Molecular and biochemical characterization of a new thermostable bacterial laccase from Meiothermus ruber DSM 1279

Article abstract of DOI:10.1039/c5ra24374b

A new laccase gene (mrlac) from Meiothermus ruber DSM 1279 was successfully overexpressed to produce a laccase (Mrlac) in soluble form in Escherichia coli during simultaneous overexpression of a chaperone protein (GroEL/ES). Without the GroEL/ES protein, the Mrlac overexpressed in E. coli constituted a huge amount of the total cellular protein, but the enzyme was localized in the insoluble fraction with no activity in the soluble fraction. Co-expression of the Mrlac with the E. coli GroEL/ES drastically improved proper folding and expression of active Mrlac in the soluble fraction. Spectroscopic analysis of the purified enzyme by UV/visible and electron paramagnetic resonance spectroscopy confirmed that the Mrlac was a multicopper oxidase. The Mrlac had a molecular weight of ～50 kDa and exhibited activity towards the canonical laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ), and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants K_m and k_cat were 27.3 μM and 325 min^-1 on ABTS, 4.2 μM and 106 min^-1 on SGZ, and 3.01 μM and 115 min^-1 on 2,6-DMP, respectively. Maximal enzyme activity was achieved at 70°C with ABTS as substrate. In addition, Mrlac exhibited a half-life for deactivation at 70°C and 75°C of about 120 min and 67 min, respectively, indicating that the Mrlac is intrinsically thermostable. Finally, Mrlac was efficient in catalyzing the removal of 2,4-dichlorophene (DCP) in aqueous solution, a trait which makes the enzyme potentially useful for environmentally friendly applications.

Full text of DOI:10.1039/c5ra24374b

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Molecular and biochemical characterization of
a new thermostable bacterial laccase from
Meiothermus ruber DSM 1279†
Cite this: RSC Adv., 2016, 6, 3910
*
D. C. Kalyani, L. Munk, J. D. Mikkelsen and A. S. Meyer
A new laccase gene (mrlac) from Meiothermus ruber DSM 1279 was successfully overexpressed to produce
a laccase (Mrlac) in soluble form in Escherichia coli during simultaneous overexpression of a chaperone
protein (GroEL/ES). Without the GroEL/ES protein, the Mrlac overexpressed in E. coli constituted a huge
amount of the total cellular protein, but the enzyme was localized in the insoluble fraction with no
activity in the soluble fraction. Co-expression of the Mrlac with the E. coli GroEL/ES drastically improved
proper folding and expression of active Mrlac in the soluble fraction. Spectroscopic analysis of the
purified enzyme by UV/visible and electron paramagnetic resonance spectroscopy confirmed that the
Mrlac was a multicopper oxidase. The Mrlac had a molecular weight of ꢀ50 kDa and exhibited activity
towards the canonical laccase substrates 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS),
syringaldazine (SGZ), and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants Km and kcat were 27.3 mM
and 325 minꢁ1 on ABTS, 4.2 mM and 106 minꢁ1 on SGZ, and 3.01 mM and 115 minꢁ1 on 2,6-DMP,
respectively. Maximal enzyme activity was achieved at 70 ꢂC with ABTS as substrate. In addition, Mrlac
exhibited a half-life for deactivation at 70 ꢂC and 75 ꢂC of about 120 min and 67 min, respectively,
indicating that the Mrlac is intrinsically thermostable. Finally, Mrlac was efficient in catalyzing the removal
of 2,4-dichlorophene (DCP) in aqueous solution, a trait which makes the enzyme potentially useful for
environmentally friendly applications.
Received 18th November 2015
Accepted 17th December 2015
DOI: 10.1039/c5ra24374b
www.rsc.org/advances
bacterial laccases have thus been found to possess distinctive
properties, such as being highly thermotolerant, maintaining
high levels of activity in neutral to alkaline conditions,7 and
Introduction
Laccases (EC 1.10.3.2) belong to the multicopper oxidase (MCO)
protein family and are produced by fungi, bacteria, plants, and
insects. Laccases couple the one-electron oxidation of various
phenolic substrates to the reduction of molecular oxygen to form
water.1 Due to the use of oxygen as electron acceptor and no need
for expensive co-factors, laccases nd applications in several
biotechnological processes including dye decoloration,2 biore-
mediation,3 and have also been suggested to be potentially useful
in lignocellulosic biofuel processing4 and lignin modication.5
To date, our understanding of laccases mainly originates
from studies on fungal enzymes. However, identication and
characterization of laccases from bacterial sources may provide
for novel functionalities. Although the production yields and
redox potential of bacterial laccases are lower than those of
fungal laccases, the bacterial laccases have numerous advan-
tageous properties that are more favorable for industrial
applications compared to fungal laccases.6 Most of the reported
having high halide tolerance.8 In contrast, the activity of fungal
laccases decays rapidly at high temperatures and pH values.9
Moreover, bacterial laccases are suitable for overproduction in
heterologous hosts like Escherichia coli, and their expression
level, stability and catalytic properties are far easier to improve
by directed evolution compared to their fungal counterparts.6,10
Thermostability is considered a key prerequisite for maintain-
ing catalytic robustness of laccases or other enzymes in many
industrial processes.11 The most well-known bacterial laccase is
CotA (an endospore coat protein) from Bacillus subtilis which
has high thermostability.12 Other thermostable bacterial lac-
cases have been found in Thermus thermophilus,13 Thermobac-
ulum terrenum,14 B. clausii, and B. coagulans.15
Chlorophenols have been widely used for a long time in
wood preservation, anticorrosive rust production, fungicides,
and pesticides.16 Chlorophenols are generally recognized as
xenobiotic pollutants that enter the environment through
various industrial operations such as pulp bleaching, pesticide
production, chlorination of water, etc.17 and industrial waste
water containing chlorophenols is harmful to human health as
a consequence of its potential carcinogenic and mutagenic
activities.18,19 Because of their carcinogenicity, toxicity and
Center for BioProcess Engineering, Dept. of Chemical and Biochemical Engineering,
Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark.
