Báo cáo khoa học: Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme pdf

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

Department of Applied Microbiology and Gene Technology, TNO Nutrition and Food Research Institute,
Zeist, the Netherlands;
3
Unite
´
IRD de Biotechnologie Microbienne Post-Re
´
colte, IFR-IBAIM, Universite
´
s de Provence
et de la Me
´
diterrane
´
e, ESIL, Marseille, France
Pycnoporus c innabarinus laccase lac1 gene was o verexpressed
in Aspergillus niger, a well-known fungal host p roducing a
large amount of homologous or heterologous enzymes for
industrial applications. The corresponding cDNA was
placed under the control of the glyceraldehyde-3-p hosphate
dehydrogenase promoter as a strong and constitutive pro-
moter. The laccase signal peptide or the glucoamylase
preprosequence of A. niger was used to target the secretion.
Both signal peptides directed the s ecretion o f laccase into the
culture medium a s an active protein, but the A. niger pre-
prosequence allowed an 8 0-fold increase in laccase produc-
tion. The identity of the recombinant protein was further
confirmed by immunodetection using Western b lot analysis
and N -terminal sequencing. The m olecular mass of the
mature laccase was 70 kDa as expected, similar to that of the

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

copper proteins or blue copper oxidases. The ascorbate
oxidase and m ammalian plasma protein ce ruloplasmin are
other enzymes that were classified in the same family and
these have been studied extensively by biochemical and
structural characterization [9]. Laccases carry generally four
copper atoms per enzyme molecule. The four copper atoms
are distributed in one mononuclear (T1) and one trinuclear
(T2/T3) domain. The T1 (type-1) copper domain confers the
blue color of the enzyme and a characteristic adsorption of
light around 660 nm. The T2/T3 d omain (type-2 and type-3
coppers) is responsible of the adsorption of light at 330 nm.
The T1 c opper domain i s the primary electron acceptor
from the reducing substrate and electrons are transferred
from this copper to the two-electron acceptor type-3 copper
pair center [10,11]. Then, t he trinuclear center, which is the
dioxygen-binding site, accepts the se e lectrons with the
concomitant reduction of the molecular oxygen. This
three-step process allows the o xidation of phenolic com-
pounds, including polyphenols, methoxy-substituted mon-
ophenols, aminophenols and a considerable range of other
compounds [7]. Metal ions, such as Fe
2+
, and many
nonphenolic compounds, such as ABTS (2,2-azino-bis-
[3-ethylthiazoline-6-sulfonate]) are o xidized by laccases [ 12].
The biological function of most laccases is yet unclear.
They have been indicated to be i nvolved i n pigment
formation, lignin degradation and detoxification [7]. Never-
theless, laccases a re very interesting tools for industrial
applications, i.e. for bleaching i n pulp and paper i ndus-

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

an hyperglycosylation increasing the molecular mass to
110 kDa as compared to the 70-kDa wild-type protein [14].
The production level of the re combinant p rotein in Pichia
was h igh enough to a llow the first structure function studies,
but too low to consider industrial approaches. In o rder to
produce large-scale level of P. cinnabarinus laccase, we
expressed the corresponding cDNA in Aspergillus niger,a
filamentous fungal host known to overproduce homologous
and heterologous proteins of industrial interest. In addition,
this heterologous expression system would allow genetic
manipulation of the laccase gene.
EXPERIMENTAL PROCEDURES
Strains, culture media
Escherichia coli JM109 (Promega, Charbonnieres, F rance)
was used for construction and propagation of vectors.
A. niger strain D15#26 (pyrg
–
) [15] was used for h etero-
logous expression. After cotransformation with vectors
containing, respectively, the pyrG gene and the laccase
cDNA, A. niger was grown on selective solid minimum
medium (with out uridine) containing 70 m
M
NaNO
3
,7m
M
KCl, 11 m
M
KH

