Available online at www.sciencedirect.com
Biochimica et Biophysica Acta 1784 (2008) 259 – 268
www.elsevier.com/locate/bbapap
Laccase of Cyathus bulleri: structural, catalytic characterization and
expression in Escherichia coli
Salony, N. Garg, R. Baranwal, M. Chhabra, S. Mishra ⁎, T.K. Chaudhuri, V.S. Bisaria
Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi 110016, India
Received 10 February 2007; received in revised form 18 October 2007; accepted 5 November 2007
Available online 22 November 2007
Abstract
Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white
rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of
some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% α-helical, 26% β-sheet and
38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher β-sheet structures.
The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this
was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was
denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under
alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The
laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be
expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Laccase; Cyathus bulleri; Circular dichroism spectroscopy; Laccase expression
1. Introduction
Lignin is a highly cross-linked aromatic polymer formed
from monomeric units of phenylpropanoid compounds. It acts
as a structural adhesive, holding cellulose microfibrils together
in the plant cell wall. Since the degradation of lignin is a major
obstacle to efficient utilization of lignocellulosic materials [1]
considerable work has been done in the area of microbial lignindegrading enzymes. The white rot fungi, which belong to
Basidiomycetes, are the only organisms capable of selectively
degrading lignin to carbon dioxide and water thereby increasing
the forage digestibility and adding to its protein content [2,3].
These fungi secrete extracellular enzymes like lignin peroxidase, managanese peroxidase and laccases, which are responsible for degradation of lignin. Laccases can oxidize phenolic
⁎ Corresponding author. Fax: +91 11 2658 2282.
E-mail addresses: [email protected], [email protected]
(S. Mishra).
1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2007.11.006
compounds, thereby creating phenoxy radicals, while nonphenolic compounds are oxidized via participation of cation
radicals in laccase mediated systems. Some white rot fungi
contain all the three classes of the lignin modifying enzymes
while others contain one or two classes of these enzymes [4].
Laccases (bezenediol: oxygen oxidoreductases [EC 1.10.3.2])
are copper containing enzymes which catalyze the oxidation of
a broad range of phenolic compounds and aromatic amines by
using molecular oxygen as the electron acceptor. They contain
four Cu (II) ions arranged in three different sites. The reaction
mechanism proposed for these enzymes is supported by the
electron transfer reactions that occur between cupric ions during
catalysis [5]. Laccases have become industrially important due
to their potential use in diverse applications which include
waste detoxification, textile dye transformation, personal and
medical care, biosensor and analytical applications [6]. For
some laccases, the substrate specificities have been extended to
non-phenolic subunits of lignin, various dyes, polyaromatic
hydrocarbons and polychlorinated biphenyls by using redox
mediators [7].
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Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268
Laccases are encoded by complex families of structurally
related genes. At least 29 fungal genes have been cloned which
include five from Trametes villosa [8,9] and Trametes
sanguinea [10], four from Rhizoctonia solani [11], three from
Basidiomycetes I-62 (CECT 20917) [12], two from Agaricus
bisporus [13], Pycnoporus cinnabarinus [14,15], Pleurotus
ostreatus [16] and one each from Neurospora crassa [17],
Coriolus hirsutus [18], Phlebia radiata [19], Coprinus
congregatus [20], Mauginiella sp [21] and Volvariella volvacea
[22]. Overall sequence identity between fungal laccases may be
low, but conservation is high within regions involved in copperbinding. Various fungal laccases have been expressed in
heterologous eukaryotic hosts such as Aspergillus oryzae
[9,23], Pichia pastoris [24–27], Saccharomyces cerevisiae
[28,29] and Trichoderma reesei [30]. Clearly, the expression of
laccases in eukaryotes also raises the question of alternative
glycosylation patterns coupled with the difficulty of large-scale
cultivation of many of these hosts, particularly the filamentous
fungi. It would be highly advantageous to express the laccase in
the bacterium Escherichia coli, for which a large number of
expression vectors are available and also cultivation strategies
for achieving high cell density fermentations.
The bird's nest fungus, Cyathus bulleri, colonizes dead
herbaceous stems, wood chips, dung, sticks and other woody
debris and has been found to be ecologically suitable for lignin
degradation [31]. We [32] and another group [33] have recently
reported on the purification of laccase from this fungus. The
enzyme exhibited some interesting catalytic properties and has
been used for decolourization of a number of reactive azo and
non-azo dyes [32,34]. In this paper, we describe some
structural and important catalytic properties of this enzyme.
We also report on the expression of C. bulleri laccase in E. coli
using a modification of the ligation-anchored (LA)-PCR
approach [35]. The expression was confirmed by zymogram
analysis making it the first fungal laccase to be expressed in
this bacterium.
