Expression and purification of an ArsM-elastin-like polypeptide fusion and its enzymatic properties

Article abstract of DOI:10.1007/s00253-019-09638-w

Enzymes could act as a useful tool for environmental bioremediation. Arsenic (As) biomethylation, which can convert highly toxic arsenite [As(III)] into low-toxic volatile trimethylarsine, is considered to be an effective strategy for As removal from contaminated environments. As(III) S-adenosylmethyltransferase (ArsM) is a key enzyme for As methylation; its properties and preparation are crucial for its wide application. Currently, ArsM is usually purified as a His-tag fusion protein restricting widespread use due to high costs. In this study, to greatly reduce the cost and simplify the ArsM preparation process, an Elastin-like polypeptide (ELP) tag was introduced to construct an engineered Escherichia coli (ArsM-ELP). Consequently, a cost-effective and simple non-chromatographic purification approach could be used for ArsM purification. The enzymatic properties of ArsM-ELP were systematically investigated. The results showed that the As methylation rate of purified ArsM-ELP (> 35.49%) was higher than that of E. coli (ArsM-ELP) (> 10.39%) when exposed to 25?μmol/L and 100?μmol/L As(III), respectively. The purified ArsM-ELP was obtained after three round inverse transition cycling treatment in 2.0?mol/L NaCl at 32?°C for 10?min with the yield reaching more than 9.6% of the total protein. The optimal reaction temperature, pH, and time of ArsM-ELP were 30?°C, 7.5 and 30?min, respectively. The enzyme activity was maintained at over 50% at 45?°C for 12?h. The enzyme specific activity was 438.8 ± 2.1?U/μmol. ArsM-ELP had high selectivity for As(III). 2-Mercaptoethanol could promote enzyme activity, whereas SDS, EDTA, Fe²⁺, and Cu²⁺ inhibited enzyme activity, and Mg²⁺, Zn²⁺, Ca²⁺, and K⁺ had no significant effects on it.

Full text of DOI:10.1007/s00253-019-09638-w

Applied Microbiology and Biotechnology
https://doi.org/10.1007/s00253-019-09638-w
ENVIRONMENTAL BIOTECHNOLOGY
Expression and purification of an ArsM-elastin-like polypeptide fusion
and its enzymatic properties
1
1
1
1
1
2,3
Changdong Ke & Hui Xiong & Chungui Zhao & Zhigang Zhang & Xiaolan Zhao & Christopher Rensing
&
1
1
Guangya Zhang & Suping Yang
Received: 15 October 2018 /Revised: 2 January 2019 /Accepted: 4 January 2019
Springer-Verlag GmbH Germany, part of Springer Nature 2019
#
Abstract
Enzymes could act as a useful tool for environmental bioremediation. Arsenic (As) biomethylation, which can convert highly
toxic arsenite [As(III)] into low-toxic volatile trimethylarsine, is considered to be an effective strategy for As removal from
contaminated environments. As(III) S-adenosylmethyltransferase (ArsM) is a key enzyme for As methylation; its properties and
preparation are crucial for its wide application. Currently, ArsM is usually purified as a His-tag fusion protein restricting
widespread use due to high costs. In this study, to greatly reduce the cost and simplify the ArsM preparation process, an
Elastin-like polypeptide (ELP) tag was introduced to construct an engineered Escherichia coli (ArsM-ELP). Consequently, a
cost-effective and simple non-chromatographic purification approach could be used for ArsM purification. The enzymatic
properties of ArsM-ELP were systematically investigated. The results showed that the As methylation rate of purified ArsM-
ELP (> 35.49%) was higher than that of E. coli (ArsM-ELP) (> 10.39%) when exposed to 25 μmol/L and 100 μmol/L As(III),
respectively. The purified ArsM-ELP was obtained after three round inverse transition cycling treatment in 2.0 mol/L NaCl at
3
2 °C for 10 min with the yield reaching more than 9.6% of the total protein. The optimal reaction temperature, pH, and time of
ArsM-ELP were 30 °C, 7.5 and 30 min, respectively. The enzyme activity was maintained at over 50% at 45 °C for 12 h. The
enzyme specific activity was 438.8 ± 2.1 U/μmol. ArsM-ELP had high selectivity for As(III). 2-Mercaptoethanol could promote
2
+
2+
2+
2+
2+
+
enzyme activity, whereas SDS, EDTA, Fe , and Cu inhibited enzyme activity, and Mg , Zn , Ca , and K had no
significant effects on it.
.
.
.
Keywords Arsenic As(III) S-adenosylmethionine (SAM) methyltransferase Elastin-like polypeptide tag Rhodopseudomonas
palustris
Introduction
Arsenic (As) contamination has become a major public health
problem in the world (Zhou et al. 2018; Zhu et al. 2014). Due
*
*
*
Chungui Zhao
chungui@hqu.edu.cn
to As being ubiquitous in nature, microorganisms have ac-
quired strategies for As detoxification, such as arsenate
[As(V)] reduction and arsenite [As(III)] oxidation and meth-
ylation as well as organic As demethylation (Zhu et al. 2017).
These mechanisms not only play a key role in driving the As
biogeological cycle (Liu et al. 2014; Rensing and Rosen
Christopher Rensing
rensing@fafu.edu.cn
Suping Yang
yangsuping@hqu.edu.cn
2
009), but also inspired people to employ these mechanisms
1
2
Department of Bioengineering and Biotechnology, Huaqiao
University, Xiamen 361021, China
to remove As, especially As volatilization by As methylation.