E-mail: am@kt.dtu.DK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra24374b
3910 | RSC Adv., 2016, 6, 3910–3918
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persistence, chlorophenols are listed among top priority control gene was performed by PCR with the primers 50-cata-
pollutants by the US EPA and the EU.20 Processes for removing tgATGAAGCTAACCCGACGC-30 50-gaatccTTATTC-
and
chlorophenols usually involve physical, chemical, electro- GGCTTCTACCTGC-30 using genomic DNA of M. ruber as the
chemical and/or biological methods.21,22 Biological degradation template. NdeI and EcoRI endonuclease recognition sites are
of chlorophenols offers advantages including high specicity, shown in lowercase. The amplied PCR product with anking
low cost and a more environmentally friendly prole than its restriction sites was rst cloned into pGEM-T easy vector and
physical or chemical counterparts.17,23
then transformed into E. coli DH5a. Plasmid DNA (with insert)
In the present paper we report the identication, expression, and pET22b+ were digested with restriction enzymes NdeI and
purication, and biochemical characterization of a new multi EcoRI to release the insert (mrlac) and to create a nick in the
copper oxidase from the thermophilic bacterium Meiothermus expression vector, respectively. The mrlac gene released from
ruber. As the active holoenzyme production was difficult using pGEM-T vector was ligated with pET22b+ vector to give pET22b-
E. coli (DE3), and most of the over-expressed recombinant Mrlac, a plasmid that was under the control of the T7 promoter.
enzyme aggregated in the inclusion body, we attempted to gain The sequence of the mrlac insert in the resulting pET22b-Mrlac
large yields of soluble and active enzyme through optimization plasmid was veried by sequencing (Macrogen, Netherlands).
of expression conditions. The expression yields in soluble active The mrlac gene was cloned in pET22b+ and transformed into
form were notably improved by chaperone (GroEL/GroES9 expression strains: E. coli BL21 (DE3), E. coli BL21 (DE3) pLysS,
(plasmid pGro7)) assisted expression in microaerobic condi- Tuner™ (DE3), E. coli C41 (DE3) and pGro7/BL21 (DE3). The
tion at 20 ꢂC. The puried laccase was also tested for its ability overnight culture in Luria-Bertani (LB) medium was diluted to
to remove the chlorinated pesticide 2,4-dichlorophene (DCP).
1 : 100 with fresh media containing ampicillin (100 mg mlꢁ1
)
and, when required, chloramphenicol and arabinose (34 mg
mlꢁ1 and 0.5 mg mlꢁ1), respectively, and incubated at 30 ꢂC
with shaking (180 rpm). Aer cells grew to an OD600 of 0.5–0.6,
the cells were induced with 0.1 mM isopropyl-b-D-thiogalacto-
side (IPTG) and supplemented with 0.2 mM CuSO4. The cells
Materials and methods
Materials
All reagents were of analytical grade and purchased from
commercial sources. Laccase from Trametes versicolor (TvLac,
EC 1.10.3.2), 2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), syringaldazine
(SGZ) and DCP were purchased from Sigma-Aldrich (St Louis,
MO, USA).
ꢂ
were then incubated at 25 C in semi-microaerobic conditions
(by turning off the shaker 4 h aer induction). Cells were har-
vested aer 20 h by centrifugation (8000 ꢃ g, 15 min, and 4 ꢂC)
and the pellets suspended in 20 mM potassium phosphate
buffer, pH 7.6, with
1 mM protease inhibitor, phenyl-
methanesulfonyl uoride (PMSF), and then sonicated on ice.