2. Materials and methods
2.1. Organism and culture conditions
C. bulleri (Brodie) 195062 obtained from Canadian Type Culture Collection
was a kind gift from Dr. R. C. Kuhad (University of Delhi, South Campus). The
strain was maintained at 26 °C on malt extract medium with the pH set to 5.2.
For laccase production, the fungus was cultivated in basal liquid (BL) medium
[36] with 2,6-dimethylaniline as inducer as described previously [32].
The amino acid composition of several laccases was computed in mol percent
from the sequences downloaded from www.expasy.org. Only full length
laccases were used in the study. The SWISS PROT ID of the proteins analysed
in this study is listed at the end in Appendix-I.
2.4. Far-UV CD spectrum of laccase
The Far-UV CD spectrum of purified laccase was determined in 10 mM
sodium cacodylate buffer, pH 5.0 in a Jasco J-810 spectropolarimeter (Jasco
Corporation, Japan). For this, the enzyme was extensively dialyzed in this buffer
at a protein concentration of 0.5 mg/ml. The instrument parameters used for the
measurement were: sensitivity—100 mdeg, wavelength—200 to 250 nm, data
pitch—0.2 nm in continuous scanning mode. The scan speed was set at 50 nm/
min with a response of 1 s and band width at 1 nm as described previously [39].
The CD spectrum obtained from the instrument had the units in mdeg which
were converted to molar residue ellipticity by using the molecular weight and
concentration of protein solution with the help of software associated with the
instrument. Secondary structural elements were calculated from the experimental CD values in deg cm2 dmol− 1 using the software K2D [40].
2.5. Influence of pH and group specific inhibitors on laccase activity
Activity and kinetic parameters of purified laccase were determined using
2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) as the substrate
[36].The purified laccase (5 μM) was incubated with different ABTS
concentrations (0.01–10 mM) in 20 mM glycine–HCl buffer (pH 2.0–3.0) or
50 mM citrate buffer (pH 4.0–6.0). The apparent maximum velocity (Vmax)
and Km were determined from the double-reciprocal plot. The higher and the
lower pKa values were found graphically from the plot of logVmax against
pH [41].
The effect of various chelating, respiratory inhibitors and sulfhydral agents
[EDTA (0.1–1 mM), sodium azide (0.005–0.05 mM), kojic acid (0.5–10 mM),
DTT (0.01–1 mM), l-cysteine (0.05 and 0.1 mM) and p-coumaric acid (1 and
5 mM)] was studied with ABTS as the substrate. The different group specific
chemical modifiers like 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) and diethyl pyrocarbonate (DEPC) were also used to investigate the
chemical nature of the amino acids involved in enzyme activity. Based on
promising lead with EDC and the sharp decline in enzyme activity at pH
values greater than 4.5, this inhibition was studied in detail. The purified
enzyme (1.5 μM) was incubated with different concentrations of EDC (50–
200 mM) at 25 °C as described [42]. Aliquots of 50 μl were withdrawn at
regular time intervals and added to 50 μl of 50 mM citrate buffer, pH 6.0 to
quench the reaction. The residual enzyme activity was measured using
guaiacol as substrate and expressed as the percentage of unmodified control.
The log of percentage residual activity was plotted against time for different
EDC concentrations.
Pseudo-first order rate constants (kapp) were determined from the slopes of
semi-logarithmic plots of residual activity against time for different EDC
concentrations. The second-order rate constant was determined from the plot of
kapp vs. EDC concentration. The order of the reaction (n) was determined as
described [43]:
logkapp ¼ logk þ n
log
½I where k is the second-order rate constant and [I], the inhibitor concentration.
2.2. Purification of laccase
Routine assay of laccase was carried out using 10 mM guaiacol as substrate
in 50 mM phosphate citrate buffer, pH 5.0 as described [37]. The enzyme was
purified by 82-fold to a high specific activity of about 4000 U/mg protein as
described earlier [32]. The purified enzyme was stored at 4 °C and used for the
studies described in this paper.
2.3. Amino acid composition and comparison with generic laccases
Samples on PVDF membrane [38] were analysed for total amino acid
composition at the Protein Chemistry Core Laboratory at Baylor College of
Medicine, Houston, Texas. The results reported are the average of three runs.
2.6. Unfolding and refolding of purified laccase in the presence of
Cu2+ and Zn2+ ions
For unfolding of the purified laccase, chemical reagents like EDTA, DTT
and guanidinium hydrochloride (GdnHCl) were used at different concentrations
(EDTA: 1–100 mM, DTT: 50–200 μM, GdnHCl: 1–6 M). The conditions i.e.
time and concentration, were optimized for complete loss of activity at the
minimum inhibitor concentration with 0.1 mg of protein in a reaction volume of
one ml. The unfolded protein was refolded using alkaline conditions for 30 min
followed by addition of 1 mM copper or zinc. Aliquots were removed at regular
short intervals of time and activity of the enzyme monitored using guaiacol as
the substrate.
Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268
2.7. RNA isolation and enrichment of poly A+RNA
Table 1
Oligonucleotides used in the study
The fungus was grown in BL medium under static conditions as described
above. On day 5 of growth, 100 μM of 2,6-dimethylaniline was added and
mycelium were harvested after 24 h. Total RNA was isolated in a single-step
method using acid guanidinium thiocyanate-phenol-chloroform extraction
method [44]. RNA was enriched for polyA mRNA using the Qiagen kit. The
mRNA preparation was visualized using denaturing gel electrophoresis to verify
the quality of the prep.
Oligonucleotide
2.8. Cloning and expression of laccase in E. coli
The strategy of cloning is described in Fig. 1 and was a modification of the
LA-PCR method [35]. Briefly, the mRNA was transcribed to cDNA by using
Superscript reverse transcriptase II (Life technologies) using oligo dT as a
primer according to the instructions. The 3′-end of the reverse transcribed single
stranded cDNA was ligated to a 5′-phosphorylated (with polynucleotide kinase),
3′-end blocked (with ddATP using terminal deoxynucleotidyltransferase) anchor
oligonucleotide (5′ T CCC TTT AGT TGA GGG TTA ATA TAA GCG GCC
GCG TCG TGA CTG GGA GCG C 3′) using T4 RNA ligase as described [35].
The resultant anchored product was subjected to PCR amplification using the
forward primer P 2 (5′ ATT AAC CCT CAA CTA AAG GGA 3′), based on the
sequence within the anchor, or, LCC-NS/LCC-ND, based on the published [32]
peptide 2 sequence, PDGFP. This peptide was hypothesized (based on
261
Sequence
LCC-CS
LCC-CD
LCC-CDT
LCC-NS
LCC-ND
Anchor
5′-GGG TTG TCG TAG TTG TA-3′
5′-GGR TTR TCR TAR TTR TA-3′
5′-CCC TCA NGG RTT RTC RTA RTT RTA-3′
5′-CCN GAT GGN TTR CCC G-3′
5′-CCN GAY GGA TTR CCA G-3′
5′-T CCC TTT AGT TGA GGG TTA ATA TAA GCG
GCC GCG TCG TGA CTG GGA GCG C-3′
P 2 (Complementary 5′-ATT AAC CCT CAA CTA AAG GGA-3′
to anchor)
P3
5′-ATC AAC TCG GCY ATC CT 3′
P4
5′-SGG GTT GTC GTA GTT GTA 3′
Where R = A/G, N = A/C/G/T, Y = C/T, S = C/G.
comparison with other published laccase protein sequences) to be towards the
N-terminus of the purified laccase of C. bulleri. These two forward primers were
used for amplification of laccase specific gene in conjunction with a number of
reverse primers. These were (LCC-CS, LCC-CD, LCC-CDT) and were
designed based on the internal peptide sequence 1 (YNYDNP) of the purified
C. bulleri laccase [32].This sequence was proposed to be towards the Cterminus of the laccase, based on comparison with published laccase sequences.
The sequences of these reverse primers are listed in Table 1. Within the reverse
primer either a termination codon was introduced (LCC-CDT) or not introduced
(LCC-CD) leaving the product that would be translated, to run into a termination
codon naturally on the plasmid vector. The PCR was performed with 2 μl
aliquots of the terminated ligation mixture in 50 μl reaction volume containing
Taq polymerase (Perkin Elmer). The reactions were run using thermal cycler
(Perkin Elmer) with a temperature program of: initial denaturation at 94 °C for
5 min, followed by 30 cycles of initial denaturation at 94 °C for 1 min, annealing
at 48 °C for 1 min and extension at 72 °C for 1 min and a final extension at 72 °C
for 10 min. Appropriate changes in annealing temperatures were made
depending on the primer sequences. The resultant products were run on 1%
agarose gel to confirm nature of amplifications. The PCR reaction products,
where amplification of high molecular weight DNA had occurred (Fig. 5, lane2)
, were purified by Clean Genei Kit (Bangalore Genei Pvt. Ltd., Bangalore) and
ligated to pCR 2.1 vector that has 3′ T overhangs. The ligation mixture was
transformed into One Shot cells (INVαF′, TOP 10 F′) (Invitrogen) as per
manufacturer's instructions. The transformants were plated on LB agar plate
containing X-Gal and 50 μg/ml kanamycin and 50 μg/ml ampicillin. The stable
white transformants were screened for the laccase gene by colony hybridization
using the same degenerate primer (LCC-CD) that was used for amplification.
The conditions for preparation of transformants for colony hybridization were as
per standard protocols [45].
