Expression of the
Pycnoporus cinnabarinus
laccase gene
in
Aspergillus niger
and characterization
of the recombinant enzyme
Eric Record
1
, Peter J. Punt
2
, Mohamed Chamkha
3
, Marc Labat
3
, Cees A. M. J. J. van den Hondel
2
and Marcel Asther
1
1
Unite
´
INRA de Biotechnologie des Champignons Filamenteux, IFR-IBAIM, Universite
´
s de Provence et de la Me
´
diterrane
´
e,
ESIL, Marseille, France;
2

native form, suggesting no hyperglycosylation. The recom-
binant laccase was pu rified in a three-s tep procedure
including a fractionated precipitation using ammonium
sulfate, and a concentration b y ultrafiltration followed by a
Mono Q column. All the characteristics of the recombinant
laccase are in agreement with those of the native laccase. This
is the first report of the production of a white-rot laccase in
A. niger.
Keywords:laccase;Pycnoporus c innabarinus; heterologous
expression; Aspergillus niger; fungal.
Laccases ( p-diphenol:O
2
oxidoreductase; E C 1 .10.3.2) are
multicopper enzymes catalyzing the oxidation of p-diphe-
nols with the concomitant reduction of molecular oxygen to
water [1]. They were first found in 1883 in the latex of the
lacquer tree Rhus vernicifera, in Japan [2]. Laccase activity
was then d emonstrated in fungi, plants and more r ecently in
bacteria [3]. Laccases are glycoproteins, usually monomeric,
although some multimeric structures were described in
Podospora anserina [4], Agaricus bisporus [5 ] and Trametes
villosa [6]. Laccases are hete rogeneous in their biochemical
properties and molecular structures. Generally, laccases
could be characterized by a molecular mass around
60–80 kDa, a pI of 3–6, a glycosylation corresponding to
10–20% of t he protein molecular mass and laccases exhibit
1–4 isozymes [7]. The optimum pH varies from 3 to 6
depending on the substrate [8]. They a re stable at temper-
ature around 50–60 °C.
Laccases belong to the group of enzymes called the blue

tries, for detoxification of recalcitrant biochemicals, for
Correspondence to E. Record, Unite
´
INRA de Biotechnologie des
Champignons Filamenteux, IFR-IBAIM, Universite
´
sdeProvenceet
de la Me
´
diterrane
´
e, ESIL, 163 avenue de Luminy, Case Postale 925,
13288 Marseille Cedex 09, France. Fax: + 33 4 91 82 86 01,
Tel.: + 33 4 91 82 86 07, E-mail: ?Abbrevia-
tions: ABTS, 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate]; IU, inter-
national units; GLA, glucoamylase; MnP, manganese peroxidase; LiP,
lignin peroxidases.
(Received 7 September 2001, revised 16 No vember 2001, accepted 20
November 2001)
Eur. J. Biochem. 269, 602–609 (2002) Ó FEBS 2002
bioconversion of chemicals o r treatment of beverages i n
agrochemical industry [3].
In our laboratory, we demonstrated , t he presence of two
isozymes, LacI and LacII, in the white-rot fungus Pycno-
porus cinnabarinus strain ss3, w hich is the monokaryotic
strain derived f rom the dikaryotic p arental strain I-937 [13].
The g ene encoding the laccase LacI was isolated and its
expression characterized (GenBank accession number
AF170093). The la ccase gene, lac1, was overexpressed
successfully in Pichia pastoris as an active protein but with

2
HPO
4
,2m
M
MgSO
4
, glucose 1% (w/v),
and trace elements (1000· stock solution consists of: 7 6 m
M
ZnSO
4
, 178 m
M
H
3
BO
3
,25m
M
MnCl
2
,18m
M
FeSO
4
,
7.1 m
M
CoCl

gene, lac1 from P. cinnabarinus (GenBankaccessionnoAF
170093) (Fig. 1). In pLac1- B, the 21 amino acids of the
laccase signal peptide were replaced by the 24 amino-acid
glucoamylase (GLA) preprosequence from A. niger.In
both constructions, the A. nidulans glyceraldehyde-3-phos-
phate dehydrogenase g ene (gpdA) promoter, the 5 ¢ untrans-
lated region of the gpdA mRNA, and the A. nidulans trpC
terminator were used to drive t he expression of the laccase
encoding sequence.
Aspergillus
transformation and laccase production
Fungal cotransformation was basically carried out as
described b y Punt & van den H ondel [16] u sing each of
the laccase expression vectors and pAB4-1 [17] containing
the py rG s election marker, in a 10 : 1 ratio. Transformants
were selected for uridine prototrophy. Cotransformants
containing expression vectors w ere selected a s described in
the following section.
In order to screen the laccase production in liquid
medium, 50 m L of culture medium containing 70 m
M
NaNO
3
,7m
M
KCl, 200 m
M
Na
2
HPO