Disrupted cells were removed by centrifugation (18 000 ꢃ g, 15
min, and 4 ꢂC) and the level of protein expression was checked
on SDS-PAGE. The soluble and active expression was obtained
only in chaperone competent cells pGro7/BL21 (DE3). Further,
to obtain the best condition of expression with high amounts of
soluble and active protein, expression conditions were opti-
mized with regard to IPTG concentration (0.1–1 mM), CuSO4
(0.1–1 mM) and at microaerobic conditions in different
Reagents for the polymerase chain reaction (PCR), Ex-Taq
DNA polymerase, genomic DNA extraction kit, and pGEM-T
easy vector were purchased from Promega (Madison, USA).
The pET22b(+) expression vector and plasmid isolation kit were
purchased from Qiagen (Hilden, Germany). Oligonucleotide
primers were obtained from Macrogen (Netherlands).
Strains and culture conditions
Meiothermus ruber DSM 1279 was obtained from DSMZ
ꢂ
temperatures (18, 20, 25 and 30 C).
(Braunschweig,
Germany).
E.
coli
strain
DH5a
(Fꢁf80dlacZDM15 D(lacZYA-argF) U169 recA1 endA1 hsdR17
(rkꢁ, mk+) supE44 thi-1 gyrA relA1), E. coli BL21 (DE3) [FꢁompT
hsdSB (rBꢁ mBꢁ) gal dcm rne131 (DE3)], E. coli BL21 (DE3) pLysS
Purication
Aer expression, cultures were harvested by centrifugation
(8000 ꢃ g, 15 min, and 4 ꢂC). The cell pellets were resuspended
in potassium phosphate buffer (20 mM, pH 7.6) containing 0.1
mM PMSF. Cell disruption was performed by sonication on
ice, and the cell debris was removed by centrifugation (8000 ꢃ g,
30 min, 4 ꢂC). Aerwards, the supernatant was incubated for 30
[FꢁompT gal hsdSB (rB mꢁBꢁ) dcm gal l(DE3), pLysS], Tuner™
ꢁ
(DE3) [FꢁompT hsdSB (rB mBꢁ) gal dcm lacY1 (DE3)] were
purchased from Novagen (Darmstadt, Germany). E. coli strain
C41 (DE3) [FꢁompT gal hsdSB (rB mBꢁ) dcm lon l(DE3)] was
ꢁ
obtained from Avidis (Saint-Beauzire, France) and chaperone
competent cell pGro7/BL21 (DE3) was purchased from TaKaRa
Bio, Inc. (Otsu, Japan). M. ruber DSM 1279 was cultivated over-
ꢂ
min at 65 C, and denatured proteins were removed by centri-
fugation (10 000 ꢃ g, 15 min, 4 ꢂC). Purication was performed
night in potato P-YE thermus medium (potato starch 5 g lꢁ1
,
¨
¨
on an Akta explorer FPLC-system (GE Healthcare, Munchen,
Germany). Ten milliliters of diluted protein solution were
loaded on a 5 ml pre-equilibrated Q-Sepharose (GE Healthcare,
peptone 5 g lꢁ1, yeast extract 0.2 g lꢁ1) at 60 C and 180 rpm.
ꢂ
Cloning and expression optimization of the M. ruber laccase
(Mrlac)
¨
Munchen, Germany) column. The column was washed with ve
column volumes of 20 mM potassium phosphate buffer, pH 7.6.
Genomic DNA from M. ruber was extracted by using the Prom- Laccase was eluted in a stepwise gradient with 0–1 M NaCl in 20
ega Wizard Genomic DNA purication kit. Amplication of the mM potassium phosphate buffer, pH 7.6. Fractions containing
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laccase activity (as measured using the ABTS assay) were pooled
and concentrated by ultraltration (membrane cutoff 30 kDa,
Millipore, USA). Protein concentration was determined using
the Bradford assay with bovine serum albumin used as a stan-
dard. Sodium dodecyl sulfate-polyacrylamide gel electropho-
resis (SDS-PAGE) was done in a Minigel-Twin (Biometra,
Goetttingen, Germany) using a 10% polyacrylamide running
gel.
Homology modeling and validation
The 3D homology model of Mrlac was generated using the Build
Homology Models (MODELER) module in Discovery Studio 4.0
(DS 4.0; Accelrys Soware Inc., San Diego, CA). The crystal
structure of Thermus thermophilus (PDB entry 2XU9) was used as
a template.26 Comparative modeling was performed to generate
the most probable structure of the query protein by alignment
with the template sequences. Sequence identity between the
target and the template was found to be 65% according to
BLAST parameters. The t of the models' sequence in their
current 3D environment was evaluated by Prole-3D Score/
Verify Protein as implemented in DS 3.1. The discrete opti-
mized protein energy score in MODELER was also calculated to
determine the quality of the protein structures. In order to
conrm consistency of the model for docking studies, model
validation was carried out using PROCHECK.