2.9. Labelling of oligonucleotide and hybridization
Fig. 1. Protocol for modified LA-PCR approach. First strand cDNA synthesis
was performed with avian myeloblastosis virus reverse transcriptase and oligo
(dT) as primer [35]. The 5′-phosphorylated, 3′ blocked anchor oligonucleotide
was ligated to first strand cDNA. Two primers, P2, specific to a sequence within
the anchor and the second reverse primer was designed based on one of the
internal peptide sequences [32] of purified laccase. The reverse primer had either
a termination codon or did not have a termination codon as described in the text.
The PCR products (solid box) were independently cloned into PCR 2.1 vector
(Invitrogen).
The oligonucleotide (LCC-CD) was labeled with [γ-32P] ATP (BRIT,
Hyderabad) using the enzyme T4 polynucleotide kinase. The reaction mixture
contained the oligonucleotide (50 pmol), 2 μl of 10× bacteriophage T4
polynucleotide kinase buffer, 5 μl of [γ-32P] ATP, 11.4 μl of sterile water and
1 μl (10 U) of the enzyme. The efficiency of the transfer of γ-32P to the
oligonucleotide was checked by measuring the amount of radioactivity using a
scintillation counter (Kontron, Switzerland). The pre-hybridization and
hybridization (∼ 5000 cpm) was carried out at 50 °C. The hybridized and
washed membrane was exposed to X-ray film (X-OMAT, Kodak) and incubated
at − 70 °C overnight. The film was developed in an automatic X-ray developer
(Kodak).
2.10. Screening of positive recombinant clones by expression
Single colonies of the transformants which gave a positive signal with the
probe were grown in LB + ampicillin (50 μg/ml) at 37 °C overnight. This was
used to start a fresh day-culture in a 25 ml flask. At OD600 of 0.5–0.6, IPTG
(final concentration 1 mM) was added for induction. The cells were harvested by
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Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268
centrifugation at 5000 g at regular intervals. The supernantant was collected
separately and the cell pellet was washed with phosphate buffered saline, pH 7.2.
The cell pellet was suspended in 50 μl SDS-PAGE buffer and directly loaded
(without boiling) on 10% SDS-PAGE. The gel was electrophoresed at 4 °C.
Activity was detected by zymogram staining (100 ml of staining solution
contained 60 mg diaminobenzidine and 30 mg NiCl2.6H2O in 50 mM sodium
acetate buffer, pH 4.5) as described earlier [32]. A control host with only the
vector and a preparation of the purified C. bulleri laccase (∼ 8 μg protein) were
run on the same gel for comparison.
Table 2
Comparison of mol % amino acid composition of Cyathus bulleri laccase with
other known generic laccases (Swiss PROT ID of laccases is provided in
Appendix-I)
2.11. Determination of the partial sequence of the laccase gene
Glu(E) + Gln (Q)
An aliquot (5 μl) each of the reverse transcribed mRNA and PCR amplified
product obtained using P2/LCC-CD (Fig. 5, lane2) was again used in a new PCR
reaction set up in a total volume of 25 μl. The primers (Takara, HPLC purified)
used were:
Phe (F)
Gly (G)
His (H)
Iso (I)
Lys (K)
Leu (L)
Meth (M)
Pro (P)
Arg (R)
Ser (S)
Thr (T)
Val (V)
Trp (W)
Tyr(Y)
(corresponding to the
published [32] internal
peptide sequence, G(?)
reported earlier was not
used in designing the
primer as in most laccases the analogous
amino acid is N)
(the complementary
sequence of which is
given below)
(corresponding to the published internal peptide sequence of C. bulleri laccase
[32]).
The reaction vial contained 0.2 mM of each dNTP, 50 pmol each of P3 and
P4 and 1.25 U of Takara Ex Taq™ (Takara Bio Inc., Japan). The conditions for
PCR amplification were initial denaturation at 94 °C for 7 min followed by
35 cycles of 1 min denaturation at 94 °C, 1 min annealing at 48 °C and 2 min
extension at 72 °C. The final extension was carried out for 10 min at 72 °C. A
10 μl aliquot was run on the gel to confirm amplification of the product. The
fragment was eluted out of the gel using Qiagen gel cleaning kit and
nucleotide sequence determined on an automated DNA sequencer (ABI prism ,
Model 3730) at the Department of Biochemistry, Delhi University South
Campus. Both P3 and P4 were used for sequencing of the fragment. The
deduced amino acid sequence was submitted to NCBI and % sequence
similarity noted with the reported laccase sequences. The top 10 similar
sequences were aligned with the Clustal V program [46] of DNA-Star (for
details, see legend to Fig. 6). The sequence of the 450 bp fragment of laccase,
reported in this paper, has been deposited in the GenBank database under
Accession No. EU195884
3. Results
3.1. Amino acid composition of purified laccase and
comparison with generic fungal laccases
The amino acid composition of the purified laccase was
compared with the mol% values obtained for other fungal
laccases (Table 2). The results indicated a high proportion
(N21.44%) of Asp, Asn, Glu and Gln and 7.91% of basic
amino acids Lys and Arg in the purified laccase. However, the
C. bulleri enzyme showed lesser percentage of hydrophobic
(Phe, Iso, Leu, Pro, Val, Tyr) amino acids. This was interesting
Amino acid
Mol% of generic laccases
Mol% of C.bulleri⁎
Ala (A)
Cys (C)
Asp (D) + Asp (N)
7.73 ± 1.02
1.10 ± 0.09
8.88 ± 1.16
7.48 ± 0.76
2.68 ± 0.78
4.40 ± 0.93
8.58 ± 1.11
5.51 ± 0.48
4.97 ± 0.58
6.71 ± 0.79
1.82 ± 0.60
9.83 ± 0.91
1.37 ± 0.34
8.34 ± 0.83
5.17 ± 0.79
7.06 ± 0.86
8.69 ± 1.28
8.81 ± 0.94
2.60 ± 0.28
4.89 ± 0.55
8.37
–
10.17
11.27
3.28
17.76 a
4.75 b
4.32
4.06
7.22
–
4.29
3.85
9.82
6.04
4.96
not determined
2.08
⁎Values represented are averages for 3 replicates. (–) = Hydrolysed and cannot
be determined from this analysis.