Oligonucleotides Sequences Restriction sites
Lac1/Afl
TTC TGA ACA TGT CGA GGT TCC AGT C AflIII
MS R F Q S
Lac1/Bgl AC AGT AAC AGA TCT GCT CAG AGG TCG C BglII
St L D S
Lac1/BssH GC CAA GCG CGC CAT AGG GCC TGT G BssHII
AIGPV
Ó FEBS 2002 P. cinnabarinus laccase gene expression in A. niger (Eur. J. Biochem. 269) 603
glucose 10% (w/v), trace elements and adjusted to pH 5
with a 1-
M
citric acid solution were inoculated by
1 · 10
6
spore sÆmL
)1
in a 300-mL flask. The culture was
monitored for 12 days at 30 °C in a shaker incubator
(200 r.p.m.). pH was adjusted to 5.0 daily with 1-
M
citric
acid. F or protein purification, 850-mL cultures w ere
prepared in 1-L flasks in the same conditions.
Screening of the laccase activity and laccase assay
Agar plate assay on selective medium (minimum medium
without uridine) with 200 l
M
ABTS were used for the
selection of transformants secreting laccase. Plates were

)1
)[6].
Activity is indicated in international units (IU) which are the
amount of laccase that oxidizes 1 lmol of s ubstrate per min.
Western blot analysis and laccase immunodetection
Proteins were electrophoresed in 10% SDS/polyacrylamide
gel according to Laemmli [19] and electroblotted onto
poly(vinylidene difluoride) membrane (Millipore) at
0.8 m AÆcm
)2
at room temperature for 2 h. Immunodetec-
tion was performed as previously described by Bonnarme
et al . [20]. The primary antibodies raised against laccase were
detected using alkaline phosphatase conjugated goat anti-
(rabbit Ig) Ig (Roche Molecular Biochemicals) at dilutions of
1 : 25 000 and 1 : 4000, respectively. Alkaline phosphatase
was color developed using the 5 -bromo-4-chloro-3-indoyl
phosphate/nitro blue tetrazolium a ssay [20].
Northern blot analysis
Total RNA was isolated at various time from biomass
aliquots of A. niger as indicated by W essels et al.[21].An
aliquot of 15 lg o f total RNA was denatured a t 6 5 °Cina
loading buffer mixture containing formamide and form-
aldehyde [22] and loaded on a 1% Tris/acetate/EDTA
agarose gel containing 6% formaldehyde [22]. After
electrophoresis, RNA was blotted onto Hybond N
+
and
UV crosslinked for 1 m in (0.6 J Æcm
)1

) was filtrated
(0.45 lm) and concentrated 6.3-fold by ultrafiltration
through a cellulose PLGC membrane (molecular mass
cut-off of 1 0 kDa) (Millipore). The medium was further
concentrated by a two-step a mmonium sulfate precipita-
tion. In the first step, ammonium sulfate was added with
stirring to a 40% (w/v) final concentration, and incubated
for 2 h at 4 °C. The precipitate was discarded by centrif-
ugation at 6000 g for 30 min The resultant supernatant was
then increased to 80% (w/v) saturation w ith ammonium
sulfate and stirred for 2 h at 4 °C. The precipitate was
collected by centrifugation at 13 000 g for 30 m in and
dissolved in 4 mL of buffer A (25 m
M
sodium acetate
buffer, pH 5.0). Ammonium sulfate was removed by an
overnight dialysis at 4 °C against buffer A. After dialysis,
the concentrate (6.4 mL) was diluted to 15 mL with buffer
A a nd loaded onto a Mono Q HR 5/5 column (Amersham
Pharmacia Biotech) equilibrated with the same buffer.
Unbound proteins were eluted with five column vol. of
buffer A. Bound proteins were then eluted with 40 mL of a
linear NaCl g radient (0–500 m
M
inbufferA)ataflowrate
of 1 mLÆmin
)1
and collected with fractions of 1 mL.
Laccase activity was eluted (3 mL) with fractions corre-
sponding to 350 m