UV-visible and EPR spectra of the Mrlac protein
The UV-visible absorption spectrum was obtained at room
temperature in 50 mM citrate phosphate buffer (pH 7.5) using
a Shimadzu UV 3100 spectrophotometer. EPR spectra were
measured with a Bruker ESP-106 spectrometer. Puried enzyme
was added to a 4 mm Wilmad EPR tube, and frozen in liquid
nitrogen. The following parameters were applied: 9.65 GHz
microwave frequency, 10 mW microwave power, 10 G modula-
tion amplitude, 100 kHz modulation frequency, 328 ms time
constant, and 82 ms conversion time.
The removal of 2,4-dichlorophene (DCP)
DCP stock solutions were dissolved in 10% acetonitrile. The
assay was done with various concentrations of laccase (0.5–1.5 U
mlꢁ1) and DCPꢂ(0.3–0.6 mM) in 50 mM citrate phosphate buffer
(pH 5.5) at 50 C. The mixture was incubated for various time
Biochemical characterization Mrlac
The activity of Mrlac was tested in 50 mM McIlvaine's buffer (pH periods under continuous vibration (450 rpm). Controls for all
2.6–8) and 50 mM Tris–HCl (pH 8–9) at room temperature experiments included boiled enzymes and no enzyme.
following the oxidation of the substrates 2,20-azino-bis(3-
The degradation of 2,4-DCP was monitored by colorimetric
ethylbenzothiazoline-6-sulphonic acid) (ABTS (3 ¼ 36 000 Mꢁ1 assay.27 The assay solution was composed of diluted sample
cmꢁ1 at 420 nm)), 2,6-dimethoxyphenol (2,6-DMP (3 ¼ 27 500 (50.0 ml), buffer solution (5.0 ml, 100 mM aqueous ammonia),
Mꢁ1 cmꢁ1 at 469 nm)), and syringaldazine (SGZ (3 ¼ 65 000 Mꢁ1 potassium ferricyanide reagent (1.0 ml, 80 mg mlꢁ1 K3Fe(CN)6
cmꢁ1 at 530 nm)). The rates of substrate oxidation were deter- in water), and 4-aminoantipyrene (4-AAP) solution (1.0 ml, 2%
mined by measuring the absorbance increase at the respective 4-AAP in water). The resulting compound was a quinone type
wavelengths, and the molar extinction coefficients (3) were ob- dye with a characteristic absorption peak at 510 nm. Absor-
tained from the literature.24,25 The activity of puried laccase bance was used to calculate the concentrations of DCP
ꢂ
towards the ABTS substrate was examined at 20–80 C and the according to a calibration curve. Data collection was controlled
optimal pH value. For testing thermal stability, Mrlac and Tvlac by the program Tecan i-control, version 1.5.14.0 (Tecan). The
ꢂ
ꢂ
ꢂ
(benchmark) were incubated at 70 C, 75 C and 80 C for 0 to removal efficiency was calculated with the following formula:
480 min. Aer incubation, samples were immediately used to
C0 ꢁ C1
Removal efficiency ¼
ꢃ 100
measure residual activities with ABTS, as described above, and
the half-time of activity loss was determined by non-linear
regression using the equation of exponential decay (a(t) ¼ a0-
exp(ꢁt ln 2/T1/2)) by OriginPro 8.5 (OriginLab Corporation,
Northampton, MA, USA). Data were collected in triplicate.
Kinetic constants of Mrlac were determined with the
substrates 2,6-DMP, ABTS and SGZ. Kinetic constants were
calculated by the Michaelis–Menten method using the Graph-
Pad Prism 5 program.
C0
where C0 was the concentration of DCP at time 0, and C1 was the
concentration aer reaction in the presence of Mrlac. The
degradation of DCP was also monitored by an Agilent 1100
HPLC system (USA) equipped with a Kinetex C-18 RP-column
(4.6 mm ꢃ 150 mm). The analytes were eluted under isocratic
conditions using a mobile phase of 60% acetonitrile, 40% water
at ow rate of 1.0 ml minꢁ1. The DCP concentration was
detected using the UV-vis detector at 230 nm.
Effects of metal ions on the activity of puried Mrlac
Results and discussion
Identication, cloning and overexpression of the recombinant
The effects of various metal ions, at a concentration of 2 mM, on
puried Mrlac activity were evaluated by separately adding
different metal ions such as Cu2+ (copper sulfate), Mg2+
Mrlac
(magnesium chloride), Zn2+ (zinc chloride), Mn2+ (manganese To identify amino acid sequences corresponding to MCOs with
sulfate), Fe3+ (ferric chloride), Ba2+ (barium chloride), Ni2+ laccase activity, we performed a blast search in the ExPASy
(nickel chloride). Laccase activity determined in the absence of proteomic server. The search for putative MCOs within diverse
any metal ion was measured as a control (100%). Activities were bacterial genomes revealed a vast number of candidate genes,
measured at optimum pH and room temperature.
particularly in Bacilli, Streptomycetes, and Pseudomonads. We
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focused on MCOs with laccase activity from thermostable
organisms. The open reading frame (ORF) of Meiothermus ruber
(accession no. YP003506102) encodes a 499 amino acid protein
with a calculated molecular mass 55.4 kDa and a pI of 8.8.