a
Most likely due to transfer conditions in Tris–glycine buffer.
b
The value is an estimate, as it could not be quantitated accurately due to
closeness to Gly peak.
in view of the fact that very low standard deviation (0.02–
1.28%) was obtained for the amino acids within the general
laccase category. The unusually high content of glycine
obtained for the laccase of C. bulleri was possibly due to
transfer conditions.
3.2. Far-UV CD spectrum of laccase
The Far-UV CD spectrum of the purified laccase is shown
in Fig. 2. The CD spectrum showed two negative troughs,
one around 220 nm and the other at 208 nm which are a
typical signature for all α, all β or α + β proteins with
different intensities corresponding to the extent of secondary
structural elements [47]. For the a/β proteins, the trough at
the shorter wavelength is always skewed towards 220 nm and
one broad negative trough is observed around 220 nm. On
the other hand, proteins with no organized structure display a
negative trough around 200 nm. The content of secondary
structure in the purified laccase was calculated from the
experimental CD values according to Yang et al. [40] based
on model peptides. It contained approximately 37% α-helix,
26% β-sheet and 38% random coil. Based on these
observations, the laccase was proposed to belong to α + β
structural class.
3.3. Effect of pH on laccase activity
The effect of pH on laccase activity was determined by
studying the kinetic parameters on ABTS. The range of pH
Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268
263
constants as a function of EDC concentration. This was
determined to be 1 × 10− 4 mM− 1 min− 1. Analysis of the
order of inactivation with respect to EDC concentration
yielded a slope of 1.32 (Fig. 4B) indicating that one molecule
of EDC binds to one molecule of enzyme when inactivation
occurred.
3.4. Unfolding and refolding of purified laccase
Fig. 2. Far-UV CD spectrum of purified laccase in 10 mM sodium cacodylate
buffer pH 5.0 at 25 °C. Protein concentration was 0.5 mg/ml. CD spectrum was
measured in a quartz cuvette of 1 cm path length and the value of ellipticity was
converted to molar ellipticity as described in the text.
selected was 2.0–6.0. The value of Vmax was calculated from
the plot of 1/V vs. 1/[s] at each pH values. The values of pKa1
and pKa2 were determined graphically as shown in Fig. 3 and
found to be 3.5 and 4.65 respectively. A sharp drop in activity
was seen at pH values lower than 3.0 and more than 4.65
suggesting titration of important catalytic groups or enzyme
instability in these pH regions. Since the enzyme has been
reported to be stable up to pH 6.0, the decline in the activity at
pH N 4.5 appeared to be linked to ionization of an important
carboxylic group (Asp/Glu).
The sensitivity of the purified laccase towards several reagents was studied. The inhibitor sodium azide (at 0.05 mM),
Kojic acid (5 mM), reducing agents DTT (1 mM), and
l-cysteine (0.1 mM) completely inhibited laccase activity.
The metal chelators like p-coumaric acid and EDTA showed
partial inhibitions which was about 60% with 5 mM pcoumaric acid and 67% with 1 mM EDTA. There was no
evident inhibition on the activity of laccase in the presence
of DEPC which couples His when it was tested in the range
of 5 μM–50 mM. However, the group specific agent EDC,
(at a concentration of 200 mM) nearly completely (80%)
inhibited laccase activity. The kinetics of inhibition was
followed with EDC at various initial concentrations of the
inhibitor (50–200 mM) and the results are shown in Fig.
