
College of Basic Science, Tianjin Agricultural University, Tianjin 300384, PR China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• A new strain Bacillus velezensis TCCC

111904 with laccase activity was identi-
fied.
• The novel laccase from Bacillus
velezensis TCCC 111904 was expressed
in E. coli.
• The recombinant laccase (rLac)
displayed high thermostability and pH
stability.
• rLac showed great tolerance against
high concentration of NaCl.
• rLac efficiently decolorized azo,
anthraquinonic, and triphenylmethane
dyes.

a r t i c l e i n f o a b s t r a c t

Article history: Laccases have a huge potential in numerous environmental and industrial applications due to the ability to oxi-
Received 24 September 2019 dized a wide range of substrates. Here, a novel laccase gene from the identified Bacillus velezensis TCCC 111904
Received in revised form 11 January 2020 was heterologously expressed in Escherichia coli. The optimal temperature and pH for oxidation by recombinant
Accepted 13 January 2020
laccase (rLac) were 80 °C and 5.5, respectively, in the case of the substrate 2,2′-azino-bis (3-ethylbenzothiazo-
Available online 15 January 2020
line-6-sulfonic acid) (ABTS), and 80 °C and 7.0, respectively, in the case of 2,6-dimethoxyphenol (2,6-DMP).
Editor: Patricia Holden rLac exhibited high thermostability and pH stability over a wide range (pH 3.0, 7.0, and 9.0). Additionally,
most of the metal ions did not inhibit the activity of rLac significantly. rLac showed great tolerance against
Keywords: high concentration of NaCl, and 50.8% of its initial activity remained in the reaction system containing 500 mM
Laccase NaCl compared to the control. Moreover, rLac showed a high efficiency in decolorizing different types of dyes in-
Bacillus velezensis cluding azo, anthraquinonic, and triphenylmethane dyes at a high temperature (60 °C) and over an extensive pH
Enzymatic characterization range (pH 5.5, 7.0, and 9.0). These unique characteristics of rLac indicated that it could be a potential candidate for
Decolorization applications in treatment of dye effluents and other industrial processes.
Dye effluents
© 2020 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: No.29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin 300457, PR China.
E-mail address: lyh@tust.edu.cn (Y. Liu).
1
Contributed equally to this work.

https://doi.org/10.1016/j.scitotenv.2020.136713
0048-9697/© 2020 Elsevier B.V. All rights reserved.
2 T. Li et al. / Science of the Total Environment 713 (2020) 136713
1. Introduction
Nowadays, a substantial amount of synthetic dyes with varied struc-
tures including anthraquinone, azo, heterocyclic, and triphenylmethane
dyes are widely applied in the textile industry, as they have certain ad-
vantages over natural pigments, such as fading resistance against expo-
sures to light, chemicals, and water (Abe et al., 2019; Pereira et al., 2019;
Wang et al., 2012). The textile industries produce a large amount of
wastewater containing spent dyes and some toxic and mutagenic/carci-
nogenic chemicals every day (Dawkar et al., 2008; Dutta et al., 2020).
Millions of liters of untreated dye effluents are discharged into the
water bodies in some underdeveloped areas because removal of these
dyes is difficult and expensive; this results in severe damage to the
aquatic ecosystems for the intense coloration of the water bodies,
which reduces the light penetration efficiency and dissolved oxygen
levels in the water (Abe et al., 2018; Dawkar et al., 2008; Lu et al.,
2012a; Olukanni et al., 2013). Physicochemical processes such as coag-
ulation, adsorption, ozonation, Fenton reaction, etc., have been
employed for the degradation and removal of dyes from the effluents
of textile industries for decades. However, these conventional methods
have some drawbacks, such as high treatment cost, addition of hazard-
ous chemical additives, complex procedures, and possible secondary
pollution to the environment (Robinson et al., 2001; Saratale et al.,
2011; Shanmugam et al., 2019). By contrast, the enzymatic decoloriza-
tion method has attracted increasing attention as it has several advan-
tages in the treatment of dye effluents over the traditional
physicochemical methods, such as lower cost, higher efficiency, simple
and eco-friendly procedures, and lower energy requirement (Husain,
2010; Imran et al., 2019; Robinson et al., 2001; Saratale et al., 2011).
Laccases, a group of copper-containing enzymes that can oxidize nu-
merous substrates, such as aromatic amines, phenolic compounds, lig-
nins, aryl diamines, and some inorganic ions, were first identified and
isolated from Rhus vernicifera and have been reported to be extensively
distributed in bacteria, fungi, and higher plants (Brenelli et al., 2019;
Forootanfar and Faramarzi, 2015; Lawrance et al., 2019; Muñoz et al.,
1997; Nejad et al., 2019; Trejo-Hernandez et al., 2001; Yaropolov
et al., 1994). The copper centers in the enzyme's active site mediate
the transfer of four single-electrons from the substrate to two oxygen
molecules, thus generating water molecules (Enguita et al., 2004).
When laccase oxidizes non-phenolic compounds (e.g. lignin), media-
tors including ABTS and HBT (1-hydroxybenzotriazole) that act as elec-
tron carriers should be added to the redox reaction system mediated by
this enzyme due to its relatively low redox potential (Christopher et al.,
2014; Munk et al., 2015; Riva, 2006; Siroosi et al., 2018).