Multiple sequence alignment, using ClustalW (http://
www.ebi.ac.uk/clustalw/), showed that Mrlac contains four
histidine-rich copper motifs which are characteristic of MCOs
(Fig. 1). Further, a relatively high sequence identity to thermo-
stable T. thermophilus laccase (65%) was found. The pET vector
is known as one of the most powerful expression systems. The
Mrlac gene was amplied from genomic DNA of M. ruber DSM
1279 and inserted into the pET22b+ expression vector under
control of the T7-phage promoter. A high level of expression of
Mrlac was achieved (Fig. S1a†), and distinct bands were detec-
ted with the expected molecular mass (ꢀ50 kDa). However,
almost the entire enzyme was collected into the insoluble
fraction as an aggregate without activity in the soluble fraction.
Purication and spectroscopic characterization of the
secreted protein
Aer expression of Mrlac under the optimized conditions, cells
were harvested and lysed by sonication. To simplify the
subsequent purication steps, the supernatant was heated to 65
ꢂC for 30 min. While most of the E. coli proteins precipitated, no
loss in activity was observed for Mrlac.
The protein was further puried using anion exchange
chromatography (Q-Sepharose). SDS-PAGE analysis of the
enzyme showed a band with a molecular weight of ꢀ50 kDa
(Fig. S1c†). When isolated, Mrlac from M. ruber has the typical
deep blue color generally observed for MCOs and the UV-visible
(UV-vis) spectrum of the protein showed a consistent intense
absorption band at ꢀ610 nm, originating from a Cys-S to T1
Cu(II)33 charge transfer transition (Fig. 2a).
Additionally in the UV-vis spectrum, a broad shoulder was
observed at ꢀ330 nm, indicative of an antiferromagnetically
coupled T3 binuclear Cu(II) pair. The EPR spectrum (Fig. 2b) of
the puried enzyme, had contributions from two paramagnetic
centers: (g ¼ 2.22/A ¼ 82 ꢃ 10ꢁ4 cmꢁ1, and g ¼ 2.24/A ¼ 192
A
common limitation of recombinant bacterial laccase
production is the formation of insoluble protein aggregates
known as inclusion bodies, and it is a well-known phenomenon
in heterologous laccase production, particularly in E. coli.10 In
order to resolve the problem of inclusion body formation, E. coli
cells were co-transformed with the pET vector containing the
Mrlac gene (T7 promoter, Amp resistance) and the pGro7 vector
encoding for GroEL and GroES chaperones (araB promoter, Chl
resistance). The use of chaperone co-expression to improve
recombinant protein production has been reported in the
literature.28–30 The co-expression of GroEL/ES prevented Mrlac
from forming inclusion bodies in the cell as demonstrated here.
The semi-microaerobic conditions also favored copper accu-
mulation in the cells.31,32 In addition to adding a copper
supplement to the medium we lowered the incubation
k
k
k
k
ꢃ 10ꢁ4 cmꢁ1) T1 and T2 Cu(II)'s, respectively. These results are
consistent with reported EPR spectrum for B. subtilis CotA34 and
other LMCOs,14,31 suggesting a similar chemical structure
around the copper sites.
Biochemical properties of Mrlac
Enzyme activities towards nonphenolic (ABTS) and phenolic
(2,6-DMP and SGZ) substrates were determined. The pH optima
of the Mrlac activities were determined to be approximately pH
5 for ABTS (Fig. 3a), pH 8 for 2,6-DMP and pH 7.5 for SGZ (data
not shown). The pH optima of Mrlac for the oxidation of ABTS,
SGZ and 2,6-DMP are similar to those of other bacterial lac-
cases.12,13,35 As shown in Table 1, Michaelis–Menten parameters
were determined for ABTS, 2,6-DMP and SGZ at their optimal
pH and 40 ꢂC. Remarkably, Km values of Mrlac towards 2,6-DMP
and SGZ were much lower than those reported for CotA from B.
subtilis36,37 and other bacterial laccases.8,15,35,38,39
ꢂ
temperature to 20 C. The synergistic effect of co-expression of
GroEL/ES and the semi-microaeration led to expression of an
active, soluble enzyme. In this way, expression of active enzyme
was signicantly improved. The highest yields of active Mrlac
enzyme (measured on ABTS) were obtained with the GroEL/ES
ꢂ
co-expression aer 20 h at 25 C in LB medium supplemented
with 0.2 mM CuSO4 and 0.7 mg mlꢁ1 arabinose under static
Fig. S2† shows the predicated laccase-aided modication of
non-phenolic (ABTS) and phenolic (2,6-DMP) substrate during
oxidative catalysis. As shown in Fig. S2a,† in rst step of
conditions (Fig. S1a and b†).