As methylation can convert highly toxic As(III) to low-toxic
volatile trimethylarsine [TMAs(III)] (Qin et al. 2006, 2009;
Zhao et al. 2015) and was found to be widespread in natural
environment; therefore, this detoxification mechanism is con-
sidered to be an effective strategy for As removal. As(III) S-
adenosylmethionine (SAM) methyltransferase (termed as
Institute of Environmental Microbiology, College of Resources and
Environment, Fujian Agriculture and Forestry University,
Fuzhou 350002, China
3
Key Laboratory of Urban Environment and Health, Institute of Urban
Environment, Chinese Academy of Sciences, 1799 Jimei Road,
Xiamen 361021, China

Appl Microbiol Biotechnol
ArsM in microbes, As MT in higher organisms) is the key
Pro-Gly-Xaa-Gly)n, where Xaa is the amino acid except
Pro (Meyer and Chilkoti 1999). The ELP fusion proteins
can be purified by using non-chromatographic technique
termed as inverse thermal cycling (ITC), that is, the ELP
fusion proteins are soluble at temperatures lower than their
3
enzyme for As methylation. Since Qin et al. (2006) identified
and characterized ArsM from Rhodopseudomonas palustris
CGA009 by heterologous expression in As sensitive
Escherichia coli AW3110, many novel arsM genes or As-
methylating microbes have been reported, such as
Cyanidioschyzon sp. 5508 (Qin et al. 2009), Methanosarcina
acetivorans C2A (Wang et al. 2014b), Pseudomonas
alcaligenes NBRC14159 (Zhang et al. 2015), Streptomyces
sp. GSRB54 (Kuramata et al. 2015), Asibacter rosenii SM-1
transition temperature (T ) and become insoluble aggre-
t
gates above the T (Li and Zhang 2014; Roberts et al.
t
2015). In this study, we cloned arsM from R. palustris
CGA009, a good model organism for studying As detoxi-
fication (Qin et al. 2006; Zhao et al. 2015), and then arsM
was fused with the ELP-tag to construct an engineered
E. coli (ArsM-ELP) with a high As resistance. ArsM-ELP
was purified by the ITC system, and its enzymatic proper-
ties including temperature, pH, temperature stability, selec-
tivity, and interference factors were investigated. This work
provided a novel, simply, non-chromatographic ArsM pu-
rification method and obtained an engineered E. coli
(ArsM-ELP) with a higher As methylation rate, which laid
a good foundation for a biotechnological application of
ArsM.
(
2
Huang et al. 2016), Synechocystis sp. PCC 6803 (Xue et al.
017), and Bacillus sp. CX-1 (Huang et al. 2018). These
works provided a better understanding of bacterial As meth-
ylation pathways. However, the majority of naturally existing
microorganisms exhibited a relatively limited ability to vola-
tilize As (Wang et al. 2014a) except for A. rosenii SM-1
(
Huang et al. 2016); therefore, many genetically engineered
As-methylating organisms harboring arsM were constructed,
such as E. coli AW3110 (Yuan et al. 2008), Sphingomonas
desiccabilis (Liu et al. 2011), Bacillus idriensis (Liu et al.
2
2
011), Pseudomonas putida KT2440 (Chen et al. 2013,
014), Bacillus subtilis 168 (Huang et al. 2015),
Saccharomyces cerevisiae (Verma et al. 2016), and
Rhizobium leguminosarum R3 (Zhang et al. 2017). These
engineered organisms displayed different capacities to volatil-
ize As. For example, engineered strain P. putida KT2440 con-
taining arsM converted 25 μM As(III) to volatile TMAs(III)
Material and methods
Strains, plasmids, and reagents
Rhodopseudomonas palustris CGA009 (ATCC BAA-98) was
obtained from the American Type Culture Collection (ATCC,
USA). Escherichia coli DH5α was obtained from Tiangen
Biochemical Technology Co., Ltd. (Beijing, China). Strain
E. coli AW3110 (DE3) (ΔarsRBC) (Carlin et al. 1995) was
a present by Professor Yongguan Zhu of the Institute of Urban
Environment, Chinese Academy of Science; pET-22b-ELP
(
10 ± 1.2%) and dimethylarsine [Me AsH] (21 ± 1.0%) (Chen
2
et al. 2014). Therefore, the As-methylating or As-volatizing
organisms would have potential application value for the bio-
remediation of As-contaminated environments.
In addition to microbes, enzymes play a main role in
bioremediation (Rao et al. 2014). Regarding ArsM, the
reported studies focused on arsM function validation
(KV F-40) (Huang et al. 2011), by Professor Guangya
8
(
2
Guo et al. 2016; Huang et al. 2016, 2018; Qin et al.
006, 2009; Verma et al. 2016; Wang et al. 2014b; Ye
Zhang of Huaqiao University. As(V) (Na AsO ) and As(III)
3
4
(NaAsO ) were obtained from Merck (Darmstadt, Germany).
2
et al. 2014; Yin et al. 2011) and the investigation on deter-
mining ArsM reaction temperature and pH (Guo et al.
Unless specified otherwise, all chemicals were purchased
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China), and all reagents used in this study were of analytical
or better grade.
2
2
016; Qin et al. 2009; Wang et al. 2014b; Xue et al.
017; Ye et al. 2014; Zhang et al. 2015). So far, the de-
tailed ArsM enzymatic properties are still poorly under-
stood. In addition, ArsM was obtained as a His-tag fusion
protein with higher cost (Guo et al. 2016; Huang et al.