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

2
,6.4m
M
CuSO
4
,6.2 m
M
Na
2
MoO
4
, 174 m
M
EDTA).
Chemicals
Restriction enzymes and Pfu DNA polymerase were,
respectively, purchased from Life Technologies (Cergy
Pontoise, France) and Promega. [a-
32
P]dCTP was pur-
chased from Amersham Pharmacia Biotech (Orsay,
France). DNA sequencing was performed by Genome
Express (Grenoble, France).
Expression vectors
Two expression vectors were constructed using a PCR
cloning approach, and the cloned PCR products were
checked by sequencing. Table 1 shows the primers, vectors,
and restriction sites used in the cloning strategy, and Table 2
lists the p rimer sequences. Constructs pLac1-A and pLac1-
B contained the laccase cDNA corresponding to the laccase

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

4
,2m
M
MgSO
4
,
Table 1. Cloning strategy. For each expression vector are indicated the name of the primers u sed for amplification of the laccase cDNA and
addition of cloning sites, recipient Aspergillus expression vector and restriction sites used i n the final cloning procedure.
Expression
vectors
Primers
Cloning
vectors
Cloning site
restriction
fragments
Cloning site
vectors
Forward Reverse
pLac1-A Lac1/Afl Lac1/Bgl pNOM102
a
AflIII–BglII NcoI–BamHI
pLac1-B Lac1/BssH Lac1/Bgl pAN52–4
b
BssHII–BglII BssHII–BamHI
a
EMBL accession number Z32701;
b
EMBL accession number Z32750.
Table 2. Oligonucleotides used for c DNA amplification an d cloning. St, stop codon. Restriction sites are u nderline d.

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

incubated for 10 days at 30 °C and checked for develop-
ment of a green color.
From liquid culture medium, aliquots (1 mL) were
collected daily and cells were removed by filtration
(0.45 lm). Laccase activity in the culture supernatant was
assayed by monitoring the oxidation of 500 l
M
ABTS at
420 nm to the respective radical (e
420
¼ 36 m
M
)1
Æcm
)1
)
[18], in the presence of 50 m
M
sodium tartrate pH 4.0 at
30 °C (standard conditions). For the stability to the pH or
the optimal pH determination, syringaldazine (17 l
M
)was
also used as the substrate by monitoring the production o f
colored quinone at 530 nm (e
530
¼ 65 m
M
)1
Æcm

)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

Æmin
)1
) The blots were
probed with a
32
P-labelled probe consisting of the laccase
cDNA and for loading control a 18S P CR amplified DNA
was used as a probe. Blotted membranes were hybridized
overnight at 65 °C in a buffer containing 0.5
M
sodium
phosphate buffer pH 7.2 with 0.01
M
EDTA, 7% (w/v)
SDS, and 2% (w/v) blocking reagent (Roche Molecular
Biochemicals, Meylan, France). The most stringent posthy-
bridization w ash consisted of a 2 · 15 min in 0.2 · NaCl/
Cit (NaCl/Cit 20 ·:0.3
M
sodium citrate buffer pH 7.0,
with 3
M
NaCl) containing 1% (w/v) SDS at 65 °C. The
blots were exposed to X-ray film (Biomax MR, Eastman
Kodak Company, Rochester, NY, USA) overnight at room
temperature.
Purification of the recombinant laccase
In order to purify the recombinant laccase from A. niger,
850 m L of culture medium (4.7 IUÆmL
)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

M
NaCl and dialyzed against buffer A.
Characterization of the recombinant laccase
Protein analysis. Protein concentration was determined
according t o Lowry et al. [23] with bovine serum albumin as
standard. P rotein purification was followed by SDS/PAGE
on 10% polyacrylamide slab gels [19]. Proteins wer e stained
with Coomassie blue. Analytical isoelectric focusing was
performed with 2.5–5.0 gradient gels using a Pharmacia
LKB Phastsystem (Amer sham Ph armacia Biotech) accord-
ing to the manufacturer’s procedure.
N-Terminal amino-acid sequence determination. The
N-terminal sequence was determined according to Edman
degradation. Analysis was carried out on an Applied
Biosystem 470A. Phenylthiohydantoin amino acids were
separated by reverse phase HPLC.
Fig. 1. Laccase gene expression vectors. F or an explanation, see
Experimental procedures and Table 1.
604 E. Record et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Temperature and pH stability of the laccase. Aliquots of
purified laccase (100% refers to 0.5 and 0.8 UÆmL
)1
,
respectively, using ABTS and syringaldazine as substrate)
were incubated at v arious temperatures for different times.
After cooling at 0 °C, laccase activity was assayed at 25 °C
in standard conditions with ABTS. The effect of the pH on
the laccase stability was studied by incubating purifie d
laccase in 50 m
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