Fig. 1 The amino acid sequence alignment of potential copper coordination sites of different fungal and bacterial laccases. Bacillus subtilis
(accession no. NP388511), Thermus thermophilus HB27 (accession no. YP005339), Meiothermus ruber (accession no. YP003506102), Lentinus
tigrinus (accession no. AAX07469), Trametes versicolor (accession no. AFM31222), Melanocarpus albomyces (accession no. Q70KY3). The
numbers 1, 2 and 3 corresponds to the coordination sites for the types 1, 2 and 3 coppers. The underlined sequence indicates the Meiothermus
ruber. Residue positions identical in all six sequences are indicated with dark green. The CLUSTAL X algorithm was used for alignment.
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form quinones (2,6-dimethoxy-p-benzoquinone). Further, olig-
omers of 2,6-DMP can form through two oxidative couplings
namely C–C dimers or C–O dimers.42,43
To determine the temperature dependence of the enzyme
ꢂ
activity, turnover studies were conducted with ABTS at ꢀ70 C
(Fig. 3b). Furthermore, thermostability was investigated by pre-
incubation of the Mrlac and Tvlac at 70, 75, and 80 ꢂC. As seen
in Fig. 3c, Mrlac showed moderate stability at elevated
temperatures, the half-life for deactivation was 120 min at 70 ꢂC,
ꢂ
ꢂ
67.1 min at 75 C and 28.1 min at 80 C.
However, the fungal laccase (Tvlac) used in this study was
not stable at elevated temperatures, the half-life for deactivation
was 5.7 min at 60 ꢂC, 2.7 min at 70 ꢂC and 2 min at 75 ꢂC
(Fig. 4d). The optimal temperature of Mrlac at approximately 70
ꢂC is identical to that observed for the CotA-laccase of B. sub-
tilis,12 but lower than the 92 ꢂC determined for the laccase of the
thermophilic bacteria T. thermophilus.13
The thermostability experiment (Fig. 3c) showed that the
new Mrlac is one of the most stable bacterial laccase with a half-
life 120 min at 70 ꢂC, in contrast to fungal laccase (Fig. 3d) and
other reported bacterial laccases.35,44,45
The stability of Mrlac is an excellent feature, especially when
compared to fungal laccases. However, the T. thermophilus lac-
case with a half-life of 868 min at 80 ꢂC is the most thermostable
laccase reported so far.13 The lower thermostability of the Mrlac
in comparison to the T. thermophilus enzyme supports results
from a previous analysis described by Miyazaki,13 in which
parameters known to affect protein thermostability have been
outlined. These include proline content (10% and 6.8% for T.
thermophilus laccase and Mrlac, respectively) and the aliphatic
index values, dened as the relative volume of the protein
occupied by aliphatic chains of alanine, valine, isoleucine, and
leucine (96.39% and 92.24% for T. thermophilus laccase and
Mrlac, respectively).
Effect of metal ions on Mrlac activity
Fig. 2 Spectroscopic properties of purified Mrlac. (a) UV/vis spectrum
of Mrlac in potassium phosphate buffer. Mrlac shows a laccase-typical
absorption spectrum. The maximum at 592 nm corresponds to type I
or blue copper and the shoulder around 330 nm is characteristic for
type 3 copper centers. (b) EPR spectrum of purified Mrlac (1.47 mg
mlꢁ1 in 50 mM phosphate buffer, pH 7.5) at 9.65 GHz microwave
frequency, 10 mW microwave power, 10 G modulation amplitude, 100
kHz modulation frequency, 328 ms time constant, and 82 ms
conversion time.
The enzyme activity was enhanced by metal ions such as Cu,
Mn, Ba (each at 1 mM), while the metals like Ni, Mg, Fe and Zn
reduced the activity considerably at a concentration of 1 mM
(Fig. S3†).
Previous research has demonstrated that Mn(II) oxidation in
several bacteria such as Leptothrix species,46 Pseudomonas
putida,47 Pedomicrobium species,48 and Bacillus species49,50 is an
enzymatic process, and many MCOs are postulated to be
directly involved. Furthermore, it was observed that Mn-
oxidizing ability was lost upon the disruption of a gene whose
sequence identies it as a MCO.48 The Mn2+ oxidation driven by
Mrlac may also have useful biotechnological applications in the
elds of biomaterials, enzyme catalysts, bioremediation, metal
recovery, and bioenergy.
oxidation, ABTS is freely oxidized by laccase to the cation radical
ABTS+c (forms intense green-blue colour).40 The cation radical
can be oxidized further to the dication (ABTS2+) (Christopher
et al., 2014).41 However, 2,6-DMP undergoes a single electron
oxidation to produce 2,6-dimethoxy-phenoxyl radical species
(forms intense red-brown colour) (Fig. S2b(I)†), that resonate
with corresponding para-radical species (Fig. S2b(II)†). It has
been postulated phenolic substrates release a proton and elec-
tron when they are oxidized to radical products by the enzy-
matic action of a laccase.42 These radicals can be react non-
enzymatically to other phenolic radical or molecular oxygen to
Overall structure description of Mrlac
The amino acid sequence of Mrlac was aligned with the laccase
of T. Thermophilus (PDB entry 2XU9) using the Align Multiple
Sequences module of DS 4.0 (Fig. S5†). A homology model of
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Fig. 3 Mrlac pH, temperature optimum, and thermostability. (a) pH-Dependent activity profile of Mrlac. The activity was measured at room
temperature using ABTS as the substrate, in 50 mM citrate phosphate buffers at the pH values indicated. (b) Effect of temperature on the activity
of Mrlac. The enzyme activity was assayed at each temperature in 50 mM citrate-phosphate buffer (pH 5.0) in the presence of 0.5 mM of ABTS.