Construction and identification of engineered E. coli
(ArsM-ELP)
2
016, 2018; Qin et al. 2006, 2009; Wang et al. 2014b;
Ye et al. 2014; Yin et al. 2011). Therefore, it is of great
significance to develop a simple, cost-effective, large-scale
ArsM preparation method and explore ArsM enzymatic
properties.
Elastin-like polypeptide (ELP), as a non-chromatographic
protein purification tag, has been widely used in various
protein purification due to its simple, low-cost, easily
large-scale preparation (Trabbic-Carlson et al. 2004). ELP
are polymers composed of repeats of the pentapeptide (Val-
The arsM (GenBank accession no. BX571963.1) was ampli-
fied by PCR from R. palustris CGA009 using the following
primers: arsM-F: 5′-CATGCATATGCCACTGACAT
GCAAGACGTGAAG-3′ and arsM-R: 5′-CCGG
GATCCCCCGCAGCAGCGCGCC-3′. The primers were de-
signed by Primer Premier 5.0 software (Premier Biosoft
International, Palo Alto, CA, USA) and synthesized by
Suzhou Genewiz Biotechnology Co., Ltd. (Suzhou, China).
The arsM fragment and pET-22b-ELP plasmid were double-

Appl Microbiol Biotechnol
digested with NdeI and BamHI and were recovered by agarose
gel electrophoresis. The gene fragment was ligated with pET-
experiments without As(III) were carried out under the same
conditions. Each experiment was repeated in triplicate. The
bacterial growth was monitored by measuring OD600. The
growth inhibition rate was used to assess the calculated
growth based on the growth of the corresponding As-free
medium.
2
2b-ELP plasmid at 16 °C for 20 h and transformed into
E. coli DH5α competent cells. DH5α cells were plated on
Luria-Bertani (LB) solid medium containing 100 μg/mL am-
picillin and incubated overnight at 37 °C. Positive clones were
screened to identify the recombinant plasmids (pET-22b-
arsM-ELP) by double restriction enzyme digestion with
NdeI and BamHI to obtain the cloned arsM. The identified
plasmid (pET-22b-arsM-ELP) was transformed into compe-
tent E. coli AW3110 (DE3) cells. The engineered E. coli
AW3110 (pET-22b-arsM-ELP) was screened for ampicillin
resistance, verified by double enzyme digestion and sequenc-
ing by Suzhou Genewiz Biotechnology Co., Ltd. (Suzhou,
China). The E. coli AW3110 (DE3) strain harboring the
pET-22b-arsM-ELP and pET-22b-ELP were named E. coli
The optimization of expression of ArsM-ELP by E. coli
(ArsM-ELP)
To enhance the protein expression level of E. coli (ArsM-
ELP), the temperature, IPTG concentration, and expression
time of ArsM-ELP were optimized by a single-factor experi-
ment. The recombinant E. coli (ArsM-ELP) was inoculated in
a 250 mL flask containing 100 mL of LB medium and cul-
tured in a shaker at 200 r/min at 37 °C until the culture reached
an OD600 of approximately 0.5~0.6. 0.8 mmol/L of IPTG was
added to induce for 12 h at 200 r/min at 16, 20, 24, 30, and
(
ArsM-ELP) and E. coli (pET-22b-ELP), respectively. The
ELP gene was ligated to the C-terminus of the arsM.
37 °C, respectively. The optimized temperature was used to
Purification and identification of ArsM
optimize IPTG concentration and the expression time of
ArsM-ELP. The total protein content was determined as de-
scribed by Bradford (1976), and the ArsM-ELP protein was
checked by SDS-PAGE and analyzed by a Gel-Pro Analyzer 4
(Media Cybernetics, Rockville, MD, USA).
A single colony of engineered E. coli (ArsM-ELP) was picked
and inoculated in 100 mL LB liquid medium containing am-
picillin (100 μg/mL), and incubated at 37 °C until optical
density at 600 nm (OD600) of 0.4~0.6. The cells were induced
by 0.2 mmol/L of isopropyl β-D-1-thiogalactopyranoside
The optimization of purification conditions
for ArsM-ELP
(
IPTG) for 16 h at 30 °C.
The total protein extract was prepared as follows: 5 mL of
cells was harvested by centrifugation and washed with
0.0 mmol/L phosphate-buffered saline (PBS) buffer
137.0 mmol/L NaCl, 2.7 mmol/L KCl, 10.0 mmol/L
Na HPO ·12H O, 2.0 mmol/L KH PO , pH 7.4), then resus-
The purification procedures of the ELP-tag fusion protein
were performed as described previously (Huang et al. 2011).
5
(
In this study, the effects of NaCl concentration, T , phase tran-
t
sition time, and total protein content on enzyme purification
effectiveness were investigated, respectively. Briefly, the cell
lysate was centrifuged at 12000 r/min for 15 min at 4 °C to
obtain the supernatant. The inverse phase transition of ELP
fusion proteins was triggered by the addition of NaCl
(1.0~2.5 mol/L); the aggregated ELP protein precipitation
was collected by centrifugation and then was resolubilized in
cold PBS buffer (pH 7.4) and ice bath for 15 min, and centri-
fuged again to remove particulate contaminants. This aggre-
gation and resolubilization process was repeated for three
times, and the purified protein content was determined as de-
scribed by Bradford (1976). The optimized NaCl concentra-
2
4
2
2
4
pended in PBS buffer. The cells were disrupted by a sonicator
Scientz-IID, Ningbo Xinzhi Biotechnology Co., Ltd.,
(
Ningbo, China) (300 W output, pulse 2 s, stop 2 s, cycle 90
times), and the supernatant was collected by centrifugation at
1
3,000×g for 25 min at 4 °C and submitted for measuring total
protein content as described by Bradford (1976) using bovine
serum albumin (Sigma, Shanghai, China) as a standard.