abilities to grow on a minimum medium plate without
uridine. For each construct, approximately 100 uridine
prototrophic transformants were obtained per microgram
of expression vector.
Cotransformants containing the laccase cDNA were
tested for laccase expression by growing on minimum
medium plates supplemented w ith ABTS. R ecombinants
expressing laccase were identified by the appearance of a
green zone around the colonies after 7–10 d ays at 30 °C.
Colored zones on plates were not observed in the case of
control transformants lacking the laccase cDNA. Thirty
positive clones w ere cultured in liquid for each construction
and then assayed at optimal day o f production Results for
laccase activity were ranging from 30–90 I UÆL
)1
(day 7) and
from 1800–7000 IUÆL
)1
(day 10), respectively, for A. niger
transformed by pLac1-A and pLac1-B. T he best clone was
selected for each construction in o rder to study the time
course of the laccase activity.
Study of the recombinant laccase production
in
A. niger
For both expression v ectors, the laccase activity was found
in the culture me dium, indicating that laccase was secreted
from A. niger. Activity was not found in the control c ulture
(transformation with pAB4-1, without pLac1). In both
cultures, mycelial dry weight increased until day 5, and

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.

Northern blot analysis was performed in order to check
the laccase gene expression during production (Fig. 4). An
18S gene probe was used as a control for the loading
difference. As seen in Fig. 2B, production of laccase by
pLac1-B increased until day 1 2. This is also supported by
continuous level of expression of the recombinant lac1
transcripts during the same growth period (Fig. 4).
Purification and characterization of the recombinant
laccase
Purification procedure. Recombinant laccase was purified
from a culture medium o f A. niger by three successive steps
(Table 3). Eight hundred and fifty millilitres of medium
0 2 4 6 8 10 12
0
5
10
15
20
0
5
10
15
20
0
50
100
150
0
5000
10000

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.

Molecular mass and isoelectric point. The homogeneity o f
the laccase was checked on an SDS/polyacrylamide gel and
the electrophoresis shows a single band of 70 kDa corre-
sponding to a purified laccase (Fig. 5). Analytical isoelectric
focusing of the recombinant laccase on a polyacrylamide gel
was performed to determine the isoelectric point. The
protein was, as the wild-type, very acidic and the pI
estimated to be 3.7.
N-terminal sequencing. The first 15 amino acids (AIG
PVADLTLTNAQV) of the recombinant laccase were
sequenced and aligned with the wild-type laccase. Results
from alignment reveals 100% identity between both
sequences confirming that t he 24-amino-acid GLA prepro-
sequence from A. niger was correctly cut off before the
mature N-terminal sequence of the protein.
Temperature and pH stability. In order to determine
temperature and pH stability, activities were measured after
various pretreatment using the standard protocol (Fig. 6 ).
As shown in Fig. 6., the recombinant protein was very
stable until 60 °C. At 65 °C, the half-time of the enzyme w as
 100 min, whereas at 75 °C, the laccase was completely
inactivated in less than 15 min. pH stability w as studied
between pH 2.5 and 5.0 and re sults showed that the
recombinant laccase was stable at pH 5.0 for at least
120 min. Below pH 5.0, the laccase activity decrea sed by
less than 10% after 180 min of incubation.
Effect of temperature and pH on laccase activity. Studies
of the recombinant laccase showed an optimal activity
between 65 °Cand70°C (Fig. 7). Testing the laccase
activity between pH 2.5 and 8 using syringaldazine as the