Thermal inactivation of (c) purified Mrlac and (d) Tvlac. Purified enzyme was incubated in vials at different temperatures. At the indicated times,
samples (10 ml) were withdrawn and tested for laccase activity at room temperature by using ABTS as substrate.
˚
Table 1 Kinetic properties of recombinant Mrlac. 3max indicates molar
extinction coefficient
The RMSD was estimated to be 0.394 A on the basis of the C-
alpha atoms (Fig. S5a†). A calculated Ramachandran plot sug-
gested that 93.7%, 5.3%, 0.5%, and 0.5% of the residues in the
derived model are in, most favored, additional allowed, gener-
ously allowed and disallowed regions, respectively (data not
shown). Altogether, 99.5% of the residues were placed in the
favored and the allowed categories combined.
3max
Substrate (Mꢁ1 cmꢁ1
)
pH Km (mM) kcat (minꢁ1
)
kcat/Km (sꢁ1 mMꢁ1
)
ABTS
2,6-DMP 27 500
SGZ 65 000
36 000
5
27.3
325
115
106
0.19
0.63
0.42
8.0 3.01
7.5 4.2
A model structure with a high percentage of residues in the
favored and the allowed categories combined are likely to
represent a good protein fold. Thus PROCHECK validated the
folding integrity of our model and indicated that the model
structure derived from the 2XU9 template was of high quality in
terms of protein fold. Laccases are a family of enzymes typically
composed of three plastocyanin-like domains (Cu-oxidase super
family domains). The sequence alignments indicated that Mrlac
features four highly homologous regions, consisting of
histidine-rich motifs that constitute the copper-binding sites.12
Mrlac was generated based on the crystal structure of the lac-
case from T. Thermophilus (Fig. S5a†).
The root mean square deviation (RMSD) between the model
and template was calculated in order to determine the reliability
of the model by superimposing the model on the template
crystal structure.
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environment. Therefore, laccases are considered to be more
promising for enzymatic removal of phenol-compounds from
polluted wastewater.20 In the present work, the degradation of
DCP by Mrlac was studied, and the kinetic data were tted to an
intermediate model (Fig. 4a). The kinetic curves followed a rst-
order reaction equation: C ¼ C0 exp(ꢁk1t), in which C0 is the
initial concentration of the reactant, C is the concentration of
reactant at time t, and k1 is the reaction constant of a rst-order
reaction. All the correlation coefficients (R2) were in the range
0.96–0.98. The kinetic reaction constant, k1, which is related to
the degradation of DCP, was 0.0020, 0.0051 and 0.015 minꢁ1
,
for laccase concentrations of 0.5, 1, and 1.5 U mlꢁ1, respectively.
The removal percentage of DCP reached ꢀ92% within 180 min
with 1.5 U mlꢁ1 laccase, so for further study the amount of
laccase was set as 1.5 U mlꢁ1. Fig. 4b shows the reaction of
Mrlac with DCP concentrations in the range 0.4–0.6 mM. At 0.4
mM, DCP was completely removed from the solution over 240
min, while 60 and 38% of the 0.5 and 0.6 mM DCP were
removed over 300 min, respectively. The HPLC analysis of
sample collected at the beginning of reaction showed one major
peak for 2,4-DCP at retention time 3.40 (Fig. S6a†). As, the
reaction progressed the concentration of 2,4-DCP in reaction
mixture decreased and emergence of additional peaks were
observed due to degradation at retention time 1.80 min, 2.48
min (Fig. S6b†).
Consistent with the ndings in other studies using laccase
and chlorophenols,52,53 we found that M. ruber laccase could
oxidize DCP. Although the highest laccase activity was obtained
at 70 ꢂC, chlorophene removal assays were carried out at 50 ꢂC.
ꢂ
It seems Mrlac is more stable at 50 C and the temperature of
industrial effluent is typically between 40 and 50 ꢂC. As dis-
played in Fig. 4a, a high removal rate (0.015 minꢁ1) of DCP was
observed at 1.5 U mlꢁ1 Mrlac with ꢀ92% of the total DCP
removed.
Fig. 4 (a) Degradation kinetics curves of DCP by Mrlac. Laccase
concentrations (U mlꢁ1) 0.5 (C); 1 (B); 1.5 (;) and predicted
concentration profiles (solid lines) using a first order reaction model.