Expressed ArsM-ELP proteins were identified by sodium-
dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-
PAGE).
As resistance in engineered E. coli (ArsM-ELP)
tions were designed to optimize T , the phase transition time,
t
and the total protein content. The purified protein purity was
ascertained by SDS-PAGE.
Single colonies of E. coli (pET-22b-ELP) and E. coli (ArsM-
ELP) were picked and inoculated in 20 mL LB medium sup-
plemented with the appropriate antibiotics, respectively, and
incubated at 37 °C overnight. Two percent (V/V) inocula were
transferred to 100 mL liquid LB medium containing
The relationship between NaCl concentration and Tt
NaCl was dissolved in 50.0 mmol/L PBS buffer (pH 7.4) to
prepare different concentration NaCl solution (0, 0.5, 1, 1.5, 2,
and 2.5 mol/L). The purified ArsM-ELP was set at a
final concentration of 1.0 mg/mL. The reaction system was
0
.2 mmol/L of IPTG and As(III) (50 and 70 μmol/L) and
incubated at 37 °C for 22 h. As(III) at 50 and 70 μmol/L were
chosen as described previously (Qin et al. 2006). Control

Appl Microbiol Biotechnol
measured by an ultraviolet-visible spectrophotometer
Specord 200, Analytical Jena, Jena, Germany). The
programed heating temperature was set to 1.0 °C/min, and
the absorbance was monitored by measuring OD350. The tem-
perature in corresponding to half of the maximum value of
added and incubated at 37 °C for 1 h. The fluorescence inten-
sity was measured at both 665 and 620 nm with excitation at
337 nm in a multi-function microplate reader (SpectraMax
M5, Molecular Devices, Silicon Valley, CA, USA). The blank
control (without As, without enzyme) and the As-free control
were set up. The homogeneous time-resolved fluorescence
(
OD350 is defined as the T (Meyer and Chilkoti 1999).
t
(HTRF) signal was calculated from the ratio of emission at
Determination of the recovery rate of purified
ArsM-ELP
665 and 620 nm. The yield of SAH was calculated from the
SAH standard curve. The enzyme activity was evaluated as
the difference in SAH content between the experimental group
and the As-free control group.
The purified ArsM-ELP was formulated into gradient enzyme
solutions containing 2.0 mol/L NaCl in 50.0 mmol/L PBS
buffer (pH 7.4) and was submitted for one round of ITC pu-
rification treatment. The protein concentration was deter-
mined and the protein recovery rate was calculated.
As volatilization of engineered E. coli (ArsM-ELP)
and ArsM-ELP
The difference in total As content before and after bacterial
The enzymatic properties of ArsM-ELP
growth was used to assess the As volatilization of E. coli
(
ArsM-ELP). 20 mL of cells of E. coli (ArsM-ELP) and
The optimal reaction time of ArsM-ELP was determined in
the presence of 5.0 μmol/L ArsM-ELP at 37 °C, pH 7.4.
Subsequently, based on the optimal reaction time, the effects
of temperature and pH on enzyme activity were investigated.
Under optimal temperature and pH conditions, the thermal
stability, substrate selectivity, and interference factors on en-
E. coli AW3110 (harboring empty plasmid) (OD600 about
0.5) was cultured in LB liquid medium containing
0.20 mmol/L IPTG and 25 μmol/L As(III). Control experi-
ments without As(III) were performed under the same condi-
tions. Each experiment was repeated in triplicate. These ex-
periments were performed by shaking at 200 r/min, at 37 °C
for 8 h and 24 h and submitted for total As measurement.
+
2+
zyme were investigated, respectively. KCl [K ], CaCl [Ca ],
2
2
+
3+
3+
FeSO [Fe ], FeCl [Fe ], Cr (SO4) [Cr ], and Na AsO
0.50 mL of the sample was digested with 6 mL of HNO with
4
3
2
3
3
4
3
[
0
[
As(V)] were selected to replace As(III) as the substrates.
a microwave digestion apparatus (Multiwave Pro, Anton Paar
GmbH, Graz Austria) for microwave-assisted digestion. The
digestion parameters were set as described previously (Zhu
et al. 2008). The digested sample was diluted to 50.0 mL
and filtered through a 0.22 μM microporous membrane. The
total As content was analyzed by inductively coupled plasma-
mass spectrometry (ICP-MS) (Agilent 7800, Agilent
Technologies, Tokyo, JHS, Japan). The As mixture standards
were used as external standard samples and measured as de-
scribed previously (Oliveira et al. 2017).
2
+
2+
.3 mmol/L of MgCl [Mg ], ZnSO [Zn ], CuSO
2 4 4
2
+
2+
2+
+
Cu ], CaCl2 [Ca ], FeSO4 [Fe ], KCl [K ], 2-
mercaptoethanol (2-Me), sodium dodecyl sulfate (SDS), and
ethylene diamine tetraacetic acid (EDTA) was used to assess
the interference effect on ArsM-ELP enzyme activity. The
enzyme activity of ArsM-ELP measured under the optimized
conditions was defined as 100%.