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

Western blot (lane 2) analysis of the culture medium. F or immuno -
detection, an tibodies raise d against Pycnoporus cinnabrinnus laccase
were used.
Laccase
18S
1 2 3 4 6 8 10 12
Table 3. Purification of the recombinant laccase.
Purification
step
Volume
(mL)
Protein
(mg)
Total activity
(IU)
Specific activity
(IUÆmg
)1
)
Recovery
(%)
Purification
(-fold)
(1) Crude extract 850.0 1365.0 4030 3.0 100 1
(2) Ultrafiltration 135.0 485.0 3790 7.8 94 3
(3) Precipitation 6.4 35.0 1400 40.0 35 13
(4) Mono Q 3.0 6.3 650 1030 16 34
Fig. 4. Nort hern blot analysis of the total RNA
isolated at various time from biomass aliquots of
A. niger transformed by pLac1-B. The laccase

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

)1
of recombinant laccase was obtained as
compared with 45 mgÆL
)1
of wild-type laccase from the
dikaryotic strain I-937 of P. cinnabarinus and 145 mgÆL
)1
from the derived monokaryotic strain ss3 of P. cinnabarinus.
In order to improve the secretion of the recombinant
laccase, the laccase cDNA was fused to the GLA prepro-
sequence and the production level markedly increased, up to
70 mgÆL
)1
. In previous work, w e have cloned and expressed
P. cinnabarinus laccase lac1 cDNA in Pichia pastoris using
the Lac1 signal peptide or that of the a-factor from
S. cerevisiae. Both constructions yielded the same level of
production, i.e.  8mgÆL
)1
[14]. In this case, the yeast
peptide signal was not more efficient for the triggering
laccase production even if the processing was correct in both
conditions. Several fungal laccase genes were already cloned
and heterologously expressed in S. cerevisiae [28], Tricho-
derma reesei [29] and Aspergillus oryzae [6,10,30]. Produc-
tion levels in yeast were quite low, i.e.  5mgÆL
)1
, though
filamentous fungal hosts allowed a production of
0

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.

1 2 3 4 5 6 7 8
0
20
40
60
80
100
Laccase activity (%)
pH
Fig. 8. Effect of the pH on the activity of the purified laccase. pH in the
range of 2.5–8 were tested with 500 l
M
ABTS (d)and17l
M
of
syringaldazine ( j) as the su bstrate.
Sd 1
94 kDa
67 kDa
43 kDa
30 kDa
20 kDa
Fig. 5. SDS/PAGE gel analysis of the pure laccase. Sd, molecular mass
standards a nd lane 1, pure recombinant laccase stained with
Coomassie blue.
Ó FEBS 2002 P. cinnabarinus laccase gene expression in A. niger (Eur. J. Biochem. 269) 607
10–20 mgÆL
)1
. The best production of recombinant laccase
was recently obtained w ith t he C oprinus c inereus laccase

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

that a difference in the carbohydrate composition could
increase the pH stability. Previously, the P. cinnabarinus
laccase produced in P. pastoris was demonstrated to have
a m olecular mass of 110 kDa instead of 70 kDa for the
native laccase, suggesting that an heterologous protein
with hyperglycosylation was produced [14]. This phenom-
enon was a lso d escribed for the Trametes villosa laccase
produced in A. oryzae [6]. Glycosylation was 0.5% of
the molecular mass of t he native laccase and and 10% for
the recombinant l accase. I n the heterologous production
of the P. cinnabarinus lac case in P. past oris [14] or the
T. villosa laccase in A. oryzae [6], additional carbohydrates
were added to the recombinant laccase, but had appar-
ently no effect on their enzymatic activity [6,14]. In our
experiment, t he recombinant laccase produced by A. niger
has the same molecular mass than the native laccase,
suggesting the absence of hyperglycosylation. For this
reason, A. niger seems to be the most adapted for fungal
laccase overproduction.
In conclusion, heterologous expression of a white-rot
fungal laccase gene was successfully performed for the first
time in A. niger. The production level allows structure–
function studies to be carried out and, in addition, the
recombinant laccase will be produced at a pilot scale level t o
improve the productivity and subsequently obtain large
protein amounts for industrial applications.
ACKNOWLEDGEMENTS
This research was supported by the European program, Quality of Life
and Manag ement o f Liv ing R esources (PELAS : (Peroxidases and
Laccases) Fungal metalloenzymes oxidizing aromatic compound of

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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