(b) Time course plot showing the degradation of various concentra-
tions of DCP (mM) 0.4 mM (C); 0.5 mM (B); 0.6 mM (;). Enzyme
dosage was 1.5 U mlꢁ1 and temperature 50 ꢂC.
Jeon et al.53 reported a maximum degradation efficiency of
ꢂ
ꢀ73% at a pH 5, temperature 30 C, time 24 h and an enzyme
and mediator (cocktail ABTS and vanillin) concentration of 3 U
mlꢁ1 and 2 mM respectively. However, Bhattacharya and
Banerjee18 reported ꢀ98% removal efficiency of 2,4-DCP at pH
6, temperature 40 ꢂC within 9 h. In another study Kim and
Nicell54 and Murugesan et al.55 used the synthetic mediators
ABTS, 1-HBT (1-hydroxybenzotriazole) and TEMPO (2,20,6,60-
tetramethoxy piperidine-1-oxyl), and the natural phenolic
compound syringic acid for triclosan (5-chloro-2-(2,4-
dichlorophenoxy)phenol) removal. However, despite their
effective removal of triclosan, it was found that the oxidized
ABTS radical were highly toxic in the Microtox toxicity test.54
Nevertheless, mediator usage for the degradation is being
limited due to their side reaction (with substrate or product)
which leads to reduced yield and productivity, high cost and
create extra pollution.5,56 It is important to remark that most of
the previous chlorophenol biodegradation studies were con-
The four highly conserved regions include “HPxHLHG” and
“HCHxxxH” motifs at domain 3 as well as “HxH” and “WYHxH”
motifs at domain 1 (x: any amino acids). The three-dimensional
structure of Mrlac built by homology modeling suggested that
residues in the T1 copper ion site present a planar triangular
coordination with two histidines (His427 and His485) and
a cysteine (Cys480), while the axial position of the T1 copper
consists of Met490 (Fig. S5b†). The T2 copper center is coordi-
nated to His430 and His105, whereas the T3 copper centers are
coordinated to three histidine residues each (His417, His432,
and His479 for T3 (a); and His107, His145, and His481 for T3
(b)) (Fig. S4c–e†).
Degradation of DCP using Mrlac
The interest in laccases has increased in recent years because of ducted over ꢀ9–24, while the Mrlac produced in the present
its ability to oxidize a variety of xenobiotic compounds in the study and under the selected conditions allowed a signicant
presence or absence of synthetic or natural redox mediators.51
reduction in chlorophenol concentration (0.4 mM) in a shorter
Unlike peroxidases, oxidation using laccases does not reaction time (4 h) and without any mediator.
require hydrogen peroxide, which is harmful to the
3916 | RSC Adv., 2016, 6, 3910–3918
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18 S. S. Bhattacharya and R. Banerjee, Chemosphere, 2008, 73,
81–85.
Conclusions
19 S. Li, X. Ma, L. Liu and X. Cao, RSC Adv., 2015, 5, 1902–1909.
20 J. Jia, S. Zhang, P. Wang and H. Wang, J. Hazard. Mater.,
2012, 205–206, 150–155.
21 S. Sharma, M. Mukhopadhyay and Z. V. P. Murthy, Sep. Purif.
Rev., 2013, 42, 263–295.
22 Z.-l. Li, J. Nan, J.-q. Yang, X. Jin, A. Katayama and A.-j. Wang,
RSC Adv., 2015, 5, 89157–89163.
23 Y. Wang, X. Chen, J. Liu, F. He and R. Wang, Environ. Sci.
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24 D. Litthauer, M. J. van Vuuren, A. van Tonder and
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25 D. Kalyani, S. S. Dhiman, H. Kim, M. Jeya, I. W. Kim and
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E. Rudino-Pinera, Acta Crystallogr., Sect. F: Struct. Biol.
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27 R. J. King, K. A. Short and R. J. Seidler, Appl. Environ.
Microbiol., 1991, 57, 1790–1792.
28 A. Kondo, J. Kohda, Y. Endo, T. Shiromizu, Y. Kurokawa,
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Bioeng., 2000, 90, 600–606.
We found that laccase from M. ruber (Mrlac) can be properly
folded and synthesized in E. coli under microaerobic conditions
during coexpression with a chaperone protein. Aer the
expression and purication step, its biochemical and kinetic
properties were studied. The spectrophotometric characteriza-
tion (UV-visible and EPR) and sequence alignment of Mrlac
showed that the enzyme has all the ngerprint spectral features
that are characteristic for laccases like MCOs. The puried
Mrlac laccase displayed the ability to oxidize chlorophenol in
a reduced reaction time compared to previously discovered
laccases without the addition of any meditator.
Acknowledgements
This research was partially supported by the Danish National
Advanced Technology Foundation via the B21st technology
platform (Grant no. 001-2011-4). The authors would like to
thank Dr Søren Brander for his help in EPR spectral analysis.
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