In the past decades, laccases have been widely applied in diverse in-
dustries, such as food industry, textile industry, polymer grafting,
laccase-mediated polymer synthesis, and cosmetic and pharmaceutical
industries (Bilal et al., 2019; Nemadziva et al., 2018; Pezzella et al., 2015;
Slagman et al., 2018). Recently, more and more laccases have been con-
tinuously investigated to expand their applications in some industrial
sectors that employ harsh conditions, such as high alkaline and temper-
ature conditions (Ausec et al., 2015; Chauhan et al., 2017; Hildén et al.,
2009; Mathews et al., 2016; Uthandi et al., 2012). For example, laccases
have been proven to effectively degrade dyes used in textile industry,
such as anthraquinonic, triarylmethane, azo, and indigoid dyes (Du
et al., 2020; Nejad et al., 2019; Couto and Herrera, 2006; Zhuo et al.,
2019).
Lignolytic fungi, also called white-rot fungi, are the most compre-
hensively studied laccase-producing microorganisms, and they have
been widely employed in the industrial production of laccase due to
their striking features, such as low cost of cultivation, extracellular se-
cretion of laccase, and high degradation ability towards lots of xenobi-
otic compounds (Ikehata et al., 2004; Shah and Nerud, 2002).
However, the optimal activity of fungal laccases is commonly observed
in an environment with low pH and temperature. This hinders the ap-
plication of fungal laccases in the treatment of textile dye effluents,
which typically have high temperatures and a wide pH range. On the
contrary, bacterial laccases could function at high temperatures and a
wide pH range (Guo et al., 2017; Kumar et al., 2016; Lončar et al.,
2016; Sharma et al., 2007). Moreover, the stability of most bacterial
laccases is considered to be better than fungal laccases. Additionally,
the influence of metal ions on the activities of bacterial laccases is rela-
tively less; indeed, bacterial laccases are not easily influenced by some
of the inhibitors (Safary et al., 2016; Sharma et al., 2007; Singh et al.,
2011). Bacillus species, which are ubiquitous in nature and able to sur-
vive harsh environments including high pH, temperature, and salt con-
centration, are favored by researchers for obtaining laccases with high
activities under harsh conditions, such as high alkaline conditions or
high temperatures. A novel laccase derived from Bacillus tequilensis
SN4 was considered to be highly suitable for industrial applications
under extreme conditions due to its thermo-alkali-stable properties
(Sondhi et al., 2014). A non-blue laccase gene identified in
B. amyloliquefaciens was heterologously expressed in Pichia pastoris suc-
cessfully. This novel enzyme was stable at pH 9.0 for 10 days and
remained activated in 200 mM salt solution (Chen et al., 2015). There-
fore bacterial laccases are apparently more suitable for decolorizing in-
dustrial textile dye effluents (Chauhan et al., 2017; Sharma et al., 2007).
The objective of this study was to find a novel laccase with high ac-
tivity under high temperatures and over a broad pH range for decolori-
zation of synthetic dyes with a high efficiency. Here, we screened a
strain with laccase activity from the forest soil of Hainan Island of
China. This enzyme was heterologously expressed in Escherichia coli
and its characteristics were further investigated.
2. Materials and methods
2.1. Strains, vectors, and reagents
E. coli JM109, E. coli BL21 (DE3), and vector pET-22b (+) were con-
served in our laboratory. ABTS, 2,6-DMP, azophloxine, Congo red, adizol
black B, reactive blue 5, reactive blue 19, crystal violet, indigo carmine,
and malachite green were provided by Sigma-Aldrich (St. Louis, MO,
USA). Restriction enzymes, Pyrobest DNA Polymerase, T4 DNA ligase,
pMD18-T vector cloning Kit, DNA Extraction Kit, Plasmid Mini Kit, and
Gel Extraction and Purification Kit were ordered from TaKaRa Bio Inc.
(Dalian, China). Other chemicals obtained from local suppliers were of
analytical grade.
2.2. Strain screening and cultivation conditions
The laccase-active strain was obtained from the forest soil of Hainan
Island of China. The strain was screened by following the procedure
used for the isolation of Klebsiella pneumoniae in our previous report,
producing a novel pH-stable laccase (Liu et al., 2017). Briefly, soil sam-
ple (10 g) was dispersed in sterile saline solution (100 mL, 0.9% NaCl).
Then, the soil suspension (1 mL) was thoroughly mixed with 5 mL
Luria-Bertani (LB) liquid medium (yeast extract 5 g/L, tryptone 10 g/L,
and NaCl 10 g/L). The enriched cells were serially diluted with sterile sa-
line water and incubated at 37 °C for 24 h on a LB agar plate containing
0.2 mM Cu2+ (copper sulfate). After adding several drops of 0.1% (w/v)
syringaldazine (SGZ)/ethanol (absolute) to the bacterial colonies, the
colonies positive for laccase activity were picked by observing the ap-
pearance of pink color (Wang et al., 2010). The positive colonies were
further purified using LB-Cu2+ agar plate, and the colony demonstrating
the darkest pure pink color was picked and identified by measuring the
laccase activity.
2.3. Identification and phylogenetic analysis of the laccase producing strain
Total DNA of the isolated cells with laccase activity was extracted to
amplify the 16S rDNA by polymerase chain reaction (PCR) using two
bacterial universal primers: F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and R:
 T. Li et al. / Science of the Total Environment 713 (2020) 136713
5′-GGTTACCTTGTTACGACTT-3′ (Suzuki and Giovannoni, 1996). The
resulting PCR fragments were inserted into the pMD18-T vector. E. coli
DH5α competent cells were then transformed with this vector. After-
wards, the 16S rDNA of the positive transformant was sequenced by
BGI Company (Beijing, China).
The isolated strain was first identified by aligning the sequence of
the 16S rDNA with data deposited in the National Center for Biotechnol-
ogy Information (NCBI) database, and performing phylogenetic analysis
by constructing a bootstrap consensus tree using the neighbor-joining
method with MEGA 6.0 software (Tamura et al., 2013). Further taxo-
nomic analysis was performed according to the instructions provided
in Bergey's Manual of Determinative Bacteriology to confirm the strain
identification (Holt et al., 1994).