ArsM-ELP enzyme activity assay
The difference in total As content was used to assess the As
volatilization rate of ArsM-ELP. The ArsM-ELP reaction sys-
tem was set at 5.0 mL containing 2.0 mmol/L SAM,
20.0 mmol/L GSH, 100.0 μmol/L As(III), and 50.0 μmol/L
ArsM-ELP. After incubating for 3 h and 12 h, 0.5 mL of
EPIgeneous™ Methyltransferase Assay kit (Cisbio
Bioassays, Bedford, MA, USA) was used to assay the enzyme
activity of ArsM-ELP. The conversion of SAM to S-
adenosylhomocysteine (SAH) was measured according to
the assay kit instruction. The reaction time was set at 30 min
sample was taken and 3% HNO was added to make a volume
3
(
Dong et al. 2015). The reaction system had a volume of
of 50.0 mL. The total As content was determined by ICP-MS.
1
5
0.0 μL of 50 mmol/L PBS buffer (pH 7.4) containing
.0 μmol/L ArsM-ELP, 10 μmol/L As(III), 20 μmol/L
SAM, and 4 mmol/L glutathione (GSH). ArsM-ELP enzyme
at 5.0 μmol/L and As(III) at 10 μmol/L were chosen as de-
scribed previously (Dong et al. 2015). SAM at 20 μmol/L was
used to assay the enzyme activity of ArsM-ELP according to
the Instruction of EPIgeneous™ Methyltransferase Assay kit
Results
Construction and identification of engineered E. coli
(ArsM-ELP)
(
Cisbio Bioassays, Bedford, MA, USA). The reactions were
The engineered E. coli (ArsM-ELP) cell was obtained by re-
combinant DNA techniques. The PCR-amplified arsM was
ligated into the pET-22b-ELP vector and heterologously
performed at 37 °C for 30 min. At the end of the reaction, the
ruthenium-labeled SAH antibody and SAH-d2 tracer were

Appl Microbiol Biotechnol
expressed in the As(III)-hypersensitive E. coli AW3110
(
(
DE3), which lacks the arsRBC operon and has no arsM
Carlin et al. 1995). The sequencing results showed that the
arsM sequence cloned into the pET-22b-ELP vector and
expressed in E. coli (ArsM-ELP) was identical to those depos-
ited in GenBank. A protein band of 49.48 kDa was visible in
E. coli (ArsM-ELP) after IPTG induction, whereas the
protein bands in E. coli (pET-22b-ELP) and in E. coli
(
(
ArsM-ELP) were not obvious without ITPG induction
control) (Fig. 1). Due to the presence of ELP at the C-
terminus of ArsM, the molecular weight of the expressed
ArsM was in accordance with the expected molecular weight
of the expressed protein (49.48 kDa), indicating that arsM was
functionally expressed.
Fig. 2 As(III) resistance of the engineered E. coli (ArsM-ELP) and the
control strain E. coli (pET-22b-ELP). The inocula of 2.0% (V/V) were
incubated for 22 h in 100 mL LB medium in the presence of 0.2 mmol/L
IPTG and different As(III) concentrations. Data shown were the mean
values (± standard deviations) obtained from three independent
experiments
As resistance and volatilization by engineered E. coli
(ArsM-ELP)
ArsM could methylate As(III) to form methylated As species
with lower toxicity and volatile As species under aerobic con-
ditions, thereby conferring As resistance to As(III)-sensitive
E. coli AW3110 (Qin et al. 2006). To examine the functional
activity of the ArsM-ELP fusion protein, we determined the
As resistance and As volatilization rate of the engineered
E. coli (ArsM-ELP) (Fig. 2). In the absence of As(III), no
significant difference (P > 0.05) in cell growth between the
engineered strain and the control could be observed. After
exposure to 50 μM and 70 μM As(III), the inhibition rate of
the cell growth displayed a difference between the control
strain (79.9% and 89.8%, respectively) and engineered strain
(
37.0% and 54.3%, respectively), indicating that the
engineered E. coli (ArsM-ELP) possessed higher As resis-
tance than the control strain in the presence of As(III).
Furthermore, the expressed ArsM-ELP fusion protein was
functionally active in vivo.
To examine the As volatilization rate of engineered E. coli
(ArsM-ELP), the difference in total As content before and
after bacterial growth was used to assess the As volatilization
rate (Fig. 3). After incubation for 8 h and 24 h, the total As
content in the medium decreased by 10.39% and 13.42%
when incubated with the engineered strain, respectively,
whereas the total As content decreased by only 0.61% and
0.53% with the control strain, respectively. These results dem-
onstrated that the engineered E. coli (ArsM-ELP) had a sig-
nificant As volatilization capacity and the ArsM-ELP fusion
protein was responsible for As(III) methylation. Therefore, we
further purified the ArsM-ELP fusion protein and studied its
enzymatic properties.
The expression of ArsM-ELP by E. coli (ArsM-ELP)
As shown in Fig. 4, the highest expression level of ArsM-ELP
(0.14 g/L) was observed at 30 °C, which accounted for 23.4%
of the total protein content (Fig. 4a2). Under optimized con-
ditions at 30 °C, 0.2~1.0 mmol/L of IPTG induced a high
expression level of ArsM-ELP of about 0.14 g/L (Fig. 4b2).
Under the optimized 30 °C and 0.2 mmol/L of IPTG
Fig. 1 SDS-PAGE analysis of ArsM-ELP in engineered E. coli (ArsM-
ELP). Lane M: protein marker. Lane 1: cell lysate of E. coli (pET-22b-
ELP). Lane 2: cell lysate of E. coli (ArsM-ELP) without IPTG induction.
Lane 3: cell lysate of E. coli (ArsM-ELP) induced by IPTG. Samples
were harvested after induction with 0.2 mmol/L IPTG at 30 °C for
1
6 h, and the cellular proteins were analyzed by SDS-PAGE

Appl Microbiol Biotechnol
conditions for ArsM-ELP expression in recombinant E. coli
(
(
ArsM-ELP) were that 0.2 mmol/L of IPTG induced E. coli
ArsM-ELP) for 16 h at 30 °C; the total protein yield reached
0.79 g/L.
The purification of ArsM-ELP
The highest yield of ArsM-ELP (9.46%) was obtained in
the presence of 2.5 mol/L NaCl after three rounds of ITC
purification (Fig. 5a). However, considering the negative
influence of high concentration of NaCl on enzymatic ac-
tivity, we selected 2.0 mol/L NaCl for subsequent experi-
ments. In the presence of 2.0 mol/L NaCl, the optimal Tt
was optimized at 32 °C (Fig. 5b). Under the above-
optimized condition, the phase transition time for ArsM-
ELP purification was optimized at 5 min (Fig. 5c). In order
to ensure the complete precipitation of ArsM-ELP, the
phase transition time was prolonged to 10 min.
Fig. 3 As volatilization rate comparison between the engineered E. coli
(
(
ArsM-ELP) and the control E. coli (pET-22b-ELP). 100 mL of culture
OD600 = 0.5) was incubated for 8 h and 24 h at 37 °C in the presence of
The effect of total protein content on ArsM-ELP purifi-
cation was investigated based on the optimal NaCl concen-
0
.2 mmol/L IPTG and 25.0 μmol/L As(III). The As volatilization rate was
assessed by the difference in total As content before and after bacterial
growth. The data shown were the mean (± standard deviation) obtained
from three independent experiments
tration, T , and phase transition time (Fig. 5d, e). The
t
6.0~16.0 mg/mL of total protein extract was selected for
ArsM-ELP purification. After three rounds of ITC, a single
protein band was observed by SDS-PAGE; the yield of
ArsM-ELP reached 9.6~10.8% of the total protein.
Two rounds of ITC were enough to satisfy the purity
condition, the high expression level of ArsM-ELP (0.25 g/L)
was observed at 16 h of culture time, accounting for 32.2% of
the total protein content (Fig. 4c2). Collectively, the optimal
Fig. 4 The effect of culture temperature (a), IPTG concentration (b), and
the expression time (c) on the ArsM-ELP amount in E. coli (ArsM-ELP).
a E. coli (ArsM-ELP) was induced for 12 h at 0.8 mmol/L IPTG; b E. coli
cultured at 30 °C and 0.2 mol/L IPTG. ρArsM-ELP represents the ArsM-
ELP amount in recombinant E. coli (ArsM-ELP). ωArsM-ELP represents
the ratio of ArsM-ELP to total protein content in recombinant E. coli
(ArsM-ELP)
(ArsM-ELP) was induced for 12 h at 30 °C; c E. coli (ArsM-ELP) was

Appl Microbiol Biotechnol
Fig. 5 Optimization of ITC conditions for ArsM-ELP purification. a
NaCl concentration; b phase transition temperature; c phase transition
time; d ITC treatment times; e total protein extract concentration: Lane
M: protein marker; Lane A–D: 16, 12, 8, and 6 mg/mL, respectively;
subscript 1, 2, and 3 represented ITC round times, respectively
requirement for ArsM-ELP since a protein yield of
ArsM-ELP was investigated (Fig. 7). Under the optimized
1
7.6~20.2% had been achieved, and the purity requirement
NaCl concentration, T , and phase transition time condi-
t
is not that stringent in practical applications. Therefore, the
ArsM-ELP content available in 1 L of E. coli culture
ranged from 0.14 to 0.16 g.
tions, a recovery rate of 85.2~96.0% for ArsM-ELP was
achieved in the range of 1.0~1.5 mg/mL ArsM-ELP after
one round of ITC treatment. A positive linear relationship
was observed between the recovery rate and increasing
The relationship between NaCl concentration and Tt
A linear negative correlation between T and NaCl concen-
t
tration was observed in the presence of 1.0 mg/mL of pu-
rified ArsM-ELP (Fig. 6); this phenomenon was consistent
with the reported literature (Huang et al. 2011; Lim et al.
2
007; Meyer and Chilkoti 1999; Reguera et al. 2007). The
Tt was 31.7 °C in the presence of 2.0 mol/L NaCl, which
was consistent with the optimized T (32 °C). The result
t
shown here (T = 31.7 °C, 2.0 mol/L NaCl) was much bet-
t
ter compared with the reported ELP transition data (T =
t
3
5 °C, 2.5 mol/L NaCl) because the lower salinity could
prevent the denaturation of the spatial structure of ArsM
caused by higher salt concentration.
The recovery rate of purified ArsM-ELP
To determine whether ArsM-ELP could be recovered and
reused, the recovery rate of different concentrations of
t
Fig. 6 The relationship between NaCl concentration and T in the
presence of 1.0 mg/mL of purified ArsM-ELP

Appl Microbiol Biotechnol
2
+
2+
2+
+
including Mg , Zn , Ca , and K had no significant effect
on the enzyme activity (P > 0.05). Under optimized condi-
tions, the specific activity of the enzyme was 438.8 ± 2.1 U/
μmol.
As volatilization rate by purified ArsM-ELP
The As volatilization by ArsM-ELP is shown in Fig. 9. The
initial value of As(III) was 99.45 ± 0.21 μmol/L. The As con-
tent in the medium decreased by 35.49% and 38.35% after
incubation for 3 h and 12 h, respectively, indicating that
ArsM-ELP catalyzed the conversion of As(III) to volatile As.
Discussion
Fig. 7 The relationship between the recovery rate and increasing
concentrations of purified ArsM-ELP
Arsenic (As) contamination affects an increasing number of
people and has been given more attention as approaches are
needed to mitigate the negative effects of this health crisis
(Zhou et al. 2018; Zhu et al. 2014). Genetically engineered
enzyme concentrations, which would be of great signifi-
cance for bioremediation of As-contaminated environments
by using ArsM-ELP.
bacteria display more efficient enrichment and immobilization
of heavy metals and are gradually emerging as an effective
tool for remediation of heavy metal pollutants (Singh et al.
2011). For example, arsenic regulatory protein gene (arsR),
The enzymatic properties of ArsM-ELP
phytochelatin gene, and metallothionein gene were used to
construct engineered E. coli which showed a high metal-
binding capacity (Ke et al. 2018; Sauge-Merle et al. 2003;
Singh et al. 2008). Recently, many As-methylating microbes
and ArsM enzyme were shown to convert highly toxic As(III)
into less toxic pentavalent or volatile methylated As species;
the volatile As species was removed from environment via
volatilization. Therefore, As methylation was considered to
be an effective method for the remediation of As contamina-
tion (Ye et al. 2012). However, except for a wild strain
displaying high As(III) volatilization (such as the A. rosenii
SM-1, Huang et al. 2016), most naturally occurring microbes
showed a limited As volatilization capacity (Wang et al.
2014a). In an attempt to obtain high As volatilizing microbes,
arsM was used to construct engineered strains (Chen et al.
2013, 2014; Huang. et al. 2015; Liu et al. 2011; Zhu et al.
2008), and the As volatilization rate of these engineered
strains was generally in the range of 0.01~13% except for
P. putida KT2440 (31%) (Chen et al. 2014). The reason for
a lower As volatilization rate might be because ArsM was
poorly expressed in vivo. In addition, As(III) could not be
transported by a specific transporter into the cells leading to
lower As(III) concentrations. However, lower concentration
of As(III) in vivo did not efficiently induce transcription of
arsM to produce sufficient ArsM for a high rate of As(III)
methylation. On the other hand, perhaps the ArsM activity
in vivo was not enough to catalyze the conversion of As(III)
to TMA(III) gas, leading only to the formation of MMA(V) or
DMA(V) intermediates. In contrast, the purified ArsM was
The ArsM-ELP is able to catalyze the demethylation of SAM
to SAH and in turn methylate As; therefore, the change in
SAH yield can reflect the enzyme activity of ArsM-ELP
in vitro. When exposed to 10 μmol/L As(III), catalytic activity
2
of ArsM-ELP increased linearly within 30 min (R = 0.9981)
(
Fig. 8a). More than 95% of enzymatic activity was observed
in the range of 25~35 °C; the SAH yield reached 648.7 nmoles
under the optimal 30 °C condition (Fig. 8b). More than 75%
and 50% of enzyme activity were retained at 35 °C and 45 °C
for 12 h, respectively. The enzyme activity was retained at
more than 60% at 55 °C for 2 h (Fig. 8c). The highest enzyme
activity was observed at pH 7.5, and the SAH yield reached
6
7
58.23 nmoles, the enzyme activity was still retained above
0% in the range of pH 5.0~9.0, indicating that the ArsM-ELP
had an even broader pH tolerance (Fig. 8d).
The substrate specificity for ArsM-ELP was also examined
under the above-optimized conditions (Fig. 8e). It was shown
that the catalytic activity of ArsM-ELP was only observed
when exposed to As (III), but not for other ions including
+
2+
2+
3+
3+
K , Ca , Fe , Fe , Cr , and even As(V), indicating that
ArsM-ELP had a narrow selectivity for substrates.
The interference factors on ArsM-ELP enzyme activity
were further investigated (Fig. 8f). It could be shown that
ArsM-ELP enzyme activity increased by14.71% after the ad-
2
+
2+
dition of 2-Me, whereas SDS, EDTA, Fe , and Cu signif-
icantly inhibited enzyme activity (decreased by 55.74%,
3
9.49%, 11.67%, and 14.71%, respectively). The other ions

Appl Microbiol Biotechnol
Fig. 8 ArsM-ELP enzymatic
properties. a reaction time curve;
b the effects of temperature on the
enzyme activity; c the thermal
stability; d the effects of pH on
enzyme activity; e substrate
selectivity; f the effects of
interference factor on enzyme
activity. *P < 0.05; **P < 0.01.
The data shown were the mean (±
standard deviation) obtained from
three independent experiments
able to contact directly with As(III), therefore leading to a high
As methylation rate. In previous studies, the determination of
As methylation was mainly used to identify the function of
putative arsM genes, and ArsM was purified as a fusion protein
using His tags (Guo et al. 2016; Huang et al. 2016, 2018; Qin
et al. 2006, 2009; Wang et al. 2014b; Ye et al. 2014; Yin et al.
harboring arsM derived from CGA009 were developed. In
this study, we fused arsM from R. palustris CGA009 with
ELP-tag and heterologously expressed it in E. coli. The
engineered E. coli (ArsM-ELP) could volatilize 10.39% and
13.42% of ~ 25 μmol/L As after 8 h and 24 h, respectively,
showing a high level of As volatilization traits compared to
most of the reported strains.
2011). Since the His-tag affinity chromatography purification
technique is cost-expensive (Kosobokova et al. 2016), the ap-
plication of ArsM purified using His-tag in remediation of As
contamination is limited. Thus, it was necessary to explore a
simple and cost-effective preparation of ArsM purification.
ArsM encoded on the genome of R. palustris CGA009 was
considered a suitable candidate for an applied As methylation
process. Since Qin et al. (2006) firstly validated the ArsM
function, many engineered bacteria (Chen et al. 2014; Liu
et al. 2011; Yuan et al. 2008) or plants (Meng et al. 2011)
Compared to the His-tag affinity chromatography, the
ELP-tag purification approach is non-chromatographic
(Meyer and Chilkoti 1999; Roberts et al. 2015), convenient,
low cost, and easy to realize at a large scale (Trabbic-Carlson
et al. 2004). So far, many recombinant proteins such as chlor-
amphenicol acetyltransferase, blue fluorescent protein,
thioredoxin, calmodulin, and xylanase have been successfully
purified by using ITC (Li and Zhang 2014; Lim et al. 2007;
Trabbic-Carlson et al. 2004). In this study, the purified ArsM-

Appl Microbiol Biotechnol
higher volatilization rate for As(III) than the engineered E. coli
(
(
ArsM-ELP), which was consistent with the reported literature
Verma et al. 2016; Yin et al. 2011). Probably As(III) is able to
react with ArsM directly in vitro, whereas the expression of
ArsM in vivo is regulated by As(III), which needed across the
cell membrane to induce and react with ArsM. In addition,
As(III)-pump protein (ArsB) would pump out As(III), the
substrate of ArsM. Therefore, the enzyme has an advantage
in the remediation of environmental As contamination com-
pared to the engineered strain. However, current studies main-
ly focused on the characterization of As-methylating mi-
crobes, less was known about using an ArsM strategy to ad-
dress As pollution. Therefore, this study is of great potential
application value for future As pollution bioremediation.
Collectively, this study provided a novel, simple, cost-
effective strategy for ArsM enzyme preparation at a large
scale. The purified, stable, specific, higher activity of the
ArsM-ELP could be obtained by using a modified ELP non-
chromatography protein purification technique. The ELP-tag
is not only used for protein purification in large scale, but also
for the protein recovery and recycles. Both purified ArsM-
ELP and engineered E. coli (ArsM-ELP) could perform
As(III) methylation, and the As(III) methylation rate by
ArsM-ELP was higher than that of E. coli (ArsM-ELP). The
ArsM-ELP would not only be used for bioremediation of As
contamination environment by As methylation, but also for
the standards’ preparation of various As methylated interme-
diate products.
Fig. 9 As(III) volatilization by ArsM-ELP. The reaction system
5.00 mL) contained 2.0 mmol/L of SAM, 20.0 mmol/L of GSH,
00 μmol/L of As (III), and 50 μmol/L of ArsM-ELP. The As volatiliza-
tion rate was measured after incubation for 3 h and 12 h. Percentage
indicates As volatilization. The data shown were the mean (± standard
deviation) obtained from three independent experiments
(
1
ELP was obtained after ITC purification in the presence of
2
.0 mol/L NaCl at 32 °C for 10 min. Both the NaCl concen-
tration and T were decreased (2.0 mol/L NaCl at ~ 32 °C)
t
compared to the reported ITC purification system (2.5 mol/L
NaCl at 35 °C, Huang et al. 2011) and the phase transition
time was shorter (~ 10 min). A good enzyme activity of puri-
fied ArsM-ELP was observed under optimized ITC
conditions.
Studies have shown that enzymes play a major role in bio-
remediation processes and were shown to have a significant
advantage over microbes and plants in remediation of heavy
metal contaminated soils (Rao et al. 2014). For example, the
conversion rate of As(III) to TMA(III) by NsArsM derived
from Nostoc sp. PCC7120 (< 2.0% of total As, after 12 h)
was higher than that of E. coli AW3110 harboring NsarsM
Funding This study was funded by National Marine Public Industry
Research (No. 201505026), by Natural Science Foundation of Fujian
Province (No. 2018J01049), and by Subsidized Project for Cultivating
Postgraduates Innovative Ability in Scientific Research of Huaqiao
University.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
(
< 1% of total As) (Yin et al. 2011). When WaArsM derived
from Westerdykella aurantiaca used As(III) (100 ppb) and
As(V) (200 ppb) as substrates respectively, 29.3% and
Ethical approval This article does not contain any study with human
participants or animals performed by any of the authors.
1
3.95% of As volatilization rate were achieved after 3 h, re-
spectively. Saccharomyces cerevisiae harboring WaarsM vol-
atilized 29.0% and 11.0% of total As when incubated with
As(III) (2 ppm) and As(V) (20 ppm) for 24 h, respectively
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
(
Verma et al. 2016). Compared to the engineered organisms,
the As(III) methylation reaction catalyzed by ArsM needed
less time and resulted in higher As volatilization, highlighting
the potential advantage of ArsM as an enzyme preparation in
the remediation of environmental As contamination. In this
study, the purified ArsM-ELP could reduce the initial As con-
tent (~ 100 μmol/L) by 35.49% after 3 h; the engineered
E. coli (ArsM-ELP) reduced 13.42% of the initial As content
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