4A. EDC caused a time and concentration dependent
decrease in activity towards guaiacol. The semi-logarithmic
plots of residual activity as a function of time were biphasic,
indicating the complexity of EDC induced inactivation. The
pattern of this inhibition can be resolved into two first order
processes with slope of longer times determining the rate of
inactivation. The pseudo-first order rate constants were
0.0038 min− 1, 0.0176 min− 1, 0.0185 min− 1 and 0.025
min− 1 at 50 mM, 100 mM, 150 mM and 200 mM of EDC
respectively. The second-order rate constant of inactivation
was determined from a plot of pseudo-first order rate
The purified laccase was subjected to unfolding using
EDTA, GdnHCl and DTT. The purified protein was incubated
with the respective denaturant for one hour and laccase activity
measured using guaiacol as the substrate. It was found that
EDTA at a concentration of 1 mM, GdnHCl at 4 M and DTT at
100 μM resulted in loss of activity of the enzyme. The time for
denaturation with EDTA, DTT and GdnHCl was optimized to
be 1 hr. Since EDTA is a metal chelating agent, at high
concentration (1 mM), it was assumed that it bound to the metal
ions, resulting in the loss of enzyme activity. The denatured
protein (with EDTA, DTT, GdnHCl and combination of the
three) diluted 100-fold with the citrate buffer (pH 5.5) did not
show much activity against guaiacol. Since laccase is a metalloprotein, the refolding experiment was done in the presence of
copper. Addition of copper at pH 5.5 led to only 45% recovery
of the original activity. Longer times of incubation for refolding
resulted in precipitate formation. However, when refolding was
attempted at an alkaline pH of 8.0 for about 30 min and copper
was added at the end of the refolding period, almost 100%
activity was recovered in 10 min after addition of copper. The
refolding was also carried out in the presence of zinc as the
atomic radii of copper and zinc are similar. The refolding of the
denatured protein (with mixture of EDTA, DTT and GdnHCl)
was done in citrate buffer (pH 5.5) and zinc was added after
60 min. In this case, 100% activity was recovered in about
5 min.
3.5. Cloning of laccase and its expression in E. coli
The LA-PCR method was used to clone the laccase gene.
The results of the PCR experiment are shown in Fig. 5A. As
Fig. 3. Effect of pH on catalytic activity of purified laccase of C. bulleri. The
enzyme was incubated with six different substrate concentrations, prepared in
buffers ranging from pH 2.0 to 6.0 and Vmax was calculated at each pH from the
double-reciprocal plots. The enzyme was stable in the pH range of 3.0–6.0.
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3.6. Partial sequence of the laccase gene
The partial sequence of the laccase gene was determined
from a PCR amplified fragment from the reverse transcribed
(RT) mRNA. Using the primers P3 and P4 (see Materials and
methods), a DNA fragment of approximately 435 bp was
amplified from the RT mRNA and from the reaction product of
lane 2, Fig. 5A. Comparison of the nucleotide sequence with the
nucleotide sequences in gene databases was made using the
BLASTN program [48] which confirmed that the amplified
sequences were laccase specific. The alignment of the deduced
amino acid sequence in the 435 bp fragment with the
corresponding laccase sequences (based on highest amino
acid sequence similarity result from NCBI search) from other
Fig. 4. (A) Kinetics of inhibition of purified laccase with EDC. The enzyme
(1.5 μM) was incubated with 50 mM (♦), 100 mM (■), 150 mM (▴), or
200 mM (■) of EDC at 25 °C. Aliquots of 50 μl were withdrawn after different
intervals of time (as indicated on x-axis) and added to citrate buffer, pH 6.0 to
quench the reaction. Residual enzyme activity was measured against guaiacol
and was expressed as the percentage of unmodified control (taken as 100%,
log100 = 2). (B) A plot of log kapp (pseudo-first order rate constants for each
EDC concentration) as a function of EDC concentration to determine n (slope,
which represents the order of the reaction) as explained in the text.
seen, using P2 and LCC-CS, no amplification was observed
(Fig. 5A, lane 1). With P2 and LCC-CD, high molecular weight
DNA was amplified (Fig. 5A, lane 2). Use of P2 and poly dT
resulted in low amplification (lane 3). Interestingly, using P2
and LCC-CDT (T: containing a termination codon in the reverse
primer) no amplification was observed (lane 4), nor did
amplification result with P2 and LCC-ND (as expected). With
the specific or degenerate gene sequences of the conserved N
and C termini being used as the primers, no amplification was
observed. High molecular weight DNA was also amplified
using LCC-ND/poly dT primers (lane 7).
About 150 stable E. coli colonies were obtained on
transforming the ligated products of lane 2 and pCR 2.1 vector.
Screening with labeled primer LCC-CD resulted in 10 colonies
giving a positive signal. These were then analyzed for
expression of laccase activity by PAGE–zymogram analysis.
The high expression of laccase by one of the clones, pLCC 5, is
shown in Fig. 5B, lane 3. The purified laccase of C. bulleri (Fig.