citrate/100 m
M
phosphate bu ffer (pH 2.5–
5.0) for 180 min at 30 °C. Aliquots were transferred in
standard reaction mixtures to determine the laccase activity
with ABTS and syringaldazine.
Effect of temperature and pH on the laccase activity.
Purified laccase (100% refers to 0.5 and 0.8 U ÆmL
)1
,
respectively, using ABTS and syringaldazine as substrate)
was preincubated at various designed temperatures (25–
85 °C) and laccase activity was then assayed at the
corresponding temperature in standard conditions. For
the pH, laccase activity was assayed in 50 m
M
citrate/
100 m
M
phosphate buffer (pH 2.5–7.0) and in 50 m
M
phosphate buffer (pH 6–8) at 30 °C. ABTS was u sed a s
the s ubstrate in both experiments and syringaldazine for
optimal pH determination.
RESULTS
Transformation and screening
In a cotransformation experiment, A. niger D1 5#26 was
transformedwithamixtureofplasmidpAB4-1andeachof
the t wo expression vectors c ontaining the laccase cDNA
from P. cinnabarinus. T ransformants were selected for their

reached a maximum of 17–18 gÆL
)1
until day 12 (Fig. 2). In
addition the pH was maintained by supplementation with
citric acid around pH 5.0. For the first construction, pLac1-
A, the laccase activity reached gradually 90 IUÆL
)1
and was
more or less stable until day 12. Using the GLA s ignal
sequence instead of the laccase one, the laccase activity
reached a maximum of 7000 IUÆL
)1
, i.e. an increase of
80-fold as compared to the first construction.
Considering these results, the expression vector pLac1-B
was selected to characterize the recombinant laccase from
A. niger .
Immunodetection of the recombinant laccase
and expression of the corresponding gene in
A. niger
Production of the r ecombinant laccase for t he construc-
tion pLac1-B was checked by electrophoresis on an SDS/
polyacrylamide gel (Fig. 3). A clear band of around
70 kDa was observed corresponding to the wild-type
laccase from P. cinnabarinus. Immunodetection of the
laccase was performed using antibodies raised against the
P. cinnabarinus laccase. The Western blot analysis show ed
a unique band corresponding to the 70-kDa protein
demonstrating that this protein is the recombinant
laccase.

A
B
Laccase activity
(IU.L
−1
)
Laccase activity
(IU.L
−1
)
Mycelial dry weight and pH
(g.L
−1
)
Mycelial dry weight and pH
(g.L
−1
)
Incubation time (days)
Fig. 2. Comparison of laccase production using either the native or the
A. niger glucoamylase signal sequence in A. niger. Activity (m), mycelial
dry weight (j) and pH ( d) are p lotted a s a function o f tim e fo r p Lac1-A
(A) and pLac 1-B (B).
Ó FEBS 2002 P. cinnabarinus laccase gene expression in A. niger (Eur. J. Biochem. 269) 605
were concentrated 6.3-fold by ultrafiltration with a r ecovery
of 94%, t hen further concentr ated by a t wo-step ammo-
nium sulfate precipitation to 6.4 mL, i.e. a 133-fold total
concentration. The resulting laccase was loaded onto a
Mono Q column to be purified with a recovery o f 16%,
yielding 6.3 m g of laccase.

substrate showed optimum activity at pH 4.0 (Fig. 8). With
ABTS, activity increased when pH decreased, suggesting a
faster oxidation of ABTS to the corresponding radical
cation ABTSÆ
+
at low pH.
Kinetic properties. The Michaelis constant was measured
from a Lineweaver–Burk plot using ABTS as a substrate
with standard conditions in the range of 0.005–10 m
M
and
was estimated to be 55 l
M
.
DISCUSSION
White-rot fungi that d egrade lignin a nd cellulose secrete a
large range of extracellular enzymes allowing the complete
degradation of wood polymers. The degradation of cellulose
is mediated by cellulase enzymes that cleave the cellulose
chains at th e end (exo-glucanases, cellobiohydrolases) or in
the middle (endo-glucanases) of a chain an d then b-glyco-
Sd 1 Sd 2
94 kDa
67 kDa
43 kDa
30 kDa
20 kDa
Fig. 3. SDS/PAGE gel and Western blot a nalysis of the laccase pro-
ductionintheP. cinnabarinnus culture medium. Sd, molecular mass
standards; SDS/PAGE st ained w ith C oomassie b lue (lane 1) an d