2.4. Cloning and homologous expression of the laccase gene
The full laccase gene was amplified from the genomic DNA using PCR
technology with the primers 5′-CGCGGATCCGATGGCACTGGAAAAA
TTTG-3′ (forward primer) and 5′-ACGCGTCGACCTGCTTATCCGTGACG
TCC-3′ (reverse primer) with BamHI and SalI sites (underlined). After
double digestion with BamHI and SalI, it was inserted into the BamHI-
SalI-linearized pET-22b (+) to construct the plasmid pET-lac. After
transforming pET-lac into E. coli BL21 (DE3), the recombinant laccase
(rLac) with a 6 × His-tag was heterologously expressed in E. coli BL21.
Briefly, a positive colony was initially grown at 37 °C for 12 h in 5 mL
of LB medium supplemented with ampicillin (100 μg/mL) on a rotary
shaker with constant shaking speed of 220 rpm. Then the starting cul-
ture (1 mL) was pipetted into 50 mL LB medium, and incubated until
the optical density at 600 nm was 0.6 to 0.8 under the above conditions.
Afterwards, the culture was supplied with 1 mM isopropyl-β-D-1-
thiogalactopyranoside (IPTG) to induce the expression of rLac for 20 h
at 16 °C. Meanwhile, E. coli BL21 cells containing the empty plasmid
pET-22b (+) were used as control.
2.5. Structure modeling of rLac
The 3-D homology model of rLac was built using the Swiss-Model
server (http://swissmodel.expasy.org/) and the PyMOL molecular
graphic system with the crystal structure of B. subtilis laccase (PDB ID:
2WSD) as the template.
2.6. Purification of rLac
The centrifugally (8000 ×g, 4 °C, 15 min) collected cells were resus-
pended in 20 mM Tris–HCl buffer (pH 7.0) containing 20 mM imidazole
and 500 mM NaCl and subsequently lysed by sonication in ice bath at
320 W with 4 s strokes and 3 s intervals. The supernatant containing
rLac was collected after centrifugation (12,000 ×g, 30 min, 4 °C), and
injected into a nickel-nitrilotriacetic acid (Ni-NTA) agarose gel column
(Shenggong, Shanghai, China), which was preequilibrated with
50 mM Tris-HCl buffer (pH 7.0). rLac was washed out using elution
buffer (20 mM Tris-HCl, 500 mM imidazole and 500 mM NaCl,
pH 7.0). Finally, the purified rLac was dialyzed and sterilized by filtra-
tion. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) was used to evaluate the molecular mass and purity of rLac.
2.7. rLac activity assay
The activity of rLac was assayed according to the method described
in our previous study (Liu et al., 2017). Briefly, ABTS and 2,6-DMP
were employed in the rLac mediated enzymatic reaction as substrates.
The absorbance was monitored at 420 nm using the coefficient of
ε420 = 36,000 M−1 cm−1 for the oxidation of ABTS prepared with a
final concentration of 6 mM in 0.1 M citrate-phosphate buffer
(pH 5.5). Similarly, the absorbance was recorded at 470 nm (ε470 =
49,600 M−1 cm−1) for the oxidation of 2,6-DMP (1.0 mM) prepared in
3
0.1 M citrate-phosphate buffer (pH 7.0). One unit of laccase activity
was defined as the quantity of laccase capable of oxidizing 1 μmol sub-
strate per minute under the assay conditions.
2.8. Characterization of rLac
The optimum temperature required by rLac to oxidize ABTS and 2,6-
DMP was estimated by performing the standard reaction at different
temperatures, ranging from 30 °C to 90 °C. To evaluate the optimal pH
for rLac using 2,6-DMP and ABTS as substrates, the enzymatic reaction
was performed in buffers of different pH values, viz. 0.1 M citrate-
phosphate buffer (pH 3.0 to 8.0) and 0.1 M glycine-sodium hydroxide
buffer (pH 9.0). The maximum activity was taken as 100% to calculate
the relative enzymatic activity.
The thermostability of rLac was determined by measuring the resid-
ual activity with ABTS and 2,6-DMP as substrates for 0 h to 2 h at various
temperatures (60 °C, 70 °C, and 80 °C). Similarly, pH stability of rLac was
assayed by calculating the residual activity after incubation at 4 °C in
buffers with different pH (pH 3.0, 7.0, 9.0) for different durations
(0–10 days). The activity of rLac without treatment was recorded as
100%.
The effect of metal ions (Cu2+, Na+, K+, Ca2+, Cu2+, Fe3+, Fe2+, Co2
+
, Zn2+, Mn2+, Ba2+, and Mg2+) and inhibitors (EDTA, SDS, L-cysteine,
dithiothreitol, and β-mercaptoethanol) on the activity of rLac were
assayed by determining the relative activity of the enzyme in the reac-
tion system containing individual effectors with ABTS as the substrate.
The laccase activity measured without any of the effectors was marked
as 100%.
2.9. Dye decolorization assay
The decolorization ability of rLac was determined by incubating the
enzyme with different types of dyes in the reaction system with ABTS,
acetosyringone, and syringaldehyde as the mediators. The final concen-
tration of the dyes were azophloxine (λmax = 530 nm), adizol black B
(λmax = 607 nm), reactive blue 19 (λmax = 590 nm), crystal violet
(λmax = 587 nm), indigo carmine (λmax = 612 nm), 50 mg/L; reactive
blue 5 (λmax = 602 nm), malachite green (λmax = 618 nm), 100 mg/L;
and Congo red (λmax = 501 nm), 200 mg/L. The reaction system (5 mL)
for decolorization contained buffer of different pH values (0.1 M citrate-
phosphate buffer, pH 5.5 and 7.0; 0.1 M glycine‑sodium hydroxide
buffer, pH 9.0), dyes, purified rLac (80 U), individual dye, and 0.1 mM
mediator. The reaction lasted for 6 h at 60 °C and the decolorization abil-
ity for each dye was evaluated by calculating the decrease in the maxi-
mum absorbance for each dye with the following equation:
decolorization (%) = [(initial absorbance) − (final absorbance)/(initial
absorbance)] × 100%, which reflected the decrease in concentration due
to the oxidation by rLac.
3. Results and discussion
3.1. Isolation and characterization of the strain with laccase activity
Given the advantages of bacterial laccases in industrial applications
over those derived from fungi and plant, such as wider pH adaptation,
higher pH stability, and thermostability, a considerable number of
novel laccases isolated from bacteria have been investigated over the
past years, especially those isolated from the genus Bacillus (Chen
et al., 2015; Lončar et al., 2013; Lončar et al., 2016; Lu et al., 2012a;
Martins et al., 2002; Zeng et al., 2016). We successfully isolated a strain
with laccase activity from a soil sample collected from Hainan Island of
China. This strain had the following morphological characteristics:
Gram-positive, rod-shaped, spore-producing, and rough-surfaced col-
ony (data not shown). The 16S rDNA of this strain was amplified, se-
quenced, and aligned using BLAST in the NCBI database. The BLAST
outcome revealed that this strain shares a high genetic identity with
4 T. Li et al. / Science of the Total Environment 713 (2020) 136713
the genus Bacillus (N99%). Besides, phylogenetic analysis showed that
the species most closely related to this strain was B. velezensis (Fig. 1).
Therefore, the obtained strain was preliminarily characterized as
B. velezensis TCCC 111904 in light of the results of 16S rDNA sequence
alignment, traditional morphological characterization, and biochemical
tests (data not shown).
3.2. Heterologous expression of laccase gene in E. coli
Nucleotide sequencing of the plasmid extracted from a positive
transformant confirmed a successful cloning. The sequence of lac has
been deposited in GenBank with the accession number MK396097; its
open reading frame contains 1536 bp that theoretically encode 512
amino acids with a molecular weight of about 58 kDa. As determined
by the alignment analysis, the protein sequence has a high identity to
other laccases from Bacillus species, including: B. vallismortis fmb 103
laccase, 77.3% identity (Sun et al., 2017); B. subtilis X1 laccase, 75.4%
(Chen et al., 2015); B. pumilus ATCC 7061 laccase, 65.9% (Ihssen et al.,
2017); and B. licheniformis ATCC 14580 laccase, 64.9% (Koschorreck
et al., 2008) (Fig. 2). Based on our literature review and analysis, this
is the first report of the laccase derived from B. velezensis. The predicted
3-D structure of rLac was built by homologous modeling using the struc-
ture of B. subtilis laccase as template (PDB ID: 2WSD) (Fig. 3). The active
center of laccases from Bacillus genus was highly conserved, and
contained four copper atoms, of which one was bound to the T1 or T2
copper center and the other two copper atoms were located in the T3
copper center (Solomon et al., 1996). Similarly, three Cu-oxidase do-
mains were also present in the active site of rLac, T1 copper center
(H419, C492, H497, and M502), T2 copper center (H105 and H422),
and T3 copper center (H107, H153, and H493/H155, H422, and H491).
The lysate of cells harboring the plasmid pET-lac showed a signifi-
cant laccase activity after induction with 0.5 mM IPTG for 20 h at
16 °C, while such phenomenon was not observed in the lysate of
E. coli BL21/pET-22b (+). A clear protein band of around 58 kDa was ob-
served after analyzing the constitution of crude protein extracted from
the culture of recombinant E. coli containing the laccase gene of
B. velezensis TCCC 111904. No analogous protein band was detected in
the lysate of the control strain E. coli BL21/pET-22b (+) (Fig. 4a).
Next, the crude rLac was purified by Ni-affinity chromatography, and a
single band of about 58 kDa was detected on a SDS-PAGE gel (Fig. 4b).
Fig. 1. Phylogenetic analysis of 16S rDNA of B. velezensis TCCC 111904 with other related Bacillus strains. The MEGA 6.0 software was used to build the bootstrap consensus tree by the
neighbor-joining method. The percentage of bootstrap sampling, derived from 1000 replications was suggested by the numbers at branch points.
Although the laccases isolated from different Bacillus species showed
comparable enzymatic characteristics, their molecular weights differed
from each other. The molecular weight (MW) of the laccase from novel
thermophilic bacterium Bacillus sp. PC-3 was 36 kDa (Sharma et al.,
2019), MW of the non-blue laccase from B. amyloliquefaciens LC02 was
around 65 kDa (Chen et al., 2015), MW of the CotA laccase from
B. subtilis 168 was about 66 kDa (Zeng et al., 2016), MW of the laccase
from B. tequilensis SN4 was 32 kDa (Sondhi et al., 2014), and MW of
the alkaline laccase from B. halodurans C-125 was around 56 kDa
(Ruijssenaars and Hartmans, 2004). Therefore, rLac isolated from
B. velezensis TCCC 111904 would be a new supplement to the laccase
family.
The specific activities of purified rLac with ABTS and 2,6-DMP as sub-
strates were 180 U/mg and 380 U/mg, respectively. However, laccases
from other Bacillus strains showed lower activities. It was reported
that the laccase from B. amyloliquefaciens LC02 exhibited an activity of
20.7 ± 1.2 U/mg towards ABTS (Chen et al., 2015), and laccase from
B. tequilensis SN4 demonstrated an activity of 299.4 U/mg towards 2,6-
DMP (Sondhi et al., 2014). Though, the laccase from B. pumilus MK001
showed a comparable activity of 182 U/mg towards ABTS (Kumar
et al., 2016).
3.3. Enzymatic characterization of rLac
rLac displayed its maximum activity for the oxidation of ABTS and
2,6-DMP at 80 °C (Fig. 5a). Moreover, rLac could maintain a good cata-
lytic ability at high temperatures and over a broad temperature range
of 60 to 90 °C. The activity of rLac was about 80% of its maximum
value when oxidizing 2,6-DMP at 90 °C. This dramatic and striking fea-
ture of rLac is different from those of fungal laccases whose optimum
temperature is between 30 and 60 °C, such as the laccase from basidio-
mycete Cerrena sp., 55 °C (Yang et al., 2015), the laccase from the white
rot fungus Cerrena unicolorstrain GSM-01, 45 °C (Wang et al., 2017), the
laccase from the white rot fungus Trametes sp. F1635, 50 °C (Wang et al.,
2018), and the laccase from Aureobasidium melanogenum strain 11–1,
40 °C (Aung et al., 2019). The optimum temperatures of some fungal
laccases isolated from thermophilic fungus are still evidently low in
comparison to those of bacterial laccases, for example, the fungal laccase
Lcc9 from Coprinopsis cinerea has an optimum temperature of 60 °C (Xu
et al., 2019). By contrast, bacterial laccases usually exhibit their best
 T. Li et al. / Science of the Total Environment 713 (2020) 136713
Fig. 2. Amino acid sequence alignment of rLac and other laccases from the Bacillus genus. The protein sequences of the other four laccases from B. vallismortis fmb 103, B. pumilus ATCC 7061, B. subtilis X1, and B. licheniformis ATCC 14580 were
downloaded from the NCBI website. The alignment was performed using DNAMAN software, and similar and identical amino acids are highlighted in solid grey and black, respectively.

5
6 T. Li et al. / Science of the Total Environment 713 (2020) 136713
Fig. 3. Predicted 3-D structure of rLac. The model was built with the PyMOL molecular
graphics system according to its sequence. The amino acid residues in the three copper
centers, Type 1 (His419, Cys492, His497, and Met502), Type 2 (His105 and His422), and
Type 3 (His107, His153, and His493/His155, His424, and His491) are highlighted in
green, blue, and yellow, respectively. The red balls stand for Cu2+ in the active site of rLac.
catalytic ability at much higher temperatures, especially those isolated
from the genus Bacillus, for example, CotA-laccase from B. pumilus strain
W3, 80 °C (Guan et al., 2014a); the laccase from a novel strain of
B. vallismortis and B. tequilensis SN4, 85 °C (Sondhi et al., 2014; Zhang
et al., 2012); the spore laccase from B. licheniformis LS04, 60 °C (Lu
et al., 2012b); and the laccase from B. licheniformis ATCC 9945a, 90 °C
(Lončar et al., 2016). It is no doubt that exceptions exist and the opti-
mum temperature of some laccases from Bacillus species is even lower
than those isolated from fungi, such as the laccase from B. subtilis
subsp. subtilis str. 168, 25 °C (Yang et al., 2012).
The thermostability of rLac was studied by evaluating its residual ac-
tivity after incubation for different lengths of time at 50, 60, 70, and
80 °C. It was found that the residual activity of rLac was around 80% of
its initial value after incubation at 60 °C for 120 min, and it remained
Fig. 4. Expression and analysis of molecular weight of rLac by SDS-PAGE. (a) Lane M:
protein marker; Lane 1: crude protein extracted from E. coli BL21/pET-lac; Lane 2: crude
protein extracted from E. coli BL21/pET-22b (+). (b) Lane 1: purified rLac. The band of
rLac is marked with black arrow.
over 85% at 50 °C. The stability of rLac evidently decreased after incuba-
tion at 70 and 80 °C, but its activity still remained 39.8% and 26.4% of the
original value after incubation for 120 min, respectively (Fig. 6a). These
results indicate that the thermostability of rLac is superior to some
laccases derived from other Bacillus species, such as Bacillus sp. ADR
(Telke et al., 2011), B. amyloliquefaciens 12B (Lončar et al., 2013), and
B. licheniformis ATCC 9945a (Lončar et al., 2016), and is comparable
with laccases from B. tequilensis SN4 (Sondhi et al., 2014), B. subtilis
168 (Zeng et al., 2016), and B. pumilus MK001(Kumar et al., 2016).
Thus the outstanding thermostability of rLac isolated from the new
strain B. velezensis TCCC 111904 might enable it for direct application
in the treatment of hot textile effluents discharged after the dyeing pro-
cess, which is usually performed at high temperatures (Dawkar et al.,
2008; Lončar et al., 2013; Yang et al., 2018).
rLac displayed high catalytic activity towards the substrates ABTS
and 2,6-DMP over a broad pH range (Fig. 5b). The activity of rLac for ox-
idation of ABTS was over 50% of its maximum value over the pH range of
5.0 to 7.0, and reached the maximum value at pH 5.5, which was consis-
tent with other laccases isolated from B. pumilus W3 (Guan et al., 2014a)
and B. vallismortis fmb-103 (Zhang et al., 2012). The optimum pH of rLac
towards ABTS was higher than most laccases from other species of the
Bacillus genus, such as B. licheniformis ATCC 9945a (Lončar et al.,
2016), B. subtilis X1 (Guan et al., 2014b), Bacillus sp. (Guo et al., 2017),
B. pumilus MK001 (Kumar et al., 2016), B. amyloliquefaciens 12B
(Lončar et al., 2013), B. clausii KSM-K16 (Brander et al., 2014), and Bacil-
lus sp. HR03 (Mohammadian et al., 2010). Besides, the optimum pH of
rLac for oxidization of 2,6-DMP was 7.0 (Fig. 5b), which was similar to
other laccases from the genus Bacillus (Guan et al., 2014a; Guan et al.,
2014b; Guo et al., 2017; Lu et al., 2012b). Thus, it is necessary to inves-
tigate the optimum pH when oxidizing different substrates using
laccase. Additionally, this interesting and outstanding feature could
broaden the applications of rLac in treating different kinds of dye efflu-
ents having various pH values. Most of the laccases from the genus Ba-
cillus exhibit this feature, which was attributed to the conformation
variation of the enzyme in different pH environments. Our study further
confirmed that the optimum pH for rLac in catalyzing particular xenobi-
otic compounds depend on its structure (Xu, 1997).
Besides, the stability of rLac in buffers of different pH values (pH 3.0,
7.0, and 9.0) was investigated by estimating its residual activity after in-
cubation for 0 to 10 days at 4 °C (Fig. 6b). rLac was quite stable over a
broad pH range, and its residual activity was still up to 65.5% of the ini-
tial value after incubation for 10 days at pH 3.0. This is a striking feature
in comparison to other laccases from Bacillus strains, which become un-
stable at pH 3.0. The laccase from B. licheniformis LS04 lost 77.12% of its
initial activity after incubation at pH 3.0 for 24 h (Lu et al., 2013). The
stability of laccase from B. subtilis cjp3 was also not good at pH 3.0,
only 20.56% of the initial activity remained after incubation for 1 h
(Qiao et al., 2017). The laccase activity of B. pumilus MK001 was almost
completely lost after 10 days incubation at pH 4.0 (Kumar et al., 2016).
Thus rLac was quite stable in the acidic environment in comparison to
the other laccases isolated from the genus Bacillus.
Surprisingly, the residual activity of rLac gradually increased in the
first two days of incubation in a buffer of pH 7.0, demonstrating a max-
imum increase of 2.79-fold in comparison to its initial activity. Although
the residual activity of rLac decreased slowly with increase in the incu-
bation time, it remained high, at 242% of the initial value after 10 days of
incubation. Similar phenomena were also observed for laccases from
other Bacillus strains, such as the activity of laccase from B. pumilus
W3 remained at approximately 130% of its original activity after incuba-
tion for 10 days at pH 7.0, and the laccase from B. licheniformis LS04 ex-
hibited a residual activity of up to 123% of the initial activity (Guan et al.,
2014a; Lu et al., 2012b). Not all bacterial laccases showed significantly
increased activity after incubation at pH 7.0, and activities of some
laccases, such as the recombinant laccase (CotA) from B. pumilus
MK001 (Kumar et al., 2016) and laccase from B. amyloliquefaciens
LC02 (Chen et al., 2015), reached below 100% of their initial activities
 T. Li et al. / Science of the Total Environment 713 (2020) 136713 7

Fig. 5. Influence of temperature (a) and pH (b) on the activity of rLac isolated from B. velezensis TCCC 111904. The activity of rLac was analyzed following the standard assay with ABTS or
2,6-DMP as substrate under each condition. All experiments were conducted in triplicate, and the values represent mean ± SD.

after incubation. The stability of rLac at pH 9.0 was almost the same to
that at pH 3.0, and its residual activity was retained at 67.6% of the initial
value after 10 days of incubation at pH 9.0. By contrast, fungal laccases,
such as laccase from Paraconiothyrium variabile (Forootanfar et al.,
2011) and Cladosporium cladosporioides NCIM1340 (Halaburgi et al.,
2011), are only stable in acidic to neutral environments. Thus, the ther-
mostability and pH tolerance of rLac would broaden its industrial
applications.
3.4. Influence of metal ions or inhibitors on activity of rLac
As presented in Table 1, most of the selected metal ions did not sig-
nificantly affect the activity of rLac, except for Mn2+, Fe2+, Fe3+, and Co2
+
, which showed more severe inhibition at a high concentration. The re-
sidual activity of rLac was only 4.5%, 22.5%, 33.2%, and 42.3% in presence
of 5 mM Mn2+, Fe2+, Fe3+, and Co2+ in the enzymatic reaction system,
respectively. Similar phenomena have also been observed for the
laccases from P. variabile in the presence of Mn2+ (Forootanfar et al.,
2011) and B. tequilensis SN4 in the presence of Fe2+ (Sondhi et al.,
2014). The activities of some laccases are more easily affected by Mn2
+
, for example, the activity of laccase from B. safensis sp. strain S31 re-
mains at only 13.2 ± 0.5% of the control after adding 1 mM Mn2+ in
the reaction system (Siroosi et al., 2018).
In a high salinity solution with over 100 mM NaCl, most fungal
laccases lose their activities because of their intrinsic sensitivity to-
wards halides (Jimenez-Juarez et al., 2005). The possible reason for
the occurrence of this inhibition effect on the laccase activity could
be that the high concentration of chloride disrupts the transfer of
electrons from substrate to T1 copper or from T1 copper to T3 cop-
per, which eventually influences the oxidation-reduction reaction
mediated by the laccase. However, rLac showed great tolerance
against high concentration of NaCl, and it retained 50.8% of its resid-
ual activity in the solution with 500 mM NaCl compared to the con-
trol. Similar results have also been reported for other bacterial
laccases, such as B. pumilus W3 (Guan et al., 2014a), and
B. vallismortis fmb-103 (Zhang et al., 2012). Therefore, laccases
from different sources have remarkably different tolerance against
NaCl (Shafiei et al., 2019; Wang et al., 2019). Hence, the high-
salinity tolerance of rLac would be much more advantageous in
treating the textile effluents, which usually contain high concentra-
tion of NaCl (Rodrigues et al., 2009).
Besides, EDTA also showed a significant inhibitory effect on the ac-
tivity of rLac, especially at the high concentration of 5 mM (Table 1).
The reason for the inhibition effect of EDTA against laccase was probably
due to the chelation of the copper ions by EDTA at the T1 copper center
(Kaushik and Thakur, 2013). This result further revealed the importance
of Cu2+ in laccase activity.

Fig. 6. Thermostability (a) and pH stability (b) of the rLac isolated from B. velezensis TCCC 111904. The stability of rLac was analyzed following the standard activity assay with ABTS or 2,6-
DMP as substrate under each treatment condition, and it is shown as the percentage of the residual activity compared to the initial value. All experiments were performed in triplicate, and
the values represent mean ± SD.
8 T. Li et al. / Science of the Total Environment 713 (2020) 136713

Table 1 wide substrate specificity, and environment friendly feature,

Influence of metal ions or inhibitors on activity of rLac. All experiments were carried out laccases have been widely studied and applied in the dye degrada-
independently for three times, and the values represent mean ± SD.
tion process in the last few decades (Nguyen and Juang, 2013).
Metal ions/inhibitors Concentration (mM) Relative activity (%) Most of the reported laccases have been isolated from fungi, and
None – 100 ± 1.5 are unstable in alkaline and high temperature environments
KCl 0.5 100.3 ± 2.1 (Sharma et al., 2007). This limits the application of fungal laccases
5 93.6 ± 0.9 in dye effluents treatment as the effluents are discharged with a
CaCl2 0.5 100.3 ± 1.2
high temperature and alkaline pH (Kapdan and Alparslan, 2005).
5 97.8 ± 1.3
MnCl2 0.5 17.7 ± 1.8 By contrast, bacterial laccases display a much higher thermostability
5 4.5 ± 0.6 and pH stability in comparison to the fungal laccases, and have a high
CuCl2 0.5 93.4 ± 2.3 potential for application in the treatment of dye effluents treatment
5 90.1 ± 1.0 (Santhanam et al., 2011; Sun et al., 2017; Yang et al., 2018). Hence, it
MgCl2 0.5 105.6 ± 1.6
5 96.0 ± 1.2
spurred many researchers to find novel bacterial laccases and evalu-
ZnCl2 0.5 102.7 ± 2.0 ate their enzymatic characteristics and practical applications
5 93.1 ± 0.9 (Dawkar et al., 2008; Guan et al., 2014a; Lu et al., 2012b; Qiao et al.,
FeCl3 0.5 79.6 ± 1.7 2017; Sun et al., 2017; Wang et al., 2010; Yang et al., 2018; Zhang
5 33.2 ± 1.2
et al., 2012). Here, we isolated a novel rLac with high pH stability
CoCl2 0.5 93.9 ± 2.2
5 42.3 ± 1.5 and thermostability from a new strain B. velezensis TCCC 111904
BaCl2 0.5 104.4 ± 0.8 (Fig. 6), and we investigated its decolorization efficiency at high
5 94.9 ± 1.3 temperature (60 °C) and over a wide pH range (pH 5.5, 7.0, and
FeSO4 0.5 77.2 ± 2.1 9.0) (Fig. 7). Three mediators (ABTS, acetosyringone, and
5 22.5 ± 1.1
syringaldehyde) and eight dyes including three azo types of dyes
SDS 0.5 86.9 ± 0.9
5 83.1 ± 2.0 (azophloxine, Congo red and adizol black B), two anthraquinonic
EDTA 0.5 71.3 ± 1.4 dyes (reactive blue 5, reactive blue 19), and three triphenylmethane
5 25.1 ± 1.8 types of dyes (crystal violet, indigo carmine, and malachite green)
NaCl 0.5 94.6 ± 2.6
were employed in the decolorization test, which was conducted in
5 91.0 ± 1.6
10 81 ± 1.0
different pH environments (pH 5.5, 7.0, and 9.0) (Fig. 7). The results
100 68.9 ± 0.9 indicated that the extent of decolorization did not reach over 27%
500 50.8 ± 1.7 when only rLac was used for dyes decolorization in the absence of
1000 0 mediators (data not shown). However, rLac could effectively decol-
Dithiothreitol 0.1 29.2 ± 1.1
orize all of the eight dyes over a wide pH range of 5.5 to 9.0 using
0.5 0
1 0 ABTS as the mediator, and the decolorization rates ranged from 42%
β-Mercaptoethanol 0.1 32.8 ± 1.6 to 94% (Fig. 7a). Compared to the decolorization efficiency of rLac
0.5 0 against the eight dyes at pH 9.0, rLac was much more effective in de-
1 0
colorizing the dyes at pH 5.5 and pH 7.0 using ABTS as mediator ex-
L-Cysteine 0.1 75.3 ± 2.7
0.5 27.1 ± 1.0
cept for reactive blue 5 (Fig. 7a). However, rLac was more powerful
1 0 in decolorizing the eight dyes using acetosyringone and
syringaldehyde as mediators in the buffers of pH 7.0 and pH 9.0
(Fig. 7b and c). Therefore, it could be concluded that rLac from the
newly identified B. velezensis TCCC 111904 can decolorize multiple
As shown in Table 1, the influence of some reported inhibitors (SDS, dyes over an extensive pH range of 5.5–9.0 using an appropriate me-
L-cysteine, dithiothreitol, and β-mercaptoethanol) on the activity of rLac diator for specific synthetic dyes. By contrast, fungal laccases can
was also investigated. Similar to the laccase from B. vallismortis fmb-103 best carry out decolorization in only an acidic environment. The
(Zhang et al., 2012), SDS affected the activity of rLac slightly. However, laccase from Trametes hirsuta could decolorize indigo carmine only
0.5 mM of dithiothreitol and β-mercaptoethanol and 1 mM of L- in acidic environment (Rodrigues et al., 2009). The optimum pH for
cysteine completely deactivated rLac, which was also observed for laccase from basidiomycete Cerrena sp. was 3.5, but its dye decolor-
other bacterial and fungal laccases (Guan et al., 2014a; Forootanfar ization ability was exhibited at pH 6.0 (Yang et al., 2015). The laccase
et al., 2011; Zhang et al., 2012). Conversely, the performance of laccases from white rot fungus Cerrena unicolor GSM-01 showed a potent de-
from different Bacillus strains is not always the same when being incu- colorizing ability against bromothymol blue, Evans blue, methyl or-
bated with the same inhibitors. Low concentration of SDS (0.1 mM) ange, and malachite green at pH 3.0, demonstrating decolorization
greatly inhibited the activity of laccase from B. safensis sp. strain S31, efficiencies of 50% to 85% (Wang et al., 2017). In conclusion, rLac
which remained only 1.3% of the control (Siroosi et al., 2018). showed a superior ability for dye decolorization in comparison to
the fungal laccases, which could only carry out decolorization in
3.5. Dye decolorization by rLac acidic conditions. Apart from the high efficiency of decolorization
in alkali conditions, rLac exhibited another an advantage in the de-
Nowadays, synthetic dyes are used in many industrial units, including colorization process, i.e., the ability to decolorize for a long duration
the textile processing industry, food and drugs production, and paper at a higher temperature (60 °C) compared to the laccases from other
manufacturing industry (Waring and Hallas, 2013). Direct discharge of species of Bacillus genus, such as B. amyloliquefaciens LC02, 40 °C
the dye effluents containing many non-biodegradable and toxic (Chen et al., 2015), B. pumilus W3, 50 °C (Guan et al., 2014a),
chemicals in water bodies t would lead to serious damage of our ecosys- B. vallismortis fmb-103, 37 °C (Zhang et al., 2012), B. subtilis X1,
tem and environment (Forootanfar et al., 2011). Thus, daily treatment of 50 °C (Guan et al., 2014b), Bacillus sp. A4, 40 °C (Guo et al., 2017),
thousands of tons of dye effluents has been huge challenge for us. B. amyloliquefaciens 12B, 50 °C (Lončar et al., 2013), B. licheniformis
Laccases, polyphenol oxidases belonging to the family of oxidore- LS04, 40 °C (Lu et al., 2013), and Bacillus sp. ADR, 40 °C (Telke et al.,
ductases, are capable of degrading a series of recalcitrant organic 2011). As the effluent discharged after the dyeing process usually
pollutants, including synthetic dyes (Bilal et al., 2019; Majeau et al., has a high temperature, it is of great importance to immediately
2010; Sondhi et al., 2018). Given their high catalytic efficiency, and efficiently decolorize the hot effluents upon their release using
 T. Li et al. / Science of the Total Environment 713 (2020) 136713 9

Fig. 7. Decolorization of different dyes by the rLac isolated from B. velezensis TCCC 111904 with mediators ABTS (a), acetosyringone (b), and syringaldehyde (c) at pH 5.5, 7.0, and 9.0. The
decolorization of each dye was performed by incubating 80 U of rLac at 60 °C. Dye symbols from 1 to 8 in the horizontal coordinates represent azophloxine, Congo red, adizol black B,
reactive blue 5, reactive blue 19, crystal violet, indigo carmine, and malachite green, respectively. All experiments were carried out in triplicate, and the values represent mean ± SD.

a laccase with high thermostability. A great deal of energy would be Acknowledgments

saved if the hot water could be used again after decolorization
(Lončar et al., 2013).
Given that most of the textile effluents are alkaline and
discharged at high temperature (Kapdan and Alparslan, 2005;
Pereira et al., 2010), use of rLac with high efficiency of decolorization
in neutral to alkaline pH at a high temperature (60 °C) in the treat-
ment of alkaline effluent containing synthetic dyes would be more
advantageous than fungal laccases.
4. Conclusion
This work was supported by the Tianjin Natural Science Foundation
(17JCYBJC23700), the National Key Research and Development Pro-
gram of China [2017YFD0201405-04], the Tianjin Correspondent Pro-
gram of Science and Technology of China (19JCTPJC52200), the
Natural Science Foundation of Tianjin Municipal Education Commission
[2017KJ183], and the Foundation of Key Laboratory of Industrial Fer-
mentation Microbiology of Ministry of Education and Tianjin Key Lab
of Industrial Microbiology (Tianjin University of Science & Technology)
(2017KF007).

The novel laccase rLac isolated from Bacillus velezensis TCCC 111904
displayed high thermostability and high pH stability over a broad pH
range. Besides, it also showed a high efficiency in decolorization of
azo, anthraquinonic, and triphenylmethane dyes at high temperature
over a broad pH range. Additionally, rLac showed great tolerance
against high concentration of NaCl. These unique characteristics indi-
cated that it could be a potential candidate for application in the treat-
ment of dye effluents and other industrial processes.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
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