5B, lane 1) served as a control and migrated along with the
protein mol wt marker of 66 . The host cells transformed with
only pCR 2.1 vector did not show any activity in the zymogram
test. The laccase expressed in E. coli appeared to be of higher
molecular weight compared to the native laccase.
Fig. 5. (A) Analysis of LA-PCR products by 0.7 % agarose gel electrophoresis.
Each PCR reaction used total cDNA as a template using different sets of primers.
Lane 1: P2/LCC-CDT, Lane 2: P2/LCC-CD, Lane 3: P2/poly dT, Lane 4: P2/
LCC-CDT, Lane 5: P2/LCC-ND, Lane 6:LCC-NS/poly dT, Lane 7:LCC-ND/
poly dT, Lane 8: λ-DNA-EcoRI/HindIII cut. (B) PAGE-zymogram analysis of
the whole cell extract of E. coli containing either the vector (lane 2) or the
construct containing truncated version of the laccase gene of C. bulleri (lane 3).
The purified laccase of the fungus was loaded in lane 1 as a positive control.
Samples were solubilized in SDS solubilization buffer (but were not boiled) and
loaded in 10% SDS-PAGE gel. Electrophoresis was performed at 4 °C. After the
run, the gel was washed a few times in buffer and activity was detected by
zymogram staining (100 ml of staining solution contained 60 mg diaminobenzidine and 30 mg NiCl2.6H2O in 50 mM sodium acetate buffer, pH 4.5). The
bands appeared within 10 min of incubation. The position of the mol wt standard
markers (Bangalore Genei) is shown on the right, Myosin, Rabbit Muscle:
205 kDa, Phosphorylase b: 97.4 kDa, Bovine Serum Albumin: 66 kDa,
Ovalbumin: 43 kDa.
Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268
265
Fig. 6. Alignment of the deduced amino acid sequence of the PCR amplified 435 bp DNA fragment with the laccase sequences from Coprinopsis cinerea, Coriolopsis
gallica, Funalia trogii, Pholiota nameko, Pleurotus pulmonarius, Pleurotus sajor-caju, Phlebia tremellosa, Rigidoporus microporus, Schizophyllum commune.
Alignment was done with Clustal V program [46]. The program introduces gaps wherever necessary to maximize matches. Matching amino acids are shown in grey
color.
fungi is shown in Fig. 6. Maximum similarity (71%) was seen
with Laccase 3 from Coprinopsis cinerea [49] and laccase from
Rigidoporus microporus [50] followed by other fungi (60–
68%) which included Coriolopsis gallica [51], Funalia trogii
[52], Pholiota nameko [53], Pleurotus pulmonarius [54], Pleurotus sajor-caju [55], Phelebia tremellosa [56], Schizophyllum
commune [57]. The sequences included the conserved copperbinding domain II of laccases.
4. Discussion
The aim of the present study was to structurally and
catalytically characterize the purified laccase from C. bulleri.
This fungus was chosen as it is ecologically specialized in the
breakdown of plant components and has been reported to be a
selective lignin degrader. It produces sufficiently high amount
of laccase (90 U/ml) which has been shown to be very effective
in decolorization of a large number of reactive dyes [32]. The
range of dyes decolorized was extended in the presence of the
redox mediator ABTS. The products of degradation of several
dyes were also found to be non-toxic for bacterial growth [34].
This prompted us to further investigate this enzyme.
On the basis of similarity of three internal peptides of the
purified laccase with other fungal laccases, it was concluded to
share significant similarity with these but on the basis of
exhibiting two different pH optima towards ABTS and
guaiacol, the enzyme appeared to be somewhat different. The
amino acid composition data indicated the mol% of charged
amino acids and their amides (Asn, Gln) to be similar to those
present in generic laccases but the proportion of hydrophobic
amino acids was found to be lower in the C. bulleri laccase. The
content of the secondary structure was estimated which
indicated that it had higher proportion of helical content and
the CD fit the α + β structure observed with synthetic peptides
containing 37% α-helix and 26% β-sheet. This was different
from the solved structures of several laccases. These enzymes
(with the full complement of copper atoms) consist mainly of
antiparallel β-barrels as observed in the T. versicolor [58–59],
266
Salony et al. / Biochimica et Biophysica Acta 1784 (2008) 259–268
Melanocarpus albomyces [60], Rigidoporus lignosus [61]
enzymes. This was also found in the copper depleted laccase
structure of Coprinus cinereus [62]. In the T. versicolor enzyme
[59], the third domain has the highest helical content with one
310-helix and two α-helices located in the connecting region
between the strands of the different β-sheets. This region in the
domain forms the cavity in which the type-1 copper is located.
However, the overall percentage of amino acids in the helical
conformation was much lower for the T. versicolor enzyme
compared to the laccase of C. bulleri. From our observations on
the CD spectra, we expect this laccase to belong to α + β
category and be structurally different from other laccases.
The purified laccase was less sensitive to metal chelation by
EDTA. In general, the inhibition by various chemical inhibitors
was similar to that exhibited by the P. cinnabarinus enzyme
[36]. One of the important findings in our work is the definitive
involvement of a carboxylic group [Asp/Glu] in laccase activity
based on modification with EDC. The drop in enzyme activity
at pH values lower than 3.0 was attributed to enzyme instability
while the drop in activity at pH N 4.5, was due to ionization of a
critical Asp/Glu residue, as indicated by the pKa2 value. The
group may have a critical structural or catalytic role as
confirmed by the results of Fig. 4A, where a strongly reacting
carboxylic group was observed leading to nearly 50% loss of
enzyme activity. The order of the reaction showed that one
molecule of EDC bound to one molecule of the enzyme, when
inactivation occurred. Based on sequence comparison of several
laccases of Ascomycete and Basidiomycetes, we have also
located an invariant Asp which could have an important
catalytic role (to be published).
Our results with unfolding and folding of C. bulleri laccase
gave some interesting insights into this metallo-enzyme for
which no information is available. The purified laccase was
denatured using EDTA at a concentration of 1 mM and with a
combination of the denaturants, complete loss of activity was
observed. The refolding of the protein was done with the
addition of copper under alkaline conditions. The whole process
was completed in 10 min and activity was regained to 100%.
The details of the experiment also indicated that addition of
copper in the initial stages of folding hindered the folding
process. Apparently, formation of an apo-enzyme (otherwise
complete except for the metal ion) was necessary which was
followed by final folding and activity regain on addition of
copper. The absolute requirement of copper was not found to be
necessary as zinc (which has a similar atomic radius to copper)
was equally effective in restoring enzyme activity. Thus, a
flexible role of the metal ion is indicated in laccase activity.
In this paper, we also describe a modification of the LA-PCR
approach for successful expression of laccase. The size of the
expressed laccase was larger than the purified laccase, due to
additional amino acids which may have been added due to
exclusion of a termination codon in the designed primer. The
exact size of the laccase expressed in E. coli could not be
determined as the protein samples were loaded without boiling
the sample. Either extra stability may have been conferred on
the laccase due to addition of extra amino acids or removal of a
part of the C-terminus may lend stability to the product. It has
been shown recently that when the C-terminus of the laccase of
M. albomyces was removed from the expression construct,
laccase production was considerably improved (six-fold)
compared to the full length construct [29]. The expression of
C. bulleri laccase in E. coli appeared to be under control of
the lac promoter. The partial sequence from the C-terminus of
the laccase was obtained which indicated that this part had fairly
high (60–71%) sequence similarity with other laccases.
However, the laccase sequence was sufficiently (about 30%)
different also, as being indicated from the amino acid
composition data.
In conclusion, some interesting and novel structural properties of a laccase are described in this paper which indicate the
“not so” critical role of copper atoms and important role of
carboxylic group containing amino acids. This study also
describes novel extension of the LA-PCR approach for general
expression of enzymes. It may be possible to produce other
laccases in E. coli as well facilitating the study of these enzymes
and their improvement for wider practical applications.
Acknowledgement
This work was supported by a grant from Department of
Biotechnology, Govt. of India, to one of the authors (S.M.).
Appendix A
The SWISS PROT ID of enzymes used in the study for
determining amino acid composition of laccases from white rot
fungi were: Q9UVQ2 (LAC I), Q9HDS9 (LCC3-1), O60199
(XA1B), Q99044 (LCC1_TRAVI), Q02497 (LAC 1-TRAHI),
Q8TFL8 (072-1), Q8TFM1 (Laccase III), Q8TG94 (Laccase
2), Q96UT7 (LAC1), O94222 (LCC2), O13448 (CVL3),
Q9P8G4 (LCC1), Q9HDQ0 (LCC1), Q8J1Y2 (LCC1),
Q96TR6 (LCC1), Q96VA5 (LCC1), O61263 (Bilirubin
oxidase), Q99056 (LCC5), Q12717 (LAC5-TRAVE),
O13456 (CVLG1), Q8TG93 (LAP1A), Q9UVQ5 (LAC1),
Q9Y781 (LCC2), Q12571 (Laccase), O13421 (POX2),
O13422 (POX3), Q8WZH9 (LELCC2), O74171 (Laccase),
Q99046 (LCC2), Q12718 (LCC2), Q96UK8 (LAC1), Q9Y782
(LCC3), Q9Y780 (LCC1), Q9HDS7 (LCC3-3), Q12739
(POX2), Q12729 (POX1), Q9UVY4 (LCCK), Q8WZI0
(LELCC2), Q8X1W3 (LCC2), Q9HFT4 (LAC4), Q12719
(Laccase 4), CAD 45377.1 (Laccase 1), AAF 06967.1, AAL
89554.1 (Laccase), Q99055 (Laccase).
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