cDNA from Pycnoporus cinnabarinnus was
used as th e probe. The 18S PCR amplified
DNA was used as the loading control.
606 E. Record et al. (Eur. J. Biochem. 269) Ó FEBS 2002
sidases that degrade the products of the cellulases [24,25].
Lignin degradation occurs through the action of oxidore-
ductases, such as manganese peroxidase (MnP), lignin
peroxidases (LiP) and laccase. These enzymes oxidize lignin
subunits via 1-electron abstractions, and this oxidation can
lead to nonenzymatic fragmentation reactions [26,27]. In the
white-rot fungus P. cinnabarinus I-937, neither lignin per-
oxidase nor manganese peroxidase were detected in lignin
degradation conditions [26]. For these r easons, we studied
P. cinnabarinus as a model t o explain the function of laccase
in wood degradation. We isolated the laccase gene from
P. cinnabarinus (GenBank accession number AF170093;
[14]) in order to obtain informations about the laccase
expression. In this work, we describe for the first time the
heterologous expression of a white-rot fungal laccase in th e
Deuteromycete A. niger. The recombinant laccase was also
purified to homogeneity and physico-chemically character-
ized in order to compare it’s properties to t hose of the wild-
type protein.
Two expression vectors were c onstructed containing the
cDNA encoding the P. cinnabarinus laccase eithe r with its
own signal peptide or fused with the GLA p reprosequence
from A. niger. Laccase activity was found in the extracel-
lular medium of A. niger cultures using both vectors, but
with a quite low production with laccase signal peptide. Less
than 1 mgÆL

20
40
60
80
100
0
50 100
150
Residual activity (%)
Time (min)
Fig. 6. Activity of the purified recombinant laccase after incubation at
various temperatures. Selected temperatures were 55 °C(d), 60 °C(j),
65 °C(m), 70 °C(r)and75 °C (+). Five hundred l
M
ABTS was used
as the s ubstrate for enzyme assay.
0
20
40
60
80
100
0 10 20 30 40 50 60 70 80 90
Laccase activity (%)
Temperature (°C)
Fig. 7. Effect o f the temperature on the activity of the purified laccase.
Various temperatures in the range of 25 °Cto85°C were tested with
500 l
M
ABTS as the substrate.

gene expressed in A. oryzae where r esults reached from 8 to
135 mgÆL
)1
[31]. I n conclusion, P. cinnabarinus lacc ase
production in A. niger was quite sa tisfactory and as this
host is perfectly adapted for industrial scale production,
next step will focus on the improvement of the production in
large-scale controlled fermentation.
The recombinant laccase was purified in a three-step
procedure and allowed to study the physico-chemical
properties of the recombinant enzyme for comparison with
native laccase. A ll the m ain characteristics of the recom-
binant enzymes, i.e. molecular m ass, pI, optimal temper-
ature and pH, stability to the temperature , N-terminal
sequence and the Michaelis constant, w ere compared to
those of the P. cinnabarinus laccase (data not shown).
N-Terminal sequence, molecular mass, and p I, are iden-
tical for both proteins, i.e. 70 kDa; pI around 3.7. The K
m
forABTSwasestimatedtobe55l
M
for the native and
the recombinant p rotein The optimal temperature varies in
the range of 65–70 °C, an d optimal pH is 4 for both
proteins. I n additio n, t he temperature stability was strictly
identical, and the pH stability seems to be higher for the
recombinant laccase as compared with the native form
(data not shown), i.e. half-time of the native is 60 min at
pH 3 instead of 10% loss of activity for the recombinant
for the same incubation time. This result could suggest

industrial interest) as well as GIS-EBL (Conseil Re
´
gional Provence-
Alpes-Coˆ te d’Azur and Conseil G e
´
ne
´
ral 1 3, France ). We thank Jea n-
Luc Robert for technical assistance i n enzymatic